2021 National Veterinary Scholars Symposium

Contents

Welcome...... 1 Program Schedule...... 2 Speaker Biographies...... 4 Award Winners...... 6 Abstracts Listed by Area of Research...... 8 Symposium Participants by College of Veterinary Medicine...... 36 Complete Abstracts (listed alphabetically)...... 51 Welcome

Welcome to the 2021 National Veterinary Summer Scholars Symposium, hosted by Iowa State University’s College of Veterinary Medicine! Even though you are joining us for this year’s symposium virtually and won’t be able to tour the nation’s first public veterinary college, I know our programming committee has put together an excellent and challenging series of sessions, keynotes and other activities for you. The NVSSS has long been the premier annual scientific gathering where veterinary students like yourself can showcase the many research accomplishments you have achieved this summer. The symposium will once again highlight the many different ways veterinary scientists advance basic and applied research to support global and human health. The essence of research is to inform action. For all of us that are involved in research, our ultimate hope is that our find- ings leads to actions that positively impacts society, no matter how small or large. Without action, there is really know purpose for research. I challenge to think about your research findings from this summer and how they someday will inform actions that make a difference. I hope your summer scholars experience was engaging and challenging. I know you join me in anticipating and from the research each of you conducted. Enjoy the symposium! Dan Grooms DVM, PhD Dr. Steven G. Juelsgaard Dean of Veterinary Medicine

Boehringer Ingelheim is delighted to continue our long-standing support of veterinary research by sponsoring this National Veterinary Scholars Symposium that will enable veterinary students and young researchers to share the results of their summer research, and we especially welcome this year’s Boehringer Ingelheim Veterinary Scholars. Now more than ever we realize the importance of developing and nurturing scientists with the skills and training to fulfill our needs for comparative animal research, animal and human food safety, and drug development if we are to continue to advance biomedical research. Eric Haaksma, Head of Animal Health Global Innovation, Boehringer Ingelheim

The AAVMC is proud to partner with Iowa State University on the presentation of the 2021 National Veterinary Scholars Symposium. Scientific explorations and advancements in the body of knowledge are more important than ever before as we face the impending disease and food security threats of the future, and veterinary medicine must play a role at the heart of that effort. This program encourages our students to gain experience, develop their skills and consider careers in this vital area of our profession. It also celebrates and illuminates the success of the important work they have accom- plished. The AAVMC is honored to be a part of it.” Andy MacCabe, CEO, American Association of Veterinary Medical Colleges

2021 National Veterinary Scholars Symposium • 1 2021 NVSS Program Schedule Wednesday, August 4 TIME Moderator: Yuko Sato, DVM, MS, Diplomate ACPV (Eastern Daylight Time) Assistant Professor at Iowa State University

11.30 AM – 11.40 AM Welcome Videos Dean Dan Grooms at Iowa State University and Boehringer Ingelheim

11.45 AM – 12.45 PM Poster Session 1

1.00 PM – 2.00 PM Keynote: Kathryn Marie Jones, DVM, PhD Associate Professor at Baylor College of Medicine, Houston TX “Addressing the Global Burden of Chagas Disease”

02.15 PM – 2.45 PM Boehringer Ingelheim Networking Session Leaning into Opportunities for Personal Development and Building your Network

3:00 PM – 4.00 PM Poster Session 2

4:15 PM – 4.45 PM Meet the Experts I – Breakout Sessions Career advice from veterinarians who work in: . • Academia . • Government . • Industry

2 • 2021 National Veterinary Scholars Symposium Thursday, August 5 TIME Debora Brito Goulart, BVetMed, MSc (Eastern Daylight Time) PhD student at Iowa State University

11.30 AM – 11.40 AM Welcome Videos Boehringer Ingelheim and the College of Veterinary Medicine at Iowa State University

11.45 AM – 12.45 PM Poster Session 3

1.00 PM – 2.00 PM Keynote: Jim Roth, DVM, MS, PhD, Diplomate ACVM Distinguished Professor at Iowa State University, Ames IA “Infectious Disease Control through Research, Policy Development, and Education”

02.15 PM – 2.45 PM Awards Presentation AVMA Excellence in Research Awards and Boehringer Ingelheim Veterinary Research Awards for Graduate Veterinarians and Veterinary Students

3:00 PM – 4.00 PM Poster Session 4

4:15 PM – 4.45 PM Meet the Experts II – Breakout Sessions Which degree should I get? Career advice from: • DVM/PhD Researchers • DVM/MSc Researchers • DVM Clinician Research Scientists without an Advanced Research Degree

Friday, August 6 TIME Moderator: Olufemi Fasina, DVM, PhD, Diplomate ACVP (Eastern Daylight Time) Assistant Professor at Iowa State University

11.30 AM – 11.40 AM Final Remarks and Boehringer Ingelheim Video Presentation

11.45 AM – 12.45 PM Poster Session 5

1.00 PM – 2.00 PM Keynote: Liara Gonzalez, DVM, PhD, DAVS Associate Professor at North Carolina State University, Raleigh, NC “Intestinal Regenerative Medicine: Targeting Key Stem Cells to Enhance Intestinal Viability”

02.15 PM – 2.45 PM Meet the Experts III – Breakout Sessions Career advice from veterinarians with research programs that focus on: • Companion . • Equines . • Production Animals

3:00 PM – 4.00 PM Poster Session 6

4:15 PM – 4.45 PM Meet the Experts IV – Breakout Sessions Career advice from veterinarians with research programs that focus on: • Pathogens and Vaccine Development . • Veterinary Epidemiology . • Diagnostic Pathology 2021 National Veterinary Scholars Symposium • 3 Keynote Speakers

Kathryn M. Jones, DVM, PhD Associate Professor Baylor College of Medicine

Presentation title & date: Addressing the Global Burden of Chagas Disease Wednesday, August 4 from 1.00 – 2.00pm EDT

Bio: Kathryn Jones is an Associate Professor in the Departments of Pediatrics, Section of Tropical Medicine, and Molecular Virology and Microbiology at Baylor College of Medicine. She is also the Director of the Pre-Clinical Testing Unit at the Texas Children’s Hospital Cen- ter for Vaccine Development, and the Technical Lead for the Chagas Vaccine Initiative. Dr. Jones received both her Doctor of Veterinary Medicine and PhD from State University. Dr. Jones’ current research projects focus on understanding mechanisms of inflamma- tion and fibrosis caused byTrypanosoma cruzi, the parasite responsi- ble for Chagas disease, and developing novel immunotherapies.

Jim Roth, DVM, PhD Distinguished Professor Iowa State University

Presentation title & date: Infectious disease control through research, policy development, and education Thursday, August 5 from 1.00 – 2.00pm EDT

Bio: Jim Roth is a Distinguished Professor in the College of Veterinary Medicine at Iowa State University and a member of the National Academy of Medicine. He received the DVM and PhD (immunology) degrees from ISU. He is the Director of the Center for Food Securi- ty and Public Health and the Institute for International Cooperation in Animal Biologics. Dr. Roth’s primary area of research expertise is immunity to infectious diseases of food producing animals. He has testified before Congressional committees on biosecurity preparedness, on efforts to address bioterrorism and agroterrorism, and on the need for vaccines for foreign animal diseases. Dr. Roth served on the National Science Advisory Board for Biosecurity from 2005 to 2014.

4 • 2021 National Veterinary Scholars Symposium Liara M. Gonzalez, DVM, PhD Associate Professor NC State University

Presentation Title and date: Intestinal Regenerative Medicine: Targeting Key Stem Cells to Enhance Intestinal Viability Friday, August 6, 1:00 – 2:00 EDT

Bio: Dr. Liara Gonzalez is an Associate Professor of Gastroenterology and Equine Surgery at North Carolina State University in Raleigh, NC. She received her veterinary degree from Cornell University College of Veterinary Medicine in 2006, completed a large animal surgery residency at NC State Veterinary Hospital and became a Diplomate of the American College of Veterinary Surgery in 2011. She remained at NC State and defended her doctoral thesis in 2014. Dr. Gonzalez’ clinical and research focus is on intestinal disease, with a particular interest in identifying improved means to determine tissue viability following severe intestinal injury as well as studying the role of intestinal stem cells in modulating mucosal barrier repair following isch- emic damage. Dr. Gonzalez hopes to apply her knowledge and expertise in advanced laboratory techniques to help solve clinically significant problems and further elucidate therapeutic targets that will ultimately improve patient survival.

2021 National Veterinary Scholars Symposium • 5 Award Winners AVMA Excellence in Research Award Recipients 2021

The AVMA is proud to recognize veterinarians and scientists for their outstanding contributions through research to advance animal and/or human health. The AVMA Council on Research annually selects 4 individuals for receipt of awards recognizing the significance of their research, as detailed by COR Chair Dr. John Middleton (https://vimeo.com/569978241/156b85af58).

Dr. Mark Peterson: Recipient of the AVMA Lifetime Excellence in Research Award recognizing a veterinary researcher based on the total impact of their lifetime achievement in basic, applied, or clinical research on the veteri- nary or biomedical professions. Dr. Peterson has spent his career in private practice at the Animal Medical Center and later the Animal Endocrinology Clinic in New York, and holds an adjunct professorship at Cornell University. He is recognized for his sustained research contributions that have furthered our understanding of feline and canine endocrinology. https://vimeo.com/569986216/938457457e

Dr. Larry Cowgill: Recipient of the AVMA Clinical Research Award which recognizes an AVMA members’ achievements in patient-oriented research, including the study of mechanisms of disease, therapeutic interventions, clinical trials, development of new technologies, and epidemiological studies. Dr. Cowgill is recognized for his sustained contributions to research in veterinary nephrology and hemodialysis. https://vimeo.com/569979270/d904b6abec

Dr. Brian Murphy: Recipient of the AVMF/Every Cat Health Foundation Award which recognizes contribu- tions advancing feline health and welfare through published research. Dr. Brian Murphy is recognized for his sustained contributions to research on feline viral diseases including FIV and FIP both of which serve as models for human diseases. https://vimeo.com/569971034/b9cd2322d2

Dr. Steven Budsberg: Recipient of the AVMA Career Achievement in Canine Research Award which honors an AVMA member’s long-term contribution to the field of canine research. Dr. Budsberg is recognized for his sustained contributions to research in canine orthopedics including use of gait analysis to refine management for osteoarthritis. https://vimeo.com/569987801/63e71bf0a8

Dr. Mark Peterson Dr. Larry Cowgill Dr. Brian Murphy Dr. Steven Budsberg

6 • 2021 National Veterinary Scholars Symposium Boehringer Ingelheim Veterinary Research Awards 2021

Boehringer Ingelheim Veterinary Research Award for Graduate Veterinarians Brittany Szafran, DVM PhD candidate in Environmental Toxicology Mississippi State University College of Veterinary Medicine Research Topic: Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice

Boehringer Ingelheim Veterinary Research Award for Veterinary Students Carley Allen DVM Candidate Class of 2023 University of Missouri College of Veterinary Medicine Research Topic: SHIP Inhibition in Canine Osteosarcoma: Evaluation of a Novel Target

2021 National Veterinary Scholars Symposium • 7 Abstracts Listed by Area of Research Abstracts are ordered alphabetically by last name.

Poster Page Last Name First Name Abstract Title # # Anatomic Pathology Au Yeung Sarah 20 Flexibly induced backlit imaging as a novel diagnostic tool for the 60 diagnosis of chronic enteropathy in cats Crawford Megan 107 Relationship between parity and reproductive disease in managed 104 African Painted Dogs (Lycaon pictus) Daigneault Emily 109 The role of bystander CD8+ T cells in Chlamydia-induced upper 105 genital tract pathology Evans Ian 141 Histologic Classification ofTursiops truncatus Regarding Age and 121 Freshwater Lesions Fuller Brenley 161 Distribution of Coccidioides spp. lesions in necropsy cases at a 131 veterinary teaching hospital Gasper Jepierah 172 Evaluation of CD8+ T cell exhaustion markers following Chlamydia 136 genital infection Howard Cory 234 Characterization of CD3+/CD20+ Large Cell Canine Lymphoma 167

Kelly Jamillah 279 Efficacy of pentosan polysulfate for treatment of retinal disease in 190 mucopolysaccharidosis IIIB Kroeger Austin 302 Optimizing an immunohistochemistry protocol to characterize the role 201 of tuft cells in porcine enteric diseases Ramsaran Leah 460 Determining the predictive value of radiographic change and ALP 280 levels for metastases in an osteosarcoma model Soulas Jaclyn 536 Diagnostic and prognostic markers of canine peripheral nerve sheath 318 tumors Starr Miranda 544 Co-localization of interleukin (IL)-17 and CD3 expression in equine 322 (Equus caballus) supporting limb laminitis Tonelli- Alanna 569 The pathology and incidence of building-caused bird mortality 335 Raylove Turner Skyler 581 Speciation of the etiologic agent of canine leproid granuloma 341 syndrome using PCR on FFPE tissue samples Wittorff Hannah 620 Evaluation of histopathologic sectioning of canine soft tissue 360 sarcomas Anatomy Felix Mia 143 A comparative anatomical study of the muscles of facial expression 122 between Lycaon pictus and other canids Greene Erica 198 Anatomy of the first mandibular molar and the mandibular canal 149 described by dog breed size and skull type Kujiraoka Amanda 305 Impact of incubation regimes on genital differentiation in hatchling 203 American alligators

8 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Mahmood Nicole 348 A new 3-D musculoskeletal model of the chimpanzee forearm, wrist, 224 and hand for studies of knuckle-walking Marsh Shelby 356 Functional anatomy of the snow leopard (Panthera uncia) tail 228

Rocco Felicia 479 Adaptions in the masticatory-associated musculature in the 290 hypercarnivorous African wild dog (Lycaon pictus) Sinclair Amanda 518 Evaluating three-dimensional kinematics of bipedal chimpanzees near 309 the walk-run transition speed Brezina Caleb 51 Are beef calves placed in a herd with familiar cohorts more resilient to 76 stress than unfamiliar calves? Coats Cassidy 92 Evaluation of accuracy of point-of-care analyzers in primates to 96 enhance monitoring in decentralized settings Fournier Zoe 155 Behavioural and physiological indicators of stress in horses 128 participating in equine-assisted psychotherapy Garcia Carmen- 168 Impact of age and weaning stress on histamine receptor subtypes 134 Maria localization and expression in GI tract Hagmaier Carlin 206 Telomere Attrition as a Biomarker for Animal Welfare in Sows: Role 153 of Types of Leukocytes and Social Hierarchy Hall Madeline 208 Developing a facial grimace scale to assess pain in goats 154

Parsons Dillin 433 Using digital enrichment to enhance parrot wellness 267

Richardson Sydney 471 Efficacy of a liposomal bupivacaine suspension (Nocita) for 286 disbudding in dairy calves Antimicrobial Resistance Adesola Ridwan 7 Antimicrobial resistance in Africa: Pets, wildlife, and humans in 54 danger Burleson Daniella 64 Comparison between qPCR and ddPCR for quantifying AMR genes in 82 enterica in swine fecal samples Casseri Eleni 76 Antibiotic resistance in dairy cattle: a foe, or no? A survey-based 88 study of dairy farmers Dharmasena Vidharshi 119 Likelihood of Infection in Veterinary Patients Treated for Sepsis in the 110 Emergency Room Dorr Mackenzie 126 Transferability of IncN and IncI plasmids among field strains of 113 and coli Ferguson Kyle 146 Development of Evidence Based Antibiotic Stewardship 123 Recommendations For Empiric Canine Skin Infections Johnson Sierra 260 Antibiotic resistance profiles ofStaphylococcus spp. isolated from 180 food matrices Jolley Ashlan 262 Impacts of the COVID-19 pandemic on antimicrobial use in 181 companion animals Kearney Allison 276 Salmonella serovars from Grenadian animals and their antibiotic 188 susceptibility pattern: A retrospective study Klatzer Emily 292 Phenotypic and genotypic characterization of antimicrobial resistance 196 genes in probiotics

2021 National Veterinary Scholars Symposium • 9 Poster Page Last Name First Name Abstract Title # # Liu Patrick 328 Efficacy of diagnostics for reducing inappropriate antimicrobial use in 214 simple canine urinary tract infections MacNeil Lauren 340 Antimicrobial Resistance of Pathogens in Lactating Dairy Cattle 220

Majlessi Elliot 349 Evolution of Staphylococcal antibiotic resistance systems across 225 gram-positive Munroe Cassandra 398 Weighted-incidence syndromic antibiograms for urine cultures in cats 249 and dogs Presher Alexandra 456 Surveillance for Salmonella and resistome profiling among 278 hospitalized wildlife patients Rafique Sonia 459 Developing a rapid plasmid DNA extraction method 280

Specht Aubrey 539 Describing the Geospatial Distribution of Antimicrobial Resistance 320 Genes in the Boston Urban Rat Population Walsh Elizabeth 593 Prescribing practices on the prevalence of methicillin-resistant S. 347 pseudintermedius infections in dogs Bacteriology de Ruyter Tryssa 115 Evaluation of inactive vaccines against Lactococcus garvieae 108 infection in rainbow trout (Oncorhynchus mykiss) Devorak Anne 118 Protective efficacy of a yeast byproduct diet against APEC challenge 109 in poultry Foley Brayden 152 Unearthing the elusive Johne’s Disease: a widespread granulomatous 126 and disseminated bacterial disease in goats Geer Allison 174 Detection of pathogenic Leptospira spp. in fish 137

Given Christopher 179 Dolphins to horses (but not seahorses) - prevalence, type, and 140 susceptibility of Staphylococcus delphini Grabner Carly 195 Pressure-induced wound formation and healing with Staphylococcus 148 aureus infection in a diabetic mouse model Gunn Morgan 204 In vitro evaluation of Staphylococcus felis as a potential probiotic 152 therapy for canine superficial pyoderma Hipkiss Hannah 225 Identification of novel miRNA isolated from serum-derived exosomes 163 as a diagnostic biomarker of bovine TB Lumba Rohini 336 DifferentiatingStaphylococcus schleiferi subspecies with MALDI- 218 TOF, biochemical testing and susceptibility Matsuoka Shelby 363 Staphylococcus pseudintermedius as a source of hospital infections 232

Reitsma Lara 469 What happens to Paenibacillus larvae spores after cessation of 285 antibiotic use in honey bee hives? Rogers Chloe 484 Detection and characterization of tarda isolates from 292 natural lakes and waters in Alabama Ruffin Robert 489 Evaluating the relative survivability of Mannheimia haemolytica in 295 various environments Silverstein Nicole 516 Characterizing non-target bacteria from animal carbapenemase- 308 producing Enterobacterales surveillance specimens Smith Olivia 531 The role of invG in Salmonella enterica adherence to and invasion of 316 human intestinal epithelial cells

10 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Tryzbiak Madison 577 Role of CT226 in Chlamydia trachomatis infection 339

Behavior Adkisson Cynthia 8 Relationship between ownership and enrichment in dogs with and 54 without separation anxiety or noise phobia Bixby Ryan 40 Dog and Adolescent Interactions Amid Animal Assisted Interventions 70

Bulloch Daegan 61 Postnatal development of vocal movements in a Peromyscus mouse 81 model Celatka Jillian 77 Prenatal Oxycodone Exposure and Pup Vocalizations 89

Cranfill Suna 106 Establishing the role of the lateral habenula in central itch processing 103

Gartner Kathleen 171 Evaluating the causes and consequences of canine food motivation 136 within the Dog Aging Project Pack cohort Guarino Katherine 203 Investigating the Association between ELS and Chronic Abnormalities 152 in Ultrasonic Vocalizations in Rats Johnson Callan 256 Relationship between personality traits and physiological indicators of 178 chronic stress in ring-tailed lemurs Maulini Jennifer 364 Efficacy and safety of long-term imepitoin treatment for canine storm 232 anxiety with individual dose titration Messina Andrea 382 Development of an online behaviour rating tool to enhance veterinary 241 students’ knowledge of equine behaviour Morse Allison 394 Effects of Veterinary Handling on Canine Behaviour and Physiology 247

Ortiz Negron Nashelys 428 Comparative distribution of VTA glutamate- and dopamine-releasing 264 terminals in the medial prefrontal cortex Wunderlich Emma 624 Social group and behavioral changes of white-tailed deer treated with 362 immunocontraceptive PZP Zayas Gabriella 633 The nose knows: Validation of an olfactory test in dogs with and 367 Santiago without nosework training Biochemistry Hommer Alexandra 232 Methionine sulfoxide reductases (MSRs) and Fe-S cluster biogenesis 166

Jorgensen Hannah 267 Off-target effects of small molecule therapeutics of spinal muscular 184 atrophy Kinkade Mackenzie 290 Effects of genetic manipulation of FASTKD proteins on mitochondrial 195 mRNA and function Rosales April 486 Identification of signaling protein RNA in the sea star egg 293 transcriptome Cardiology Adam Em 4 Identification and validation of alleles for cardiac arrhythmias and 52 atrial fibrillation with ECG analysis Benavides Estefania 33 O-GlcNAc-Ca2+-calmodulin kinase activity on K-channel isoform 67 within atrial and ventricular cardiomyocytes Cerrato Alexandra 78 In vitro cardioprotective effects of epigallocatechin-3-gallate 89

2021 National Veterinary Scholars Symposium • 11 Poster Page Last Name First Name Abstract Title # # Chen Katherine 83 Determining cardiomyopathies and cardiac disease via Vertebral Heart 92 Score in a large population of rabbits Chinchayan Cristina 86 Optimizing gene editing approaches in Doberman Pinscher fibroblasts 93 by targeting PDK4 and DCM Colangelo Jennifer 94 Cardiac extracellular matrix remodeling in horses with cardiac 97 pathology Decker Andrew 116 The Role of Piezo1 in Pre-Adipocyte Differentiation 108

Dolan Samantha 123 Retrospective evaluation of the indication for and outcome following 112 sildenafil administration in cats Garcia Tatiana 169 Exposure-response of amlodipine on the renin-angiotensin- 135 Marrero aldosterone system and blood pressure in healthy cats Hardgrove Caitlin 215 Genetics of supraventricular arrhythmias caused by accessory 158 pathways in Labrador Retrievers Karp Shelby 273 Evaluation of echocardiographic findings and cardiac biomarkers in 187 cats eating different types of cat foods Klasing Jessica 291 Reduced cardiac fibrosis and improved function in a novel model of 196 HFpEF using the 5’ SL mutant mice Ngo Lena 411 The effects of chronic nicotine exposure on cardiac remodeling and 256 arrhythmia susceptibility in a rabbit model Paci Nicolette 431 Effects of early life stress on the aged myocardium: potential sex 266 differences in hypertrophy and fibrosis Peterson Jessica 445 Validation of standard Doppler cuff sizes in dogs 273

Samra Gunimat 494 Increased Infiltration of Innate Immune Cells Associates with AVF 297 Maturation Failure Scott Samantha 502 Role of canine hemangiosarcoma-associated extracellular vesicles in 301 promoting tumor angiogenesis Sedighim Sharona 503 Hydrogel Templates as Cardiac Matrix Mimic for Tissue Engineering 302

Weiss Brandon 604 The role of calmodulin-dependent protein kinase II nitrosylation in 352 cardiac ischemia-reperfusion injury Clinical Pathology Allen Brittany 9 Developing and optimizing a flow cytometry-based assay for feline 55 infectious peritonitis virus Amblard Sarah 14 Pathology associated with triploid oyster gametogenesis 57

Choi Diana 88 Creation and validation of Mycobacterium tuberculosis granuloma 94 recognition software Krellwitz Kyle 299 A novel flow cytometric assay to assess platelet desialylation in canine 200 immune thrombocytopenia Lai Poyu 310 The value of a leukogram for preoperative patient assessment in 205 shelter dogs Lambert Jessica 311 Investigating diagnostic utility of agarose cell blocks prepared from 206 hepatic and splenic aspirates Li Emma 321 Novel diagnostics for sepsis: optimizing Fountain FlowTM Cytometry 211 and testing the RapidBacTM Vet immunoassay

12 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Ma Crystal 338 Evaluation of Protein X as a potential urine biomarker for MPGN in 219 dogs with proteinuric CKD McGlynn Andrew 374 Examining urine pH, specific gravity, and urine protein over time in 237 healthy dogs Olmsted Zoe 422 Searching for chronic wasting disease (CWD) in the southeastern 261 United States Reid Heather 468 Characterizing the association of proteinuria with canine and feline 284 periodontal disease Snipes Bryanna 534 Determining correlations between blood glucose, saliva glucose, and 317 pH in small and large animal patients Ullom- Rebecca 583 Preliminary evaluation of intravenous iron sucrose in healthy cats 342 Minnich Weaver Danielle 599 Pathophysiology of tau hyperphosphorylation and glial inflammation 350 in aged cats Willis Kaitlynn 614 Hepatocyte ploidy in dogs with hepatocellular carcinoma and hepatitis 357

Dermatology Billings Phillip 39 The use of mefenoxam to treat cutaneous and gastrointestinal 70 pythiosis in dogs: a retrospective study Faddoul Emma 142 Dog with MRSP pyoderma converted to MSSP after Staphylococcus 121 pseudintermedius vaccine administration Nguyen Jeffrey 413 Developing canine atopic dermatitis biomarkers 257

Reed Miriam 467 Evaluating accuracy and precision of Next-Generation Sequencing 284 using organisms causing canine otitis externa Epidemiology Ashley Elizabeth 18 Investigating relationships between prey preference and infectious 59 disease in southern sea otters Botkin Sarah 46 Lack of efficacy of routine and enhanced hygiene practices in 73 removing rotavirus A from rubber piglet mats Cappo Kyle 71 Clostridioides difficile in horses hospitalized with colic: nosocomial 86 pathogen or innocent bystander? Doran Alexandra 125 Variation in tick species abundance between two collection methods at 113 three central Illinois study sites Edache Stephen 131 Use of a yeast fermentation-based probiotic to reduce Salmonella 116 prevalence in lymph nodes of dairy cattle Fumia Heather 162 Mathematical modelling to explore the role of environmental factors 131 in avian outbreaks in snow geese Heinen Lilli 220 Evaluation of predictive models to determine outcome based on data 160 known at first treatment for BRD in cattle Kiesel Gabriela 283 Using epidemiologic tools and methods to assess the role of adverse 192 weather events in PRRS and PED outbreaks Miller Grace 386 Spatio-temporal analysis of raccoon rabies animal cases in eastern 243 North America Nakatsu Charlize 403 Heavy Metal Associated Resistance in Avian Pathogenic Escherichia 252 coli (APEC)

2021 National Veterinary Scholars Symposium • 13 Poster Page Last Name First Name Abstract Title # # Orsini Gabrielle 426 Surveillance for coronaviruses in North American bats 263

Peterson Breck 444 Epidemiological study of avian pathogenic 272 serogroups in Georgia poultry Seminara Jackson 505 Calcium metabolism and associated patterns of milk constituents in 303 early lactation dairy cows Snell Savannah 533 Novel vaccine inactivation method using UV and riboflavin preserves 317 major influenza surface antigens Tartaglia Dillon 561 Temporal relationship of fever and positivity on nasal and blood PCR 331 in a 2016 outbreak of EHM in California Taylor Anna 562 Epidemiology and risk factors for Sarcocystis neurona infection in 331 juvenile California sea lions from 2012-17 Wang Christine 595 Risk Factors for Antimicrobial Resistance among Campylobacter coli 348 from Agricultural Swine Populations Wang Wenjun 596 Quantifying Disease Transmission Risk in Infants Using Video Data 348

Equine Medicine and Surgery Benton-Levith Elizabeth 34 Correlation between sepsis scores and arterial blood pressures and 67 their variability in anesthetized foals Brauer Kiley 50 Genome-wide association study of degenerative suspensory ligament 75 desmitis in the Peruvian horse Cassale Nicole 75 Autologous biologic therapies for treatment of full thickness 88 cutaneous wounds in horses Clifford Abigail 91 Comparison of microstructural values of equine proximal sesamoid 96 bones at different CT resolutions Dugan Conner 129 Prevalence of upper respiratory pathogens in Colorado Front Range 115 rescue horses Elcombe Megan 133 Prevalence of the RAPGEF5 c.2624C>A variant associated with EFIH 117 in the United States Thoroughbred population Graber Kami 194 Exploring clinical use of electroencephalography to differentiate 147 depth in horses - a pilot study Horton Julia 233 Endocrine responses to mare’s milk in neonatal foals 167

Jones Hannah 265 Effects of Dexmedetomidine on Intestinal Epithelial Stem Cell 183 Expression in Equine Intestinal Strangulation Kepley Abby 281 Optimization of a Qubit fluorometer assay for the quantification of 191 cell-free DNA in equine plasma Killian Breanna 284 Development of a consistent induced lameness model in the horse 192

Maresca- Hailey 351 Dynamic insulin response to acclimation of a high carbohydrate diet 226 Fichter in normal and insulin dysregulated horses Marter Nicole 357 Incidence of palmar process fractures in the distal phalanx of Arabian 229 foals Mohiuddin Ava 389 Thermographic evaluation of the ventral abdomen and jugular vein in 245 healthy horses Morales Saira Mary 391 Surfactant protein D in serum and bronchoalveolar lavage fluid from 246 asthmatic horses and age-matched controls

14 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Mozeika Christina 397 Plasma and tissue disposition of ceftazidime after multiple intravenous 249 doses in healthy neonatal foals O’Donnell Kerry 418 Evaluation of intestinal alkaline phosphatase as a novel therapeutic for 259 equine intestinal epithelial injury Patel Meera 434 ACTH stability in frozen equine plasma 267

Robertson Hannah 478 Hypothalamic-Pituitary-Adrenal Axis Assessment in Healthy Foals 289 Utilizing Vasopressin Stimulation Test Snyder Hailey 535 Estrogens and Their Association with Illness in Hospitalized Foals 318

Summers Jamie 555 Vitamin D and parathyroid hormone during hospitalization in 328 critically ill foals Windham Jennifer 617 Racehorse Safety in Louisiana (2009-2021) 359

Zoul Jordyn 637 Evaluation of neuropathic pain mediators in acute and chronic 369 laminitis Exotic Pets and Zoo Animal Medicine Bonadie Kayla 43 Developing a thromboelastography assay in Elasmobranchs 72

Conner Jessica 98 Development & standardization of cardiac diagnostic parameters of 99 the bearded dragon (Pogona vitticeps) Dicharry Jena 121 A comparison of Amazon parrot blood gas values on the i-STAT and 111 EPOC Ferchaw Hunter 145 Hematology reference intervals for zoo-housed neonatal giraffe calves 123 (Giraffa camelopardalis) Fukushima Courtney 160 The effects of passive vs. active enrichment on the stress levels of ball 130 pythons Golden Olivia 183 20-year trend in type of references used for small mammal drug 142 dosages in an exotic formulary Hamilton Emily 210 Efficacy of appetite stimulants in normorexic and hyporexic 155 chinchillas Hamlin Nicole 211 Phenotypic and functional characterization of mesenchymal stem cells 156 from flamingo, polar bear, and tiger Javier Sirah 253 Contents and efficacy of quick start nitrifying bacteria aquaria 177 supplements John Shannon 255 Efficacy of lidocaine cream vs EMLA (lidocaine-prilocaine) cream for 178 pain-free venipuncture in rabbits Lex Zoey 320 Determining the pharmacokinetic properties of meloxicam in barred 210 owls (Strix varia) undergoing rehabilitation Lucich Dylan 334 Comparing temperatures at various rectal depths to alternative sites in 217 New Zealand white rabbits Peterson Macy 446 Pharmacodynamic evaluation of subcutaneous methadone in African 273 pygmy hedgehogs (Atelerix albiventris) Powers Vanessa 454 Survey of dental conditions and management in zoological animals 277

Rasys Ashley 462 Editing the chicken genome by microinjecting CRISPR reagents into 281 unfertilized oocytes

2021 National Veterinary Scholars Symposium • 15 Poster Page Last Name First Name Abstract Title # # Walter Benjamin 594 Behavioral impact and pharmacokinetics of transdermal fentanyl in 347 healthy corn snakes Immunology Abraham Taylor 1 Immune tesponse of thiamine deficient chinook salmon 51 (Oncorhynchus tshawytscha) to columnaris disease Barber Cassandra 29 Messenger RNA therapy to induce expression of bovine cathelicidins 65 to combat respiratory disease pathogens Brown Chalise 54 Interrogating MARCKS inhibition as a strategy to alter bovine 77 neutrophil responses to S. typhimurium Bryson Andreya 58 Effects of E. coli Heat-labile enterotoxins on immune cell-cell 79 communication mediated by exosomes Burk Ariela 62 Host-pathogen interactions during wound repair in zebrafish 81

Cao Jennifer 70 Canine osteosarcoma, a spontaneous large animal model for solid 85 tumor CAR T cell therapy Carswell Imani 73 The effects of intravenous opioids on leukocyte oxidative burst and 87 phagocytosis in dogs Clark Samantha 90 Evaluating the oncolytic potential of HSV-1 mutants (VC2, 95 VC2GMCSF; VC2PGDH) using murine breast cancer model Combs Olivia 97 Nuclear factor-kB inducing kinase (NIK) regulates macrophage 99 metabolism to support GBM growth and invasion Coughlin Laura 104 Establishing serum biomarkers of Mycobacterium tuberculosis 102 infection in Asian elephants DeJohn Abigail 117 Stimulator of Interferon Genes (STING) is essential for TLR-mediated 109 upregulation of Type I IFN genes Ellis Ilene 135 Defining the inflammatory cytokine profile in acute tendon injury to 118 enhance mesenchymal stem cell therapy Ellison Lauren 136 Transfection of bovine preputial keratinocytes for expression of 118 antibody against Tritrichomonas foetus Esala Micah 139 Immune response elicited by implantation of a novel polyanhydride- 120 based single dose vaccine Forker Grace 154 Boosting host immunity against bacterial otitis media though 127 disruption of pathogen mediated immunomodulation Gbore Damilola 173 Effect of water extract fromEuglena gracilis on granulocytic 137 differentiation and activation of promyeloblasts Glenn Nicole 180 Defining Tissue Expression and Isotype of Ultralong CDR3 140 “Cattlebodies” by Real Time qPCR Goldsmith Malaycia 184 Increased immune responses by intradermal and intranasal 142 immunization with a novel adjuvant in mice Gollnick Hailey 186 IL-27 Inhibits the anti-Mycobacterium tuberculosis Activity of 143 Primary Human Macrophages Gorrell Kaitlin 192 Characterization of host immune responses to Clostridium perfringens 146 affecting broiler chickens Gretler Sophie 200 Toll-Like Receptor (TLR)9-signaling and its effects on B cell receptor 150 (BCR) signaling in B-1 cells Haines Laurel 207 Osteosarcoma exosomes selectively home to the lung and elicit pro- 154 tumorigenic changes in resident lung cells

16 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Henry Mikaela 222 Development of a novel vaccine for Feline Enteric Coronavirus using 161 recombinant Lactobacillus acidophilus Hull Morgan 238 Anti-Bovine Serum Albumin IgG antibody production in horses post- 169 vaccination Jackson Nathan 248 Role of Estradiol in Intestinal Ischemia and Reperfusion Injury 174

Jasenski Jessica 252 Monocyte-derived dendritic cell signaling response following 176 Cryptosporidium parvum exposure Jimenez Daniela 254 The role of Complement Receptors (CR) 1 and 2 in Borrelia 177 burgdorferi infection Johnson Davida 257 Cross-protective potential of an vaccine against 179 E. piscicida in Oreochromis niloticus Kao Leeza 272 Assessing the pharmacodynamics of the novel antibiotic, bovine 186 lactoferrin (bLF) in chickens Knight Hannah 295 Immunomodulation from epigenetic reprogramming improves health 198 and production efficiency in aquaculture Krichbaum Hannah 300 Effects of Holstein genotype on innate immune response to 200 intramammary administration of E. coli Kumar Ayush 307 Electron microscopic characterization of recombinant Lactobacillus 204 acidophilus strains for vaccine development Lemons Margaret 315 Equine neutrophil extracellular trap formation in response to ex vivo 208 antigenic stimulation Leverance Kayla 316 Acute Effects of Repetitive Traumatic Brain Injury on Neuroimmune 208 Responses Lin Amy 325 Investigating the mechanism behind a phenotypic drift in lupus-prone 213 mice Lyles Dominique 337 Effect of chicken IgY on Staphylococcus aureus growth in vitro 219

Maguire Daniel 346 Assessing PACAP as an EGC stimulant in teleosts 223

Maslyn Kara 361 Evaluating the effects of telmisartan in healthy dogs as a preclinical 231 model for Shar-Pei fever McCall Jayden 366 Identification of protective antigens from African Swine Fever Virus 233 proteome McCurdy Raelyn 370 Impact of formalin baths and jellyfish A.( aurita & C. capillata) on 235 gill pathology in Atlantic salmon (S. salar) McGinley Jared 373 Assessment of an experimental PCV2d vaccine in the PCV2d 237 challenge model McNabney Danielle 375 Immunometabolic profiling ofTpl2-/- innate immune cells 238

Mertz Madison 381 Method optimization for studying effects of cannabinoids on canine 241 PBMC immune responses Moncrief Michele 390 Development of a novel mRNA vaccine platform for Bovine 245 Respiratory Syncytial virus Morse Benjamin 395 Effects of 25(OH)D3 on the transcriptome and cytokine production of 248 canine peripheral blood mononuclear cells Nayak Arpita 404 Improving neonatal dairy calf mucosal immune response via 252 micronutrient supplementation

2021 National Veterinary Scholars Symposium • 17 Poster Page Last Name First Name Abstract Title # # O’Day Shannon 417 Immune responses elicited in calves administered the conditionally- 259 licensed vaccine Olivares Jasmine 421 Esophageal T-cell infiltration in a pig model for Eosinophilic 261 Esophagitis (EoE) Peauroi Elise 437 The novel innate immune-antagonistic effects of the multifunctional 269 ectromelia virus C15 protein Perez Luisanny 440 Evaluating B7-H3 as a tumor antigen target in canine osteosarcoma 270 for CAR T cell therapy Perez Jaimes Martin 441 Delivery routes of antimicrobials against Mycobacterium tuberculosis 271

Perkins Heather 442 Effects of methadone on leukocyte cytokine production in dogs 271

Petersen Lynsey 443 Cerebrospinal fluid proteomic profiling for biomarker discovery in 272 canine non-infectious meningoencephalitis Phillip Lauren 448 The effects of castration and early weaning on long-term immune 274 responses Razmara Aryana 464 Optimization of expansion techniques for adoptive NK cell transfer in 282 dogs with cancer Russek Joshua 490 Chemotactic and adhesion properties of equine CD172a+ peripheral 295 blood eosinophils Salvosa-Chua HermanMax 493 Characterization of immune responses to LPS in bone marrow derived 297 dendritic cells from the chickens Sarkan Kate 497 Presence and Quantity of Influenza Virus-Specific Natural IgM 299 Antibody in Human Umbilical Cord Blood Sipple Nicole 519 Immunomodulatory activity of mesenchymal stem cell derived 310 extracellular vesicles on canine mononuclear cells Sistig Lorena 520 Development of Anti-TNFa VHHs for immunotherapy of chronic 310 inflammatory diseases in humans and canines Skalecki Shannon 522 Assessment of anti-viral and anti-inflammatory effects of 311 Mesenchymal Stem Cells in herpesviral infections Slaughter Megan 524 Evaluation for anti-erythrocyte and anti-platelet antibodies in healthy 312 dogs administered lokivetmab Steinhilber Megan 546 Involvement of the CCR9-CCL25 axis in CD8+ T cell migration into 323 the brain Swales Ella 556 Effect of mRNA-expressed antibodies onin vitro attachment of T. 328 foetus to bovine preputial keratinocytes Szostek Adrianna 557 Monoclonal antibody optimization and nanoparticle vaccine 329 development for Streptococcus pneumoniae Tarsillo Justine 560 IgE and sCD14 as Potential Biomarkers for Culicoides 330 Hypersensitivity in Icelandic Horses Tobin Victoria 567 Interrogating the gene regulatory network underlying pro-T cell fate 334 allocation using CRISPR and scRNA-seq Topka Jessica 570 Modulation of chicken macrophage function by microbial catabolites 335 of tryptophan Trogdon Mary-Grace 576 Optimization of Qubit dsDNA high sensitivity assay for quantification 338 of cfDNA in equine abdominal fluid Tupik Juselyn 579 A “sub-Lyme” system: Uncovering the protective role of anti- 340 inflammatory NLRX1 against Lyme disease

18 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Turn Jeffrey 580 Modulation of chicken and equine peripheral lymphocytes using 340 honeybee apitherapy Veith Brayden 587 T cell activation induced by dendritic cell infection with 344 Cryptosporidium parvum in serum-free media Villasenor Adriana 591 Canine osteosarcoma cells induce pro-inflammatory gene expression 346 in macrophages Wilson Alec 615 Predominance of fungal colonies in the feces of canines with diabetes 358 mellitus versus nondiabetic control dogs Winiarski Caroline 618 Borrelia burgdorferi colonization of gastrointestinal tract mucosa in 359 Mus musculus Woodfint Rachel 623 Serine protease inhibitor supplementation to enhance host response to 362 Intranasal and intraperitoneal vaccines Yungmeyer Austin 630 Ex vivo heme-induced NETs in canine neutrophils as a model for 365 NETs in canine immune-mediated hemolytic anemia Zhu Cong 636 Examination of canine peripheral blood NK cells 368

Laboratory Animal Medicine Acevedo Stephanie 2 Effect of a Warmed Environment and IVC Supplemental Heat on 51 Mouse Behavior, Breeding and Body Temperature Cannon Brinley 69 Pharmacokinetics and efficacy of high and low dose meloxicam in 85 mice Davis Camryn 113 Comparison of agents to maintain hydration of Yucatan minipig skin 107

Fink Brent 149 Can activity monitors improve our ability to assess outcomes and 125 improve animal welfare in research settings? Freeman Layne 157 Efficacy of Meloxicam as an in Mice Surgical Procedures 129

Harris Shanice 217 Evaluation of aseptic technique, analgesia and anesthesia protocols in 159 video-based articles for rodent surgery Wilson Rachel 616 Bacterial burden, moisture content, and mouse preference of DietGel 358 76A over time Zenilman Shoshana 634 Efficacy of alfaxalone vs tricaine methanesulfonate as immersive 367 anesthetics in rainbow trout Microbiomes Donovan Erin 124 Characterizing the maternal contribution to the gut microbiome of 112 one-week-old mice Edwards Kerri 132 Development of fecal IgA assay for detection of segmented 116 filamentous bacteria infection in laboratory mice Ford Tamara 153 Effect of feeding sugar beet pulp on microbial butyrate production in 127 healthy adult horses Guan Jimmy 202 Characterizing the microbiome of chickens fed NSPase producing 151 Aspergillus spp. Hickey Amber 224 Self-Assembly of Complex Polymicrobial Infections 162

Kazmierski Luke 275 A point-of-care microbiome-based diagnostic for canine gut health 188

Li Jingyi 322 An exploratory research of the Common Loon gut microbiome 211

2021 National Veterinary Scholars Symposium • 19 Poster Page Last Name First Name Abstract Title # # McAdam Tiera 365 Fecal bacterial microbiota and antibiotic-induced changes of the 233 microbiome in ferrets (Mustela putorius furo) Meena Beverly 376 The porcine gut microbiome: Understanding effects of age, sex, breed, 238 facility location and birthing method Mehta Tarun 377 The Fecal Metabolomic Profile of Dogs Transitioning from a Chicken 239 Raw Meat Diet to a Plant-Based Diet Osterland Alexandra 430 Comparative microbiome analysis of Eptesicus fuscus and Myotis 265 grisescens Piorkowski Jill 449 Pilot Study on the Healthy Canine Blood Microbiome 275

Piva Milan 450 Assessment of zoonotic parasites and bacteria in fecal samples of 275 shelter dogs Riha Jordan 472 Colonization and effect of Nissle 1917 E Coli probiotic on the healthy 286 feline microbiome Salerno Matthew 492 Standardized fecal capsulation for canine fecal transplants does not 296 alter microbial community structure Seilhamer Nikki 504 Characterization of vendor-dependent differences in the juvenile and 302 adult mouse ileal lumen and mucosa Smith Katelyn 528 Comparison of the fecal microbiome of feral and domesticated cats 314

Spilker Eric 541 Effect of chemotherapy agents on the diversity of the fecal 321 microbiome of canine lymphoma patients Willis Erin 613 Novel biomarkers of canine gastrointestinal health 357

Young Jared 628 Recoverability of metagenome assembled genomes within the fecal 364 microbiome of commercial swine Oncology Allen Carley 10 SHIP inhibition in canine osteosarcoma: Evaluation of a novel target 55

Badamo Dalia 22 Investigating the role of Caveolin-1 in cellular response to drug- 61 induced oxidative stress Bailey Lindsey 24 Image analysis for monitoring in vitro migration of feline oral 62 squamous cell carcinoma cells Bankler-Jukes Devon 27 Impact of tumor heterogeneity on the stromal landscape in a mouse 64 model of pancreatic ductal adenocarcinoma Broussard Rebecca 52 Anticarcinogenic effects of whole blueberry extract in MDA-MB-231 76 cell line Callisher Jessica 68 Prognostic significance of CD204+ tumor-associated macrophages in 84 canine cutaneous melanocytic neoplasms Cody Gena 93 Chemotherapeutic efficacy of carfilzomib in canine glioma cells in- 97 vitro Consales Blaire 100 Characterization of microglia in canine brain metastatic melanoma 100

Davis Elaina 114 Immunomodulatory effects of high-frequency irreversible 107 electroporation on canine primary lung cancer Drescher Matthew 127 Defining the PD1+ and PDL1+ populations in response to targeted 114 therapy treatment in a murine melanoma model

20 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # English Abigail 138 Inhibition of canine glioma cells by MAP4K4 knockdown and 119 treatment with the metastasis inhibitor metarrestin Ferraro Emma 147 Retrospective study of fish cancer prevalence and therapies 124

Godwin Taylor 182 Macrophages and prostate cancer racial disparities 141

Granger Julie 196 Next generation sequencing revealed heterogeneity between spatially 148 distinct PanINs Hagen Jennifer 205 Expression of LAMA4, PDPN, CAV-1, and ERG as potential 153 biomarkers for canine splenic hemangiosarcoma Herron Victoria 223 Evaluation of the oral and tumor microbiome of dogs and cats with 162 oral cancer Hung Clarey 239 Investigating Sirtuin 5 (SIRT5)-mediated regulation of macrophage 170 polarization in the tumor microenvironment Kawabata Jo 274 Investigating target antigen density influence on canine CAR-T 187 functionality Kenney Sarah 280 The Stressful Effect of Propranolol on Macrophages 190

Kliewer Maya 293 Preliminary investigation of the role of the EphA2 receptor in the 197 oncogenic fitness of canine osteosarcoma Kuhn Molly 304 The effects of gestational intermittent hypoxia on breast cancer 202 development in female offspring Luker Madison 335 Targeting the MARCKS/AXL axis to combat pro-tumor macrophage 218 polarization in cancer progression Madison Elsie 342 Evaluation of chitosan alginate nanoparticles for camptothecin 221 delivery to colorectal cancer cells Maguire Maria 347 Interrogating the role of Src inhibition in DMD-mutant osteosarcoma 224

Martin Hannah 359 A biomaterial-based delivery system for immunotherapeutic cytokines 230

Massaro Nickolas 362 Examining CD8+ T cell localization in estrogen receptor positive and 231 negative obese breast cancer metastasis Meyers Walter 383 Stance analysis and activity tracking in six dogs with osteosarcomas 242 undergoing palliative radiation therapy Mihailovich Alison 385 A Western diet promotes non-small cell lung cancer and affects PD-1- 243 based immune checkpoint inhibition Newberry Victoria 410 Investigating feline endogenous retroviruses as immunotherapy targets 255 for squamous cell carcinoma Nguy Symphony 412 Assessing changes in myeloid derived suppressor cells in canine 256 melanoma patients treated with immunotherapy Noall Lucie 415 BRAF mutations in feline tumors 258

Ojeda Vivian 419 Intramedullary stents as a device to prevent post-radiation fractures in 260 canine patients with osteosarcoma Poon Andrew 451 Metabolic analysis of canine and murine osteosarcoma cells in 276 comparison with mesenchymal stromal cells Ringhofer Justin 473 Evaluating canine aryl hydrocarbon receptor’s role in osteosarcoma 287 progression, metastasis and chemoresistance

2021 National Veterinary Scholars Symposium • 21 Poster Page Last Name First Name Abstract Title # # Rinker Cody 474 Repurposing b-blockers as anti-cancer immunotherapeutics to target 287 myeloid derived suppressor cells Ryan Declan 491 Colorectal cancer in rhesus macaques and the hunt for 296 hypermethylation Sanfacon Brittney 495 The impact of TP53 missense and truncating mutations on protein 298 function and tumor progression in canine OSA Schermacher Maxwell 501 Circulating tumor DNA analysis in canine osteosarcoma patients 301

Shaffert Kathryn 508 Lysine demethylase inhibition decreases proliferation in canine 304 osteosarcoma Smith Sidney 532 Broadening the spectrum of exotic animal species neoplasia: an 316 analysis of Rollins Laboratory cases Stoyle Brianna 548 Effects of receptor tyrosine kinase inhibition on canine glioma cell 324 lines in vitro Thomas Corrine 563 Bone’s black hole: comparing the osteolytic mechanisms of 332 hemangiosarcoma of the bone with multiple myeloma Tyler Kyleigh 582 NRP2 signaling in malignant peripheral nerve sheath tumors 341

Urbina Christian 584 Isolating canine natural killer cells for development of a novel 342 inhibitory SCT Velez Raisa 589 In vivo radiation dose-response relationships for a syngeneic 345 Contreras orthotopic murine model of oral carcinoma Watts Remy 598 Microenvironmental immune effects of stereotactic body radiotherapy 349 and immunotherapy in canine solid tumors Westbrook Madeleine 610 Evaluation of prednisolone pharmacokinetics and toxicity in dogs 355 with lymphoma or immune-mediated disease Willis Emily 612 Stereotactic radiation therapy (SRT) outcomes on the treatment of 356 canine nasal tumors One Health Cords Olivia 103 Optimizing surveillance of flaviviruses in free-ranging non-human 102 primates Elliott Stephanie 134 Investigating beaver dam analogues as a novel method to reduce 117 Cryptosporidium and Giardia load in wet meadows Gorman Myranda 191 Comparing Methodologies for the Detection of Leptospira in 146 Environmental Samples Ida Jennifer 242 Social aspects of antimicrobial use, resistance, and regulation in dairy 171 farming in Alberta, Canada Kim Kyoo Young 286 Salmonella serotype surveillance in rodent species inhabiting poultry 193 farms Knap Christopher 294 Evaluation of benefits and challenges of temporary veterinary clinics 197 in remote northern Canadian communities Langfeldt Antonia 312 Per- and polyfluoroalkyl substances (PFAS) and animals: A review 206

Le Annie 314 Understanding the interactions of pets and their owners with Saint 207 Kitts and Nevis wildlife Levitskiy Alexander 317 Deleterious health effects of harmful algal blooms on fishing 209 communities in Lake Victoria, Kenya

22 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Logue Mary 330 Use of the rOmpB gene to detect strain-level variation in Rickettsia 215 rickettsii in the Arizona region Maestas Jesse 343 Dermatologic and respiratory health among pets from in inner-city 222 homes with human asthma Mithani Sarah 388 A survey of pet-owners to identify potential barriers to access 244 veterinary care in the Cumberland Gap Region Newberne Holly 409 Using DAPI-PI staining to determine Cryptosporidium parvum oocyst 255 viability compared to qPCR quantification Pray Hannah 455 Microplastics and per- and polyfluoroalkyl substances (PFAS) in 278 bottlenose dolphins along Mississippi’s coast Prown Rowan 457 Heavy metal fish: a band of potential health threats for local 279 Philadelphian fish consumers Roe Nicolette 482 Exploring the role of Rhipicephalus sanguineus as a vector for Rocky 291 Mountain spotted fever in Arizona Rosado Braiam 485 Prevalence of TB and FMDV Among Animals Selected from 293 Ramos Slaughterhouses in Mbarara District in Western Uganda Staves Madison 545 Relationship between fecal microbiota and Cryptosporidium shedding 323 in calves Valdes Sabrina 585 Detection of tick-borne pathogens by immunohistochemical (IHC) 343 staining Wei Chuyan 602 Physiological synchronization between horse and human in EFP for 351 veterans with PTSD Wojtas Hunter 621 Mosquito diversity, abundance, and virus associations of Binder Park 361 Zoo in Battle Creek, Michigan Ophthalmology Banooni Dustin 28 Development and validation of a novel canine visual acuity 64 measurement protocol and procedure Bradford Madeline 47 The impact of netarsudil on endothelial regeneration in a murine 74 model of Fuch’s endothelial corneal dystrophy Gomez Zaira 188 Visual Acuity in Python regius 144

Halsmer Emily 209 A clinical study of ophthalmic parameters of the Quaker parrot 155 (Myiopsitta monachus) Hisey Erin 226 Characterization of antimicrobial peptides expressed by the equine 163 ocular surface and amniotic membrane Jones Katie 266 Wildfires causing poor air quality and its association with infected 183 corneal ulcerations in dogs Kimes Austin 288 Utilizing Peptide Amphiphile Micelles (PAMs) to improve genetic 194 therapy delivery for healing Corneal Fibrosis Liu Joshua 327 Investigating the effects of storage methods on rabbit corneas 214

McDaniel Kylie 371 Cataloging retinal lesions in Asiatic black bears (U. thibetanus) and 236 Malaysian sun bears (H. malaynus) Ortiz Fullana Maria 427 Visual Performance of Dogs in Low Light Levels 264

Raphtis Vanessa 461 Ocular Pulse Amplitude in normal beagles and beagles with 281 ADAMTS10-Open-Angle-Glaucoma (ADAMTS10-OAG)

2021 National Veterinary Scholars Symposium • 23 Poster Page Last Name First Name Abstract Title # # Rodts-Palenik Olivia 481 Efficacy and safety of the Rho-kinase inhibitor ripasudil (Glanatec) in 291 canine ADAMTS10-open-angle glaucoma Treadwell Caroline 573 Effect of autologous serum on corneal fibrosis in equine corneal 337 stromal fibroblasts Orthopedics Bianchi Catarina 37 Influence of chondrodystrophy on intervertebral disc mineralization 69 and vertebral geometry in the dog Carlson Jaclyn 72 CollagenV Haploinsufficiency Results in Delayed Healing and Altered 86 Wound Matrix Post-Injury in Murine Tendons Chase Kayla 81 Quantitative MRI measures of the biochemical and biomechanical 91 properties of canine intervertebral discs Chevalier Jacqueline 84 ThrombinatorTM thrombin fibrin sealant retains BioCartilage in 92 congruent, high-load cartilage defects Dalton Cambrie 110 Minimally invasive repair of acetabular fractures in dogs: an ex-vivo 105 feasibility study Gagliardi Rachel 164 The effect of BIO-PLY, a novel PRP lysate, on synoviocyte/ 132 chondrocyte co-culture supernatant viscosity Gagnon Nicole 165 Radiation lowers humeral cortical bone density but also osteoarthritis 133 incidence in non-human primates Gallego Alondra 167 The effects of Losartan use in dogs on stifle joint pain and progressive 134 osteoarthritis following TPLO Hattendorf Jenna 219 Evaluating orthopedic injuries during the Yukon Quest 160

Hynes Jessica 241 Safety and efficacy of a novel anti-DEK aptamer treatment for 171 osteoarthritis using a canine model Kerlin Brendan 282 Ex vivo evaluation of a novel tibial tuberosity advancement surgical 191 guide for the treatment of canine CrCLD Kobluk Landon 297 A Comparison of COX Inhibition and Soluble Epoxide Hydrolase 199 Inhibition in Murine Osteoarthritis Kuldell Caroline 306 Analysis of microRNA expression in chondrocytes as an indicator for 203 regenerative potential Lanza Julien 313 Osteoinductive properties and toxicity of a chitosan-based hydrogel 207 for spinal fusion application Long Sidney 332 Anti-inflammatory/anabolic factors IL1Ra and IGF-I are enriched in 216 equine bone marrow concentrate versus PRP MacKenzie Zoe 339 Assessment of repeatability of preoperative planning for the tibial 220 tuberosity advancement procedure Quiles-Torres Christian 458 Transduction efficacy of viral vector serotypes in canine joint cells for 279 osteoarthritis Shelton Emily 510 PPAR induction of Angiopoietin-like 4 in osteoblasts and its 305 interaction with hypoxia Smith Anna 526 Developing bioresorbable orthopaedic implants to apply increasing 313 loads throughout healing Tripp Payton 574 Evaluation of 3D printed Tendon Scaffolds for tissue engineering 337

Veenema Max 586 Evaluating accuracy of two 3D surface scanners when compared to 343 anatomical measurements of canine pelvic limbs

24 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Webster Meghan 600 Active cell fractions in bone marrow aspirate concentrate (BMAC) for 350 improved treatment of osteoarthritis Welsh Casey 607 CRAPP: A microCT-based method for P2 fracture scoring in a murine 354 Down Syndrome model Womack Sydney 622 Multispecies proteomics reveals potential biomarkers for osteoarthritis 361

Ya Deau Amelie 625 Developing 3D-printed osteotomy guides to improve accuracy of 363 surgical correction of angular limb deformities Zetterholm Haley 635 Efficacy of chitosan and polycaprolactone biomaterials as antibiotic 368 delivery vehicles in osteomyelitis Other Allen Tiffani 12 Sequencing of Infectious Bronchitis Virus 56

Awad Alexandra 21 Attitudes towards companion animals in Egypt 61

Badgett Samantha 23 Probabilistic genotyping in DNA mixtures of canine “bully breeds” 62 using DogFiler Baker Carol 25 Development of highly specific chicken IgY- based immunoassays for 63 the detection of Staphylococcal leukotoxins Baker Julia 26 Molecular identification of vectors and pathogens using 63 mobile, third-generation sequencing Blanco Cristina 41 Nanopore-based adaptive sequencing for mosquito-borne disease 71 surveillance Brathwaite Morgan 49 Impact of military chemical simulants on wicking of canine hair 75

Chawner Emma 82 A retrospective study of absence seizures in canines 91

Chi Jacqueline 85 Gestational sleep apnea induces autism-relevant behavioral and 93 neuronal aberrations in the offspring Christie Anneka 89 Dental and temporomandibular joint pathology of the Island Fox 95 (Urocyon littoralis) Cole DaZane 95 Investigating Bovine Anaplasmosis via analysis of ticks from Ohio 98 livestock Coonrod Sarah 102 The impact of maternal dietary deficiencies in folic acid and choline 101 on offspring neurological health Echizenya Shiori 130 Face mask debris - potential risk to face mask wearers 115

Elwood Stella 137 Client Perceptions of an Educational Partnership to Provide Low Cost 119 Veterinary Care to an Underserved Area Ettenger Simon 140 What is our impact? Examining accessible veterinary services at 120 Western University of Health Sciences’ CVM Flanagan Amanda 150 Determining the effect of neuron-derived extracellular vesicles on 125 axonal regeneration of dorsal root ganglia Flora Tasman 151 A review of botanical anti-Inflammatories for the treatment of 126 osteoarthritis Frese Rylie 158 Differentiation of PK resistant prion protein allelotypes in 129 heterozygous sheep with classical scrapie Gardner Jasmine 170 Alterations in the and Immune Responses Associated 135 with NADF Induced by PFOS 2021 National Veterinary Scholars Symposium • 25 Poster Page Last Name First Name Abstract Title # # Goldstein Savannah 185 Restoration of spermatogenesis after germ cell transplantation into 143 irradiated recipient piglet testes Gomes Dimitria 187 Methods of Separation and Characterization of L1CAM-associated 144 Extracellular Vesicles; A Systematic Review Hamza Anam 213 An interactive, equine neurology case simulator to improve problem- 157 solving skills Huber Hanah 236 Molecular mechanisms of antiviral nanoparticles against SARS- 168 CoV-2 Kaloss Alexandra 270 Cell specific EphA4 alters immune cell recruitment and vascular 185 remodeling following acute ischemic stroke Keilholz Amy 277 Cervical inflammatory-associated marker expression in rats following 189 respiratory motor neuron death Kim Lily 287 Maternal microbiota modulates a Fragile X-like Syndrome in 194 offspring mice Kuehnl Ashley 303 The effect of YY1 PcG function on hematopoietic stem cell 202 development Li Shumin 323 De novo alpaca reference genome assembly and genomics of minute 212 chromosome syndrome Lieberman Seth 324 Circumscribing laser cuts attenuate chronic focal cortical seizure 212 propagation in mice Lokaisingh Sarah 331 Effects of communication training on shared decision†making in 216 companion animal practice Lopez Catherine 333 A porcine gracilis myocutaneous flap model for Vascularized 217 Composite Allotransplantation: technique and tips Maggio Maria 345 Generation and characterization of a knock out Cyp2d gene cluster rat 223 model Marsh Jordan 355 Diet alters systemic biomarkers associated with the obese equine 228 phenotype: diagnostic and therapeutic value Merichko Danielle 380 The effect of the COVID-19 pandemic on student performance at 240 WesternU CVM Mykins Julia 401 The role of the gut microbiome on neurogenesis and cortical 251 development in germ free piglets Nagler Brendan 402 ATP7A, ATP7B, and RETN genotypes in Labrador retrievers with and 251 without copper associated hepatitis Nelson Caitlyn 407 Utilization focused evaluation to develop a sustainable program of 254 outcomes assessment Ortiz-Rivera Milton 429 Comparative analysis of osteocytes-canalicular networks among 265 vertebrates and their role in bone adaptation Pees Savannah 438 In vitro modeling of canine myotendinous junction using induced 269 pluripotent stem cells Reddy Alexandra 466 A novel xenotransplantation mouse model to study mammary gland 283 regulation and pathogenesis Roach Daana 476 Effect ofTTC37 gene variants on intestinal epithelial barrier function 288 in THES patients Rosa-Padilla Natalia 487 Effects of Porcine Intestinal Normothermic Machine Perfusion and 294 Cold Storage on Epithelial Barrier Integrity

26 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Shinn Grace 514 The Role of Advanced Glycation End Products in Neuroinflammation 307 and Neurodegeneration Spiker Justine 540 Measuring Axonal Damage in Drosophila Model of TBI 320

Torres Saez Ashley 571 Bead enrichment of prion protein using RT-QuIC for diagnosis of 336 Chronic Wasting Disease in live animals Troconis Eileen 575 Female dorsal raphe serotonin responses to sexual stimuli 338 Gonzalez Wegrzyniak Nicole 601 Interrogating the role of hydrogel electrical and physical properties on 351 neural cell behavior Weisberg Ilana 603 Evaluating the confidence and competency of team communication 352 skills gained via BLS and ALS training course Wildermuth Hannah 611 Inhibition of fibrotic mediators in partially obstructed mouse bladders 356 by administration of microRNA-29a Winn Emily 619 The molecular clock gene, CLOCK, in the human placenta 360

Zayas Hanna 632 Manipulation of DNp63 and Fosl1 modulate the regenerative capacity 366 of pulmonary progenitors Parasitology Barber Hannah 30 Search of acaricidal bacteria for the control of the lone star tick 65 (Amblyomma americanum) Barrow Ruth 31 Characterizing the roles of the Toxoplasma gondii calcium binding 66 proteins, TgCalnuc and CaM2 Brown Andrew 53 Evaluation of urine for Dirofilaria immitis antigen detection in dogs 77

Cacciatore Kyle 66 Seroreactivity of tick-immunized calves to five recombinantD. 83 andersoni proteins Chase Ellis 80 The prevalence of endoparasites of the Cuban treefrog and 90 Greenhouse frog in Florida Cubero Niurka 108 CircRNA expression in parasitic nematodes 104 Martinez Danks Hannah 112 Opening a can of (lung)worms: molecular characterization of 106 Dictyocaulus of North American bison DiGate Cameron 122 Transcriptional response of Aedes aegypti Malpighian tubules to 111 silencing of the Toll pathway inhibitor Cactus Hampton Naomi 212 Optimizing in vitro biochemical assays predictive of drug resistance in 156 Dirofilaria immitis microfilaria Keller Stefan 278 Anti-tick vaccine development for Anaplasma marginale 189

King Lindsey 289 Prevalence of B. burgdorferi in I. scapularis: comparison between 195 ticks collected from pets and the environment Lahar Rachael 309 Cryptosporidium phosphoenolpyruvate carboxylase as a molecular 205 target for novel potent antiparasitic drugs Marquez Ixzacil 354 Molecular components of oocyst formation in Cryptosporidium 227 parvum Martin Brady 358 Changes in antigen, antibody, and microfilaria in dogs during 229 Dirofilaria immitis infection and treatment

2021 National Veterinary Scholars Symposium • 27 Poster Page Last Name First Name Abstract Title # # Moscoso Erica 396 Evaluating treatments for bovine trichomonosis in a natural host 248 model Negron Veronica 405 Real-time PCR compared to modified Knott’s for detecting heartworm 253 microfilariae in routine heartworm testing Pope Catherine 452 Detection of Brugia malayi antigen in experimentally infected dogs 276 using a commercial heartworm test Rawdon Madison 463 A survey of parasites affecting wild fish populations in lakes across 282 Uganda Reardon Kiera 465 Parasites in Captive Felids at the Uganda Wildlife Education Center 283 and Their Zoonotic Implications Rogers Bridgette 483 Identification of tick salivary gland antigens recognized by protective 292 immune sera using a cDNA library Rowley Max 488 Characterization of immunoreactive cDNA expression library clones 294 of Baylisascaris procyonis Setzer Katelyn 506 Prevalence of Heterobilharzia americana in normal dogs from 303 Louisiana Tomlinson Trey 568 Surveillance of Borrelia burgdorferi in white-footed mice 334 (Peromyscus leucopus) in Pennsylvania Pharmacology and Toxicology Bergen Alexandra 35 Validation of a primary thyroid cell line to study feline 68 hyperthyroidism Buchanna Ireyon 59 Hypertension is associated with glucocorticoid excess and insulin 80 resistance in aged SHRSP rats Chiusano Alexandra 87 Differential effects of acetaminophen-induced cytotoxicity on 94 fibrinogen expression by mouse hepatocytes Coleman Jasmine 96 Genetic analysis of feline cannabinoid receptors - a pilot study 98

Cook Taylor 101 Serotonin production and signaling in mouse trophoblast giant cells 101

Fritz Bailey 159 Pharmacokinetics of acetylsalicylic acid and salicylic acid in dairy 130 cattle George Serena 176 PFAS bioaccumulation and associated wild fish and human health 138 impacts in the Detroit River, Michigan USA Gray Mackenzie 197 Improving the clinical efficacy of butorphanol for analgesia in dogs 149

Hoffmann Silke 230 Pharmacokinetics of grapiprant and effects on TNF alpha 165 concentrations following oral administration to horses Janey Madison 250 Understanding how the food preservative tBHQ can be associated 175 with Th2-meditated food allergies Kahn Brooke 269 Platelet activation by metabolites of clopidogrel 185

Kim Jessica 285 Role of cholecystokinin in immune-exhaustion of mice fed a Western- 193 style diet Kries Kelly 301 Micromanaging resistance: miRNA expression and chemosensitivity 201 in testicular germ cell tumors McCluney Tyana 369 Elimination kinetics of subcutaneously administered eprinomectin in 235 plasma and milk in dry-off dairy cattle

28 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Nguyen Sarah 414 Longitudinal assessment of structural brain alterations in a mouse 257 model of Gulf War Illness Okpechukwu Ebube 420 Chemerin: What Can We Learn 260

Penticoff Hailey 439 Understanding alpha-synuclein aggregation propensity in exotic, 270 wildlife, and zoo animals Sang Xiaoyang 496 Deliver CRISPR Cas9/sgRNA to respiratory epithelial cells 298

Sastry Lakshmi 498 Evaluating systemic exposure of drug detection dogs to narcotics 299 while performing their duties Scarlett Tasha 500 Dietary Omega-3 Fatty Acid Attenuates Silica-Triggered Autoimmune 300 Disease in16-Week-Old Lupus-Prone Mice Smiley Alaunie 525 Grape toxicosis in dogs: in vitro studies 313

Smith Lilly 529 The pharmacokinetics of a non-invasive (needle-less) administration 315 of flunixin meglumine in grower pigs Suarez Jessica 552 Evaluating basal arrhythmia incidence in a colony of telemetered non- 326 human primates Thomas Haley 564 Protein modification by oxidative fatty acid degradation products in a 332 mouse model of Alzheimer’s Disease (AD) Tucker Jackson 578 The effect of peroxynitrite on fibrinogen binding to integrins 339 expressed by THP 1 monocytic cells Yamada Sean 626 The Pharmacokinetics of Meloxicam and its effects on COX in Ball 363 Pythons (Python regius) Physiology Brunner Allison 57 Spectroscopic Characterization of Collagen and Biomechanical 79 Changes in Alcohol- Induced Osteoporosis in Mice Bukovec Katherine 60 History of eccentric contractions in Duchenne muscular dystrophy 80

Caldwell Madison 67 Glial cell inhibitor fluoroacetate suppresses enteric glial activity 84 marker GFAP in ischemia-injured jejunum Daly Kaitlin 111 Investigation of intrinsic nervous system dysfunction of the 106 gallbladder in dogs with mucocele formation Gibson Laurielle 178 Differential gene expression analysis of newborn mouse kidneys 139 lacking Prkaca (PKAca) Gluck Ryan 181 Central and peripheral contribution to heat stress intolerance in wild 141 type and MH susceptible mice Harper Briana 216 Defining the strain-specific effects of high-fat diet on intestinal 158 epithelial architecture in FVB mice Huff Denton 237 Myostatin deletion preserves glucose regulation, muscle function, and 169 endothelial function in type 1 diabetes Kobitter Emma 296 Butyrate supplementation rescues neural deficits in adult offspring in 198 rat model of gestational sleep apnea Lin Yimei 326 Extracellular vesicles and their role in promoting cell differentiation 213

Mamane Seryna 350 Comparison of moisture analysis methods using canned dog food 225

Marino Nicholas 353 Inflammation induced by gram-positive peptidoglycan may increase 227 apnea frequency in newborn rats 2021 National Veterinary Scholars Symposium • 29 Poster Page Last Name First Name Abstract Title # # Meredith Bryanna 379 Characterization of CFTR protein expression in gallbladder 240 epithelium of dogs with mucocele formation Myers Mikaela 400 Evaluation of diet-induced obesity on mRNA and ion channel 250 expression in murine hypoglossal motoneurons Nordenstahl Josefina 416 Immunohistochemical evaluation of the modular vascularity in the 258 inferior colliculus of the mouse Richards Laura 470 In vitro effects of oxycodone and nitric oxide synthase inhibition on 285 fetal rat striatal neuronal morphology Robben Nicole 477 Effect of mosquito salivary proteins in animal endothelial physiology 289

Rodriguez Mateo 480 Role of polyamine inhibitors in pheochromocytoma/paraganglioma 290 cancers with SDHB mutation Smith Emma 527 Effects of exercise on gastrointestinal tissue in an accelerated aging 314 murine model Sullivan Elizabeth 554 Evaluating Ovine Models for Human Reprometabolic Syndrome 327

Thompson- D. Angela 565 Renal effects of diltiazem continuous infusion in healthy dogs 333 Butler Timmerman Sarah 566 Developing a model of aldosterone producing adenomas for ablation 333 therapy to eliminate hyperaldosteronism Vernham Constance 590 Skeletal muscle fiber type changes contribute to muscle weakness in 345 sheep hypophosphatasia Production Animal Medicine Bradley Rachel 48 Development of antigen based diagnostic ELISA for Mycoplasma 74 hyopneumoniae Burke Erin 63 The effects of multimodal during disbudding on 82 health and performance in neonatal goat kids Fenster Lindsey 144 Mass Antimicrobial Administration and Multi-Drug Resistance in 122 Bovine Bacterial Respiratory Pathogens Hand Michelle 214 The effect of ruminal fluid transfaunation onCryptosporidium parvum 157 shedding in calves Hoerth Alexandria 228 Improving detection of digital dermatitis in beef cattle using computer 164 vision and portable devices Isensee Paige 244 Evaluation of lesions associated with condemnation of livers from 172 Missouri cattle Jantzen Derek 251 Ultrasound analysis of Holstein calf mammary gland development 176

Johnson Remie 259 Locomotor activity in nutrient restricted primiparous beef cows during 180 the final 72 hours prepartum Menn Tanzania 378 Validation of a novel goat kid health scoring system 239

Myers Madison 399 Cannabinoids modulate adipogenesis and lipogenesis in periparturient 250 dairy cows Nelson Joel 408 Effects of steroid implants and differing tall fescue endophyte 254 (Acremonium coenophialum) levels on cattle Painter Miranda 432 An insight to Ornithobacterium rhinotracheale through a time space 266 investigation in turkeys

30 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Skaggs Brittany 521 The use of Oxford Nanopore Technology as a single test to identify 311 and characterize infectious bronchitis Wangler Courtney 597 Differentiation of H3N2 IAV-S antigenic sites by RT-qPCR 349

Wesley Kaitlyn 609 Chronic pen outcomes based on diagnosis and the economic loss due 355 to bovine respiratory disease SARS-CoV-2 and Animal Health McClain Sierra 368 Evaluation of antibodies against SARS-CoV2 Spike proteins in dogs: 234 an analysis of exposure over the pandemic Adams Josetta 6 Longitudinal Assessment of SARs-CoV-2-Specific T Cell Response in 53 Convalescent Orthodox Jewish Adolescents Griffin Kristen 201 p38 MAPK and NF-kB pro-inflammatory pathways are upregulated 151 during SARS-CoV-2 infection Johnston Dylan 261 Therapeutic Intervention of SARS-CoV-2: Isolation and Purification 181 of SARS-CoV-2 Nucleocapsid Protein Jonas Maya 263 Establishing the stability of a MVA viral vector pancoronavirus 182 vaccine motif via serial passaging Joung Yujin 268 Comparative analyses of adjuvants and immunization routes on 184 antibody induction against the RBD of SARS-CoV-2 Patton Titus 435 Evaluating the basis for severe inflammation caused by pathogenic 268 human coronaviruses Spangenberg Caroline 537 Assessment of viral load and cytokine gene expression in lung tissues 319 from SARS-CoV2-infected mice Shelter Medicine Blowers Zelda 42 Supporting the human-animal bond among homeless and housing 71 insecure pet owners in Philadelphia, PA Cashel Abigail 74 Demographics of community cats presented to a veterinary clinic over 87 a ten-year span Goss Sarah 193 Reasons for relinquishment to animal shelters survey study 147

Gremling Megan 199 Reasons for Relinquishment to Animal Shelters: A Survey Study 150

Lewis Kendall 319 Mortality rate and risk factors for anesthesia-related deaths of rabbits 210 in an Arizona McCallin Ashlee 367 Pyometra management in the private practice setting 234

Porcher Tenley 453 The impacts of COVID-19 on high-quality, high-volume, spay-neuter 277 clinics Zavota Gianna 631 An exploratory study of stress behavior in dogs 366

Small Animal Medicine and Surgery Allen Megan 11 Differences in physiologic parameters in sled dogs and shelter dogs 56 undergoing anesthesia Applebaum Jennifer 15 Cytochrome P450 reaction phenotyping of hydroxyitraconazole 58 metabolism in canine liver Arendt Tessa 16 Analysis of computed tomography derived laryngotracheal 58 dimensions and correlation with body weight in rabbits

2021 National Veterinary Scholars Symposium • 31 Poster Page Last Name First Name Abstract Title # # Arne Allison 17 Effect of oral gabapentin on directly obtained ambulatory systemic 59 arterial blood pressure in healthy cats Beard Mary 32 Bladders and biofilms: chronic urinary tract infections in dogs 66

Berlin Dena 36 Incidence of gallbladder sludge in cats and correlation with routine 68 laboratory parameters Biehl Kathryn 38 Does closed glove exchange result in bacterial contamination of the 69 surgeon’s hands? Bonce Daniel 44 Retrospective evaluation of whole-body CT findings and prognostic 72 outcomes in canine trauma patients Boothby Grace 45 Accuracy of volume or weight measurement of cooked homemade 73 diets for dogs Diaz Amanda 120 Alpha-enolase as a biomarker for early diagnosis of acute kidney 110 injury and chronic kidney disease in cats Gabaldon Cristina 163 Surgical approaches to the canine liver hilus: a comparison of five 132 techniques and evaluating diaphragmotomy Gerras Jordan 177 Evaluation of contamination risk associated with forced-air warming 139 units in canine surgery Hoel Meghan 227 Comparing standard urine collection techniques to Peezy midstream 164 device in a canine model Hunter Pauline 240 Prevalence of Intestinal Helminths in Dogs and Cats and History of 170 Anthelmintic and Preventive Products Usage Jachec Emily 245 Clinical use of ursodiol in feline medicine 173

Jackson Elizabeth 246 Safety and Efficacy of Low-Dose Radiation Therapy as a Modality for 173 the Management of Feline Chronic Rhinitis Jacobson Sarah 249 Correction formula determination for estimated glomerular filtration 175 rate in puppies with and without XLHN Johnson Maddison 258 Risk factors associated with short-term perioperative anesthesia- 179 related deaths of dogs and cats in Arizona Kratzer Gabriella 298 Cost comparison of home-cooked diets vs. commercial therapeutic 199 diets for dogs with gastrointestinal disease Levy Samuel 318 Clinicopathologic findings in dogs diagnosed with coccidioidomycosis 209

Morgan Michael 392 Canine perineal hernia and comorbidities: a retrospective study 246

Pettibon Erin 447 Evaluation of hematological and biochemical profile trends in patients 274 with thyroid disorders Simon Katherine 517 Development of a novel point-of-care prognostic test of neural injury 309 for dogs Suero Monica 553 Analysis and culture results of free-catch urine samples in dogs 327

Tarbutton Jordan 559 Markers of endothelial injury in canine trauma patients: a pilot study 330

Vine Kristopher 592 Canine otitis externa diagnosis and treatment 346

Wells Jamie 606 A novel surgical approach to laryngeal collapse in brachycephalic 353 canines Yamamoto Kirby 627 Reverse Trendelenburg and the incidence of gastroesophageal reflux 364 (GER) in Isoflurane anesthetized dogs 32 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Theriogenology Adams Daniella 5 Effect of maternal obesity on placental, fetal, and maternal outcomes 53 in a mouse model of preeclampsia, BPH/5 Alvarez Alyssa 13 The effects of liquid storage and cryopreservation on the morphometry 57 of the West Indian manatee spermatozoa Gonzalez Gabriella 189 Glucocorticoid receptor in 1st trimester human trophoblast cells as 145 tool to study GR signaling in placentation Harrison Sydney 218 Characterizing Anatomy and Predicting Function of the Male Florida 159 Softshell Turtle Phallic Glans Jones Adrian 264 A Retrospective Study (2008-2018) of Canine Pyometra in Grenada, 182 West Indies Min Seunga 387 Co-Localization of Glucocorticoid Receptors and KDM1A in four 244 placenta types Nelson Alexis 406 Early identification of canine benign prostatic hyperplasia using 253 prostatic fluid metabolomic biomarkers O’Malley Brianna 423 Non-surgical Approach to Sterilizing Avian Species Using a Domestic 262 Chicken Model Roach Alexis 475 Nonhuman Primate pregnancies at advanced maternal age show 288 markers of poor metabolic and cardiovascular health Scarberry Nicole 499 Crocodylus niloticus penile glans malformation: a case study for 300 consideration of breeding soundness Sexton Brigid 507 Deslorelin-induced infertility affects expression of selected 304 antioxidant enzymes in the testis of dogs Siegel Katelyn 515 Barriers to female fertility in xylosyltransferase II deficient mice 308

Stallings Bryan 543 Platelet-rich plasma lysate as a treatment for biofilm associated 322 placentitis Stoddard Serena 547 Factors affecting vitrification of canine epididymal spermatozoa 324

Virology Asrat Makda 19 Surveillance for novel coronaviruses in West African bats 60

Browning Matthew 56 Selenium as a Potential Antiviral for PRRSV Infection 78

Chalmin Alyssa 79 Platelet-endothelial associations are dependent on CD40/CD40L 90 interactions in the context of EcoHIV Conry Megan 99 Antiretroviral therapy effects on Feline Immunodeficiency Virus 100 salivary viral load Cover Sarah 105 Screening and characterization of coronaviruses in veterinary patients 103

Dufour Rachel 128 The contributions of different functions of herpes simplex virus ICP22 114 to viral replication Henry Caitlin 221 Prevalence and pathology of equine parvovirus-hepatitis in New York 161 racehorses Hoffman Justin 229 A Role for EBOV VP40 PPxY Motif in Regulating Tight Junction 165 Complexes of the BBB and Corneal-Epithelium Huang Callista 235 First detection of a bat coronavirus in a New England big brown bat 168

2021 National Veterinary Scholars Symposium • 33 Poster Page Last Name First Name Abstract Title # # Jackson Jared 247 Regulation of host gene expression by an emerging paramyxovirus 174

Kang Danyue 271 Inflammatory markers of respiratory syncytial virus in the cotton rat 186

Kumar Ayush 308 Sex specific LRRK2 modulation of inflammation during 204 neuroinvasive viral infection in transgenic mice Loessberg Emma 329 Susceptibility of mosquito and bird cells to Usutu virus 215

Marino Morgan 352 Phylogenomic assessment of feline herpesvirus type 1 (FHV-1) in 226 cheetahs (Acinonyx jubatus) Midkiff Amanda 384 Defining the Hbz/YBX1 interaction and its role in HTLV-1 242 pathogenesis Paulson Alexandrea 436 Investigating recombinant mammalian orthoreovirus expressing 268 HER2 peptides as a HER2+ breast cancer vaccine Shaw Laura 509 Variation of in-vitro bronchiolar attachment of zoonotic H1 influenza 305 A viruses of swine Shepley- Ariel 511 Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and 306 Mctaggart Promotes Egress of VP40 VLPs Skelly Robert 523 Senecavirus protective immune response and transmission in 312 replacement gilts on persistently infected farms Stubbs Eric 551 Optimizing the detection of persistent parvovirus infections in zoo- 326 housed red pandas (Ailurus fulgens) Takaesu Tiare 558 Epitope based vaccine design: targeting HIV fusion peptide and 329 membrane proximal external region Tortella Ty 572 Setting the standard: optimization of diagnostic testing methods for 336 avian bornavirus Velazquez Jossette 588 Characterizing infectivity of Spondweni virus and Zika virus isolate in 344 human placenta and neuronal cell lines Weng William 608 Experimental infection of ferrets with different SARS-CoV-2 lineages 354

Young Kirsten 629 Can Influenza A Virus infect sialic acid knock out mammalian cells 365 through artificial antibody receptor? Wildlife Conservation Acquaviva Mariana 3 Anticoagulant rodenticide residue levels in liver samples from a 52 Maine Canada Lynx population Brown Kristen 55 Analysis of commonly encountered toxins in avian patients presenting 78 to a wildlife rehabilitation service Buzzell Joseph 65 Retrospective analysis of Avitrol toxicity in rock pigeons (Columbia 83 livia) presenting to a wildlife service Fieglar Angelica 148 Efficacy of tiletamine/zolazepam with butorphanol, medetomidine and 124 azaperone for the immobilization of bison Franzak Jacob 156 Melanomacrophage centers as a measure of lead toxicity in Lepomis 128 megalotis from southeast Missouri Galeuzzi Olivia 166 Endoparasite survey of foxes and coyotes on Prince Edward Island, 133 Canada Gentzkow Morgan 175 Biological and hematological profiles of bighorn sheep Ovis( 138 canadensis)

34 • 2021 National Veterinary Scholars Symposium Poster Page Last Name First Name Abstract Title # # Goodwein Abrielle 190 Characterization of injuries associated with fishing gear in California 145 seabirds Hojsak Jillian 231 Erosion of paint-coated lead fishing gear in a simulated loon gizzard 166

Indeglia Vanessa 243 Assessing bat diversity and abundance in Massachusetts using 172 acoustic monitoring Macquisten Ella 341 The effect of reproductive status on in vitro embryo production in 221 bison Maestas Jesse 344 Determining sub-clinical effects of lead exposure on southern 222 California turkey vultures (Cathartes aura) Martone Carly 360 A genomics approach to characterize white coat color genes in 230 American bison, Bison bison McGimsey Malea 372 Assessing northern sea otter (Enhydra lutris kenyoni) immune 236 function in Alaska Morris Clarice 393 Domestic Animal Injuries and their Impacts on Vulnerable Wildlife 247 Species in central Ohio Orluk Julia 424 Temporal comparison of plasma total mercury in Steller sea lions 262 (Eumetopias jubatus) of the Aleutian Islands Orrico William 425 Herpesvirus prevalence in neotropical bat species across disrupted 263 landscapes in northwestern Ecuador Sherinian Rachel 512 Bat activity and diversity across three microhabitats on a university 306 campus in North Grafton, Massachusetts Shields Morgan 513 MiteBusters: The Survival, Control, and Identity of Respiratory 307 (Halarachnidae) in Sea Otter Facilities Smith Nicole 530 A citizen science pilot study: results from a survey about urbanized 315 parrots in Point Loma, San Diego Sparks Jessica 538 The effect ofFusarium spp. on cold-stunned Kemp’s ridley sea turtles 319

Springer Kelsey 542 Preliminary investigation of deleterious alleles in peregrine falcons, 321 golden eagles, and zebra finches Straw Stephanie 549 Vocal characteristics of an Atelopus zeteki breeding colony 325

Struck Madeline 550 Determining the threshold of serum progesterone to diagnose 325 pregnancy in moose using ROC Analysis Weiss Paige 605 Validating behavioral indications of stress in red-tailed hawks 353

2021 National Veterinary Scholars Symposium • 35 Symposium Participants by College of Veterinary Medicine

Vet School Last Name First Name Poster # Atlantic Veterinary College Bailey Lindsey 24 Atlantic Veterinary College Brown Kristen 55 Atlantic Veterinary College Buzzell Joseph 65 Atlantic Veterinary College Fieglar Angelica 148 Atlantic Veterinary College Fournier Zoe 155 Atlantic Veterinary College Galeuzzi Olivia 166 Atlantic Veterinary College Guarino Katherine 203 Atlantic Veterinary College Hamza Anam 213 Atlantic Veterinary College Johnson Callan 256 Atlantic Veterinary College Knap Christopher 294 Atlantic Veterinary College MacKenzie Zoe 339 Atlantic Veterinary College MacNeil Lauren 340 Atlantic Veterinary College Maguire Daniel 346 Atlantic Veterinary College McCurdy Raelyn 370 Atlantic Veterinary College Messina Andrea 382 Atlantic Veterinary College Morse Allison 394 Atlantic Veterinary College Watts Remy 598 Atlantic Veterinary College Wei Chuyan 602 Auburn University CVM Coleman Jasmine 96 Colorado State University CVM Cao Jennifer 70 Colorado State University CVM Conry Megan 99 Colorado State University CVM Diaz Amanda 120 Colorado State University CVM Dugan Conner 129 Colorado State University CVM Fink Brent 149 Colorado State University CVM Gonzalez Gabriella 189 Colorado State University CVM Haines Laurel 207 Colorado State University CVM Hattendorf Jenna 219 Colorado State University CVM Henry Mikaela 222 Colorado State University CVM Jones Katie 266 Colorado State University CVM Kerlin Brendan 282 Colorado State University CVM Kuldell Caroline 306 Colorado State University CVM Lopez Catherine 333 Colorado State University CVM Marsh Jordan 355 Colorado State University CVM Maslyn Kara 361 Colorado State University CVM McAdam Tiera 365 Colorado State University CVM McGimsey Malea 372 Colorado State University CVM Min Seunga 387 Colorado State University CVM Ojeda Vivian 419

36 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # Colorado State University CVM Olmsted Zoe 422 Colorado State University CVM Orluk Julia 424 Colorado State University CVM Orsini Gabrielle 426 Colorado State University CVM Paci Nicolette 431 Colorado State University CVM Perez Luisanny 440 Colorado State University CVM Perez Jaimes Martin 441 Colorado State University CVM Rinker Cody 474 Colorado State University CVM Sanfacon Brittney 495 Colorado State University CVM Slaughter Megan 524 Colorado State University CVM Snell Savannah 533 Colorado State University CVM Sullivan Elizabeth 554 Colorado State University CVM Tarbutton Jordan 559 Colorado State University CVM Weaver Danielle 599 Colorado State University CVM Webster Meghan 600 Colorado State University CVM Weisberg Ilana 603 Colorado State University CVM Westbrook Madeleine 610 Cornell University CVM Casseri Eleni 76 Cornell University CVM Chevalier Jacqueline 84 Cornell University CVM DeJohn Abigail 117 Cornell University CVM Drescher Matthew 127 Cornell University CVM Flanagan Amanda 150 Cornell University CVM Gomes Dimitria 187 Cornell University CVM Granger Julie 196 Cornell University CVM Harper Briana 216 Cornell University CVM Henry Caitlin 221 Cornell University CVM Hommer Alexandra 232 Cornell University CVM Hung Clarey 239 Cornell University CVM Ida Jennifer 242 Cornell University CVM Kim Lily 287 Cornell University CVM Levitskiy Alexander 317 Cornell University CVM Lieberman Seth 324 Cornell University CVM Lin Yimei 326 Cornell University CVM Liu Patrick 328 Cornell University CVM Morse Benjamin 395 Cornell University CVM Presher Alexandra 456 Cornell University CVM Ramsaran Leah 460 Cornell University CVM Seminara Jackson 505 Cornell University CVM Steinhilber Megan 546 Cornell University CVM Tarsillo Justine 560 Cornell University CVM Troconis Gonzalez Eileen 575 Cornell University CVM Urbina Christian 584 Cornell University CVM Womack Sydney 622 Cornell University CVM Zenilman Shoshana 634 Cummings SVM at Tufts University Acquaviva Mariana 3 2021 National Veterinary Scholars Symposium • 37 Vet School Last Name First Name Poster # Cummings SVM at Tufts University Awad Alexandra 21 Cummings SVM at Tufts University Bixby Ryan 40 Cummings SVM at Tufts University Cashel Abigail 74 Cummings SVM at Tufts University Celatka Jillian 77 Cummings SVM at Tufts University Choi Diana 88 Cummings SVM at Tufts University Coughlin Laura 104 Cummings SVM at Tufts University Dharmasena Vidharshi 119 Cummings SVM at Tufts University Dolan Samantha 123 Cummings SVM at Tufts University Elwood Stella 137 Cummings SVM at Tufts University Hojsak Jillian 231 Cummings SVM at Tufts University Huang Callista 235 Cummings SVM at Tufts University Indeglia Vanessa 243 Cummings SVM at Tufts University Karp Shelby 273 Cummings SVM at Tufts University Li Jingyi 322 Cummings SVM at Tufts University Maguire Maria 347 Cummings SVM at Tufts University McDaniel Kylie 371 Cummings SVM at Tufts University Munroe Cassandra 398 Cummings SVM at Tufts University O’Malley Brianna 423 Cummings SVM at Tufts University Orrico William 425 Cummings SVM at Tufts University Richards Laura 470 Cummings SVM at Tufts University Scott Samantha 502 Cummings SVM at Tufts University Shaffert Kathryn 508 Cummings SVM at Tufts University Sherinian Rachel 512 Cummings SVM at Tufts University Sistig Lorena 520 Cummings SVM at Tufts University Specht Aubrey 539 Cummings SVM at Tufts University Wells Jamie 606 Cummings SVM at Tufts University Wunderlich Emma 624 Cummings SVM at Tufts University Zavota Gianna 631 Federal University of Agriculture, Makurdi Edache Stephen 131 Iowa State University CVM Brezina Caleb 51 Iowa State University CVM Cubero Martinez Niurka 108 Iowa State University CVM Esala Micah 139 Iowa State University CVM Frese Rylie 158 Iowa State University CVM Garcia Marrero Tatiana 169 Iowa State University CVM Jorgensen Hannah 267 Iowa State University CVM Joung Yujin 268 Iowa State University CVM Kelly Jamillah 279 Iowa State University CVM Klatzer Emily 292 Iowa State University CVM Krellwitz Kyle 299 Iowa State University CVM Kroeger Austin 302 Iowa State University CVM McGinley Jared 373 Iowa State University CVM Moncrief Michele 390 Iowa State University CVM Moscoso Erica 396 Iowa State University CVM Nelson Alexis 406 38 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # Iowa State University CVM Painter Miranda 432 Iowa State University CVM Paulson Alexandrea 436 Iowa State University CVM Powers Vanessa 454 Iowa State University CVM Reed Miriam 467 Iowa State University CVM Richardson Sydney 471 Iowa State University CVM Skaggs Brittany 521 Iowa State University CVM Skelly Robert 523 Iowa State University CVM Takaesu Tiare 558 Iowa State University CVM Wesley Kaitlyn 609 Kansas State University CVM Barber Hannah 30 Kansas State University CVM Cassale Nicole 75 Kansas State University CVM Fritz Bailey 159 Kansas State University CVM Gray Mackenzie 197 Kansas State University CVM Hall Madeline 208 Kansas State University CVM Hampton Naomi 212 Kansas State University CVM Harris Shanice 217 Kansas State University CVM Heinen Lilli 220 Kansas State University CVM Huber Hanah 236 Kansas State University CVM Hull Morgan 238 Kansas State University CVM Jackson Nathan 248 Kansas State University CVM Lai Poyu 310 Kansas State University CVM Li Shumin 323 Kansas State University CVM McCall Jayden 366 Kansas State University CVM Meyers Walter 383 Kansas State University CVM Nelson Joel 408 Kansas State University CVM O’Day Shannon 417 Kansas State University CVM Robben Nicole 477 Kansas State University CVM Thompson-Butler D. Angela 565 Kansas State University CVM Timmerman Sarah 566 Kansas State University CVM Tomlinson Trey 568 Kansas State University CVM Ullom-Minnich Rebecca 583 Kansas State University CVM Weng William 608 Lincoln Memorial University CVM Davis Elaina 114 Lincoln Memorial University CVM Geer Allison 174 Lincoln Memorial University CVM Halsmer Emily 209 Lincoln Memorial University CVM Mithani Sarah 388 Lincoln Memorial University CVM Morales Saira Mary 391 Louisiana State University SVM Adams Daniella 5 Louisiana State University SVM Badamo Dalia 22 Louisiana State University SVM Benton-Levith Elizabeth 34 Louisiana State University SVM Broussard Rebecca 52 Louisiana State University SVM Cerrato Alexandra 78 Louisiana State University SVM Clark Samantha 90 Louisiana State University SVM Dicharry Jena 121 2021 National Veterinary Scholars Symposium • 39 Vet School Last Name First Name Poster # Louisiana State University SVM Dufour Rachel 128 Louisiana State University SVM Freeman Layne 157 Louisiana State University SVM Godwin Taylor 182 Louisiana State University SVM Lex Zoey 320 Louisiana State University SVM Marino Morgan 352 Louisiana State University SVM Ortiz Negron Nashelys 428 Louisiana State University SVM Patel Meera 434 Louisiana State University SVM Quiles-Torres Christian 458 Louisiana State University SVM Setzer Katelyn 506 Louisiana State University SVM Tripp Payton 574 Louisiana State University SVM Valdes Sabrina 585 Louisiana State University SVM Veenema Max 586 Louisiana State University SVM Willis Kaitlynn 614 Louisiana State University SVM Windham Jennifer 617 Michigan State University CVM Acevedo Stephanie 2 Michigan State University CVM Allen Megan 11 Michigan State University CVM Buchanna Ireyon 59 Michigan State University CVM Chiusano Alexandra 87 Michigan State University CVM Crawford Megan 107 Michigan State University CVM Decker Andrew 116 Michigan State University CVM Ferchaw Hunter 145 Michigan State University CVM Gallego Alondra 167 Michigan State University CVM Garcia Carmen-Maria 168 Michigan State University CVM Hamlin Nicole 211 Michigan State University CVM Hickey Amber 224 Michigan State University CVM Hipkiss Hannah 225 Michigan State University CVM Howard Cory 234 Michigan State University CVM Hynes Jessica 241 Michigan State University CVM Jackson Elizabeth 246 Michigan State University CVM Janey Madison 250 Michigan State University CVM Kahn Brooke 269 Michigan State University CVM Langfeldt Antonia 312 Michigan State University CVM Majlessi Elliot 349 Michigan State University CVM Maresca-Fichter Hailey 351 Michigan State University CVM McClain Sierra 368 Michigan State University CVM Myers Madison 399 Michigan State University CVM Nagler Brendan 402 Michigan State University CVM Nayak Arpita 404 Michigan State University CVM Okpechukwu Ebube 420 Michigan State University CVM Penticoff Hailey 439 Michigan State University CVM Phillip Lauren 448 Michigan State University CVM Rafique Sonia 459 Michigan State University CVM Raphtis Vanessa 461 Michigan State University CVM Skalecki Shannon 522 40 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # Michigan State University CVM Spilker Eric 541 Michigan State University CVM Suarez Jessica 552 Michigan State University CVM Tucker Jackson 578 Michigan State University CVM Winn Emily 619 Michigan State University CVM Wojtas Hunter 621 Midwestern University CVM Berlin Dena 36 Midwestern University CVM Bulloch Daegan 61 Midwestern University CVM Callisher Jessica 68 Midwestern University CVM Carswell Imani 73 Midwestern University CVM Coonrod Sarah 102 Midwestern University CVM Daigneault Emily 109 Midwestern University CVM Felix Mia 143 Midwestern University CVM Fuller Brenley 161 Midwestern University CVM Gasper Jepierah 172 Midwestern University CVM Hand Michelle 214 Midwestern University CVM Jasenski Jessica 252 Midwestern University CVM Javier Sirah 253 Midwestern University CVM Johnson Maddison 258 Midwestern University CVM Kim Jessica 285 Midwestern University CVM Levy Samuel 318 Midwestern University CVM Lewis Kendall 319 Midwestern University CVM Logue Mary 330 Midwestern University CVM Mahmood Nicole 348 Midwestern University CVM Marsh Shelby 356 Midwestern University CVM McCallin Ashlee 367 Midwestern University CVM Mihailovich Alison 385 Midwestern University CVM Myers Mikaela 400 Midwestern University CVM Perkins Heather 442 Midwestern University CVM Peterson Jessica 445 Midwestern University CVM Reid Heather 468 Midwestern University CVM Rocco Felicia 479 Midwestern University CVM Roe Nicolette 482 Midwestern University CVM Rosales April 486 Midwestern University CVM Russek Joshua 490 Midwestern University CVM Sinclair Amanda 518 Midwestern University CVM Smith Emma 527 Midwestern University CVM Spiker Justine 540 Midwestern University CVM Staves Madison 545 Midwestern University CVM Straw Stephanie 549 Midwestern University CVM Tonelli-Raylove Alanna 569 Midwestern University CVM Veith Brayden 587 Midwestern University CVM Wilson Alec 615 Mississippi State University CVM Baker Carol 25 Mississippi State University CVM Barber Cassandra 29 2021 National Veterinary Scholars Symposium • 41 Vet School Last Name First Name Poster # Mississippi State University CVM Beard Mary 32 Mississippi State University CVM Brunner Allison 57 Mississippi State University CVM Ellison Lauren 136 Mississippi State University CVM Evans Ian 141 Mississippi State University CVM Ferguson Kyle 146 Mississippi State University CVM Grabner Carly 195 Mississippi State University CVM Knight Hannah 295 Mississippi State University CVM Mertz Madison 381 Mississippi State University CVM Pray Hannah 455 Mississippi State University CVM Rawdon Madison 463 Mississippi State University CVM Reardon Kiera 465 Mississippi State University CVM Rosado Ramos Braiam 485 Mississippi State University CVM Sparks Jessica 538 Mississippi State University CVM Swales Ella 556 Mississippi State University CVM Turner Skyler 581 Mississippi State University CVM Zetterholm Haley 635 North Carolina State University CVM Amblard Sarah 14 North Carolina State University CVM Badgett Samantha 23 North Carolina State University CVM Bonadie Kayla 43 North Carolina State University CVM Brown Chalise 54 North Carolina State University CVM Browning Matthew 56 North Carolina State University CVM Caldwell Madison 67 North Carolina State University CVM Daly Kaitlin 111 North Carolina State University CVM Ellis Ilene 135 North Carolina State University CVM Ferraro Emma 147 North Carolina State University CVM Gagliardi Rachel 164 North Carolina State University CVM Gagnon Nicole 165 North Carolina State University CVM Gerras Jordan 177 North Carolina State University CVM Gorrell Kaitlin 192 North Carolina State University CVM Goss Sarah 193 North Carolina State University CVM Gremling Megan 199 North Carolina State University CVM Jolley Ashlan 262 North Carolina State University CVM Jones Hannah 265 North Carolina State University CVM Kepley Abby 281 North Carolina State University CVM Lambert Jessica 311 North Carolina State University CVM Maulini Jennifer 364 North Carolina State University CVM McCluney Tyana 369 North Carolina State University CVM Meredith Bryanna 379 North Carolina State University CVM Newberry Victoria 410 North Carolina State University CVM O’Donnell Kerry 418 North Carolina State University CVM Olivares Jasmine 421 North Carolina State University CVM Ortiz Fullana Maria 427 North Carolina State University CVM Roach Alexis 475 North Carolina State University CVM Robertson Hannah 478 42 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # North Carolina State University CVM Rosa-Padilla Natalia 487 North Carolina State University CVM Scarlett Tasha 500 North Carolina State University CVM Simon Katherine 517 North Carolina State University CVM Smith Lilly 529 North Carolina State University CVM Smith Sidney 532 North Carolina State University CVM Stallings Bryan 543 North Carolina State University CVM Velez Contreras Raisa 589 North Carolina State University CVM Villasenor Adriana 591 North Carolina State University CVM Walsh Elizabeth 593 North Carolina State University CVM Wang Christine 595 North Carolina State University CVM Zayas Santiago Gabriella 633 Ohio State University CVM Allen Brittany 9 Ohio State University CVM Cole DaZane 95 Ohio State University CVM Horton Julia 233 Ohio State University CVM Jachec Emily 245 Ohio State University CVM Kang Danyue 271 Ohio State University CVM Kratzer Gabriella 298 Ohio State University CVM Li Emma 321 Ohio State University CVM Long Sidney 332 Ohio State University CVM Ma Crystal 338 Ohio State University CVM McGlynn Andrew 374 Ohio State University CVM Midkiff Amanda 384 Ohio State University CVM Morris Clarice 393 Ohio State University CVM Riha Jordan 472 Ohio State University CVM Salerno Matthew 492 Ohio State University CVM Shields Morgan 513 Ohio State University CVM Snyder Hailey 535 Ohio State University CVM Summers Jamie 555 Ohio State University CVM Tyler Kyleigh 582 Ohio State University CVM Wang Wenjun 596 Ohio State University CVM Wittorff Hannah 620 Ohio State University CVM Woodfint Rachel 623 Oklahoma State University CVM Foley Brayden 152 Oklahoma State University CVM Golden Olivia 183 Oklahoma State University CVM Griffin Kristen 201 Oklahoma State University CVM Huff Denton 237 Oklahoma State University CVM John Shannon 255 Oklahoma State University CVM King Lindsey 289 Oklahoma State University CVM Lucich Dylan 334 Oklahoma State University CVM Martin Brady 358 Oklahoma State University CVM Patton Titus 435 Oklahoma State University CVM Siegel Katelyn 515 Oklahoma State University CVM Tryzbiak Madison 577 Oklahoma State University CVM Willis Erin 613 2021 National Veterinary Scholars Symposium • 43 Vet School Last Name First Name Poster # Oniris Nantes Lanza Julien 313 Ontario Veterinary College Chawner Emma 82 Ontario Veterinary College Kumar Ayush 307 Ontario Veterinary College Kumar Ayush 308 Ontario Veterinary College Lokaisingh Sarah 331 Ontario Veterinary College Poon Andrew 451 Oregon State University CVM Flora Tasman 151 Oregon State University CVM Gentzkow Morgan 175 Oregon State University CVM Matsuoka Shelby 363 Oregon State University CVM Sipple Nicole 519 Purdue University CVM Clifford Abigail 91 Purdue University CVM Davis Camryn 113 Purdue University CVM Fumia Heather 162 Purdue University CVM Goldsmith Malaycia 184 Purdue University CVM Graber Kami 194 Purdue University CVM Ortiz-Rivera Milton 429 Purdue University CVM Rowley Max 488 Purdue University CVM Smiley Alaunie 525 Purdue University CVM Willis Emily 612 Royal Veterinary College Cover Sarah 105 Royal Veterinary College Hagen Jennifer 205 Royal Veterinary College Young Kirsten 629 St Georges University SVM Jones Adrian 264 St Georges University SVM Kearney Allison 276 Stephens College Harrison Sydney 218 Stephens College Kujiraoka Amanda 305 Texas A&M University CVM Biehl Kathryn 38 Texas A&M University CVM Brown Andrew 53 Texas A&M University CVM Burleson Daniella 64 Texas A&M University CVM Cannon Brinley 69 Texas A&M University CVM Combs Olivia 97 Texas A&M University CVM Danks Hannah 112 Texas A&M University CVM Gartner Kathleen 171 Texas A&M University CVM Given Christopher 179 Texas A&M University CVM Glenn Nicole 180 Texas A&M University CVM Gunn Morgan 204 Texas A&M University CVM Jacobson Sarah 249 Texas A&M University CVM Lumba Rohini 336 Texas A&M University CVM Martone Carly 360 Texas A&M University CVM Negron Veronica 405 Texas A&M University CVM Parsons Dillin 433 Texas A&M University CVM Ruffin Robert 489 Texas A&M University CVM Tortella Ty 572 Texas A&M University CVM Vernham Constance 590 44 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # Texas A&M University CVM Welsh Casey 607 Texas A&M University CVM Wilson Rachel 616 Tuskegee University CVM Bryson Andreya 58 Tuskegee University CVM Gardner Jasmine 170 Tuskegee University CVM Gibson Laurielle 178 Tuskegee University CVM Hunter Pauline 240 Tuskegee University CVM Johnson Sierra 260 Tuskegee University CVM Lyles Dominique 337 Tuskegee University CVM Madison Elsie 342 Tuskegee University CVM Pettibon Erin 447 Tuskegee University CVM Rogers Chloe 484 Tuskegee University CVM Snipes Bryanna 534 Tuskegee University CVM Stoddard Serena 547 Tuskegee University CVM Vine Kristopher 592 University College Dublin Spangenberg Caroline 537 University of California - Davis SVM Abraham Taylor 1 University of California - Davis SVM Adkisson Cynthia 8 University of California - Davis SVM Ashley Elizabeth 18 University of California - Davis SVM Asrat Makda 19 University of California - Davis SVM Au Yeung Sarah 20 University of California - Davis SVM Bianchi Catarina 37 University of California - Davis SVM Bradford Madeline 47 University of California - Davis SVM Christie Anneka 89 University of California - Davis SVM Colangelo Jennifer 94 University of California - Davis SVM Consales Blaire 100 University of California - Davis SVM Cords Olivia 103 University of California - Davis SVM Echizenya Shiori 130 University of California - Davis SVM Elcombe Megan 133 University of California - Davis SVM Elliott Stephanie 134 University of California - Davis SVM Gluck Ryan 181 University of California - Davis SVM Gretler Sophie 200 University of California - Davis SVM Hardgrove Caitlin 215 University of California - Davis SVM Hisey Erin 226 University of California - Davis SVM Hoffmann Silke 230 University of California - Davis SVM Jimenez Daniela 254 University of California - Davis SVM Johnson Davida 257 University of California - Davis SVM Liu Joshua 327 University of California - Davis SVM Luker Madison 335 University of California - Davis SVM Ngo Lena 411 University of California - Davis SVM Petersen Lynsey 443 University of California - Davis SVM Razmara Aryana 464 University of California - Davis SVM Ringhofer Justin 473 University of California - Davis SVM Ryan Declan 491 University of California - Davis SVM Shelton Emily 510 2021 National Veterinary Scholars Symposium • 45 Vet School Last Name First Name Poster # University of California - Davis SVM Stoyle Brianna 548 University of California - Davis SVM Stubbs Eric 551 University of California - Davis SVM Taylor Anna 562 University of California - Davis SVM Tobin Victoria 567 University of California - Davis SVM Wegrzyniak Nicole 601 University of California - Davis SVM Weiss Brandon 604 University of California - Davis SVM Yungmeyer Austin 630 University of Florida CVM Alvarez Alyssa 13 University of Florida CVM Bergen Alexandra 35 University of Florida CVM Billings Phillip 39 University of Florida CVM Bonce Daniel 44 University of Florida CVM Chase Ellis 80 University of Florida CVM Chinchayan Cristina 86 University of Florida CVM Dalton Cambrie 110 University of Florida CVM Dorr Mackenzie 126 University of Florida CVM Gabaldon Cristina 163 University of Florida CVM Mamane Seryna 350 University of Florida CVM Porcher Tenley 453 University of Florida CVM Rodriguez Mateo 480 University of Florida CVM Smith Olivia 531 University of Florida CVM Suero Monica 553 University of Florida CVM Tartaglia Dillon 561 University of Georgia CVM Allen Tiffani 12 University of Georgia CVM Arne Allison 17 University of Georgia CVM Barrow Ruth 31 University of Georgia CVM Boothby Grace 45 University of Georgia CVM Brathwaite Morgan 49 University of Georgia CVM Cody Gena 93 University of Georgia CVM Devorak Anne 118 University of Georgia CVM English Abigail 138 University of Georgia CVM Fenster Lindsey 144 University of Georgia CVM Forker Grace 154 University of Georgia CVM Gollnick Hailey 186 University of Georgia CVM Jackson Jared 247 University of Georgia CVM Kim Kyoo Young 286 University of Georgia CVM Lemons Margaret 315 University of Georgia CVM McNabney Danielle 375 University of Georgia CVM Mohiuddin Ava 389 University of Georgia CVM Mozeika Christina 397 University of Georgia CVM Nakatsu Charlize 403 University of Georgia CVM Nguyen Sarah 414 University of Georgia CVM Peterson Breck 444 University of Georgia CVM Pope Catherine 452 University of Georgia CVM Rasys Ashley 462 46 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # University of Georgia CVM Sang Xiaoyang 496 University of Georgia CVM Sarkan Kate 497 University of Georgia CVM Szostek Adrianna 557 University of Georgia CVM Trogdon Mary-Grace 576 University of Georgia CVM Turn Jeffrey 580 University of Ibadan Adesola Ridwan 7 University of Ibadan Gbore Damilola 173 University of Illinois CVM Applebaum Jennifer 15 University of Illinois CVM Botkin Sarah 46 University of Illinois CVM Cacciatore Kyle 66 University of Illinois CVM Doran Alexandra 125 University of Illinois CVM Greene Erica 198 University of Illinois CVM Kries Kelly 301 University of Illinois CVM Lahar Rachael 309 University of Illinois CVM Marquez Ixzacil 354 University of Illinois CVM Nordenstahl Josefina 416 University of Illinois CVM Thomas Corrine 563 University of Illinois CVM Wangler Courtney 597 University of Minnesota CVM Adam Em 4 University of Minnesota CVM Baker Julia 26 University of Minnesota CVM Blanco Cristina 41 University of Minnesota CVM Bradley Rachel 48 University of Minnesota CVM Burke Erin 63 University of Minnesota CVM Chase Kayla 81 University of Minnesota CVM Coats Cassidy 92 University of Minnesota CVM Kenney Sarah 280 University of Minnesota CVM Kiesel Gabriela 283 University of Minnesota CVM Kobluk Landon 297 University of Minnesota CVM Krichbaum Hannah 300 University of Minnesota CVM Leverance Kayla 316 University of Minnesota CVM Menn Tanzania 378 University of Minnesota CVM Miller Grace 386 University of Minnesota CVM Rodts-Palenik Olivia 481 University of Minnesota CVM Springer Kelsey 542 University of Minnesota CVM Struck Madeline 550 University of Minnesota CVM Velazquez Jossette 588 University of Minnesota CVM Weiss Paige 605 University of Minnesota CVM Yamamoto Kirby 627 University of Minnesota CVM Young Jared 628 University of Minnesota CVM Zhu Cong 636 University of Missouri CVM Allen Carley 10 University of Missouri CVM Cook Taylor 101 University of Missouri CVM Donovan Erin 124 University of Missouri CVM Edwards Kerri 132 2021 National Veterinary Scholars Symposium • 47 Vet School Last Name First Name Poster # University of Missouri CVM Ford Tamara 153 University of Missouri CVM Franzak Jacob 156 University of Missouri CVM Herron Victoria 223 University of Missouri CVM Isensee Paige 244 University of Missouri CVM Johnson Remie 259 University of Missouri CVM Keilholz Amy 277 University of Missouri CVM Keller Stefan 278 University of Missouri CVM Killian Breanna 284 University of Missouri CVM Kimes Austin 288 University of Missouri CVM Kinkade Mackenzie 290 University of Missouri CVM Klasing Jessica 291 University of Missouri CVM Le Annie 314 University of Missouri CVM Meena Beverly 376 University of Missouri CVM Morgan Michael 392 University of Missouri CVM Nelson Caitlyn 407 University of Missouri CVM Nguy Symphony 412 University of Missouri CVM Noall Lucie 415 University of Missouri CVM Osterland Alexandra 430 University of Missouri CVM Piva Milan 450 University of Missouri CVM Rogers Bridgette 483 University of Missouri CVM Scarberry Nicole 499 University of Missouri CVM Seilhamer Nikki 504 University of Missouri CVM Smith Katelyn 528 University of Missouri CVM Topka Jessica 570 University of Missouri CVM Torres Saez Ashley 571 University of Missouri CVM Treadwell Caroline 573 University of Missouri CVM Winiarski Caroline 618 University of Pennsylvania SVM Adams Josetta 6 University of Pennsylvania SVM Bankler-Jukes Devon 27 University of Pennsylvania SVM Benavides Estefania 33 University of Pennsylvania SVM Blowers Zelda 42 University of Pennsylvania SVM Cappo Kyle 71 University of Pennsylvania SVM Carlson Jaclyn 72 University of Pennsylvania SVM Chalmin Alyssa 79 University of Pennsylvania SVM Cranfill Suna 106 University of Pennsylvania SVM de Ruyter Tryssa 115 University of Pennsylvania SVM DiGate Cameron 122 University of Pennsylvania SVM Hagmaier Carlin 206 University of Pennsylvania SVM Hoffman Justin 229 University of Pennsylvania SVM Kawabata Jo 274 University of Pennsylvania SVM Kazmierski Luke 275 University of Pennsylvania SVM Maggio Maria 345 University of Pennsylvania SVM Peauroi Elise 437 University of Pennsylvania SVM Prown Rowan 457 48 • 2021 National Veterinary Scholars Symposium Vet School Last Name First Name Poster # University of Pennsylvania SVM Roach Daana 476 University of Pennsylvania SVM Sastry Lakshmi 498 University of Pennsylvania SVM Shepley-Mctaggart Ariel 511 University of Pennsylvania SVM Silverstein Nicole 516 University of Pennsylvania SVM Smith Anna 526 University of Pennsylvania SVM Soulas Jaclyn 536 University of Pennsylvania SVM Starr Miranda 544 University of Pennsylvania SVM Thomas Haley 564 University of Pennsylvania SVM Ya Deau Amelie 625 University of Pennsylvania SVM Zayas Hanna 632 University of Tennessee CVM Faddoul Emma 142 University of Tennessee CVM Gorman Myranda 191 University of Tennessee CVM Mykins Julia 401 University of Wisconsin SVM Arendt Tessa 16 University of Wisconsin SVM Banooni Dustin 28 University of Wisconsin SVM Brauer Kiley 50 University of Wisconsin SVM Burk Ariela 62 University of Wisconsin SVM Chi Jacqueline 85 University of Wisconsin SVM George Serena 176 University of Wisconsin SVM Hamilton Emily 210 University of Wisconsin SVM Hoel Meghan 227 University of Wisconsin SVM Hoerth Alexandria 228 University of Wisconsin SVM Jantzen Derek 251 University of Wisconsin SVM Jonas Maya 263 University of Wisconsin SVM Kobitter Emma 296 University of Wisconsin SVM Kuehnl Ashley 303 University of Wisconsin SVM Kuhn Molly 304 University of Wisconsin SVM Marino Nicholas 353 University of Wisconsin SVM Martin Hannah 359 University of Wisconsin SVM Massaro Nickolas 362 University of Wisconsin SVM Pees Savannah 438 University of Wisconsin SVM Peterson Macy 446 University of Wisconsin SVM Schermacher Maxwell 501 University of Wisconsin SVM Walter Benjamin 594 University of Wisconsin SVM Wildermuth Hannah 611 University of Wisconsin SVM Zoul Jordyn 637 Virginia-Maryland Regional CVM Bukovec Katherine 60 Virginia-Maryland Regional CVM Kaloss Alexandra 270 Virginia-Maryland Regional CVM Lin Amy 325 Virginia-Maryland Regional CVM Loessberg Emma 329 Virginia-Maryland Regional CVM Newberne Holly 409 Virginia-Maryland Regional CVM Reddy Alexandra 466 Virginia-Maryland Regional CVM Sexton Brigid 507 Virginia-Maryland Regional CVM Tupik Juselyn 579 2021 National Veterinary Scholars Symposium • 49 Vet School Last Name First Name Poster # Western College of Veterinary Medicine Goldstein Savannah 185 Western College of Veterinary Medicine Kliewer Maya 293 Western College of Veterinary Medicine Macquisten Ella 341 Western College of Veterinary Medicine Reitsma Lara 469 Western College of Veterinary Medicine Shaw Laura 509 Western University of Health Sciences Chen Katherine 83 Western University of Health Sciences Conner Jessica 98 Western University of Health Sciences Ettenger Simon 140 Western University of Health Sciences Fukushima Courtney 160 Western University of Health Sciences Gomez Zaira 188 Western University of Health Sciences Goodwein Abrielle 190 Western University of Health Sciences Guan Jimmy 202 Western University of Health Sciences Johnston Dylan 261 Western University of Health Sciences Kao Leeza 272 Western University of Health Sciences Maestas Jesse 343 Western University of Health Sciences Maestas Jesse 344 Western University of Health Sciences Marter Nicole 357 Western University of Health Sciences Mehta Tarun 377 Western University of Health Sciences Merichko Danielle 380 Western University of Health Sciences Nguyen Jeffrey 413 Western University of Health Sciences Piorkowski Jill 449 Western University of Health Sciences Salvosa-Chua HermanMax 493 Western University of Health Sciences Samra Gunimat 494 Western University of Health Sciences Sedighim Sharona 503 Western University of Health Sciences Shinn Grace 514 Western University of Health Sciences Smith Nicole 530 Western University of Health Sciences Yamada Sean 626

50 • 2021 National Veterinary Scholars Symposium Complete Abstracts Immune response of thiamine deficient chinook salmon (Oncorhynchus tshawytscha) to columnaris disease

Taylor Abraham, Diem Thu Nguyen, Zeinab Yazdi, Heather Bell, Anne Todgham, Mark Adkinson, Rachel C. Johnson, Carson Jeffers, Scott Foott, Nann A. Fangue, Esteban Soto

University of California, Davis, CA (Abraham, Nguyen, Yazdi, Bell, Todgham, Johnson, Jeffers, Fangue, Soto), California Department of Fish and Wildlife, Rancho Cordova, CA (Adkinson), NOAA (Johnson), US Fish and Wildlife Service (Foott)

High morbidity and mortality of Chinook salmon (Oncorhynchus tshawytscha) fry has been reported in California’s Central Valley associated with thiamine deficiency (TD). Thiamine is an essential B vitamin that is required by all living cells for energy metabolism and neurological function. Flavobacterium columnare is a Gram-negative bacteria that causes columnaris disease in fish resulting in substantial losses in global aquaculture production. Little is known on the effect of TD on the immune system of salmon and the potential it may have on disease susceptibility, as well as the effects on the immune response. Although there is growing awareness that both TD and F. columnare outbreaks are emerging diseases to salmon on the U.S. West Coast, it is unknown if fish that survive from low/intermediate thiamine eggs will experience latent mortality due to susceptibility to endemic diseases such as columnaris. The main objective is to investigate the pathogenesis of TD, particularly focusing our attention on the immune response and susceptibility of thiamine-deplete and -replete fry to columnaris diseases. To investigate this, Winter run Chinook salmon fry hatched from thiamine-deplete (n = 120) and -replete (n = 120) salmon with known thiamine concentrations were challenged with F. columnare, using an immersion challenge model of infection. The immune responses mounted in the gills and spleen were investigated through quantitative reverse-transcription PCR, and bacterial loads in the tissues were estimated by qPCR. This study can help provide insights into the pathogenesis of these diseases and provide information to regulatory bodies to guide management in ways to mitigate the effects these diseases have in salmonid populations.

Research Grant: This work was funded by California Department of Fish and Wildlife Proposition 1 Research Program. Student Support: California Department of Fish and Wildlife Statewide Fish Disease Research Program

Effect of a Warmed Environment and IVC Supplemental Heat on Mouse Behavior, Breeding and Body Temperature

Stephanie Acevedo, Jacquelyn M Del Valle, F. Claire Hankenson

College of Veterinary Medicine and Campus Animal Resources, Michigan State University, East Lansing, MI

It is essential to minimize variables related to laboratory mice for study success and experimental reproducibility. One extrinsic variable is the “cold-stress” endured by mice in standard housing conditions with room temperatures of 20-268C, which are much cooler than the mouse thermoneutral zone (TNZ), the preferred temperature range (29-348C) in which mice can maintain normal body temperatures with little energy expenditure. Studies have noted impacts of chronic “cold- stress”, the resulting physiological variance in mice, and its potential to affect this animal model in emulating human physiology in disease states. In this pilot study, we evaluated mouse behavior, breeding success, and body temperature when ambient temperature (Ta) was increased (29.48C) and supplemental heat (378C) was provided to a portion of the cage floor via a digitally controlled heat source. Mice were divided by sex (n = 4 per group) with continuous access to two interconnected IVC cages; one of these cages received supplemental heat. Mice were then placed in breeding pairs (n = 4 pairs) in single IVC cages, two with heat plate access. Cage-side videography was utilized for data collection. With increased Ta, we expected that animals would engage in more sedentary behaviors during the light cycle, as “cold-stress” encourages hyperactivity, and decline in nest quality and pup size/weight associated with decreased demand for heat conservation and fat stores. Preliminary results indicate that mice with supplemental heat have decreased nest scores and occupy non-heated cage quadrants. Providing a range of temperatures close to the TNZ within the mouse microenviron- ment allows rodents to self-select preferred temperatures throughout the circadian cycle.

Research Grant: NIH Grant 5T35OD016477-19, the Nathan Brewer Endowment, and Campus Animal Resources within the Office of Regulatory Affairs Student Support: NIH Grant 5T35OD016477-19

2021 National Veterinary Scholars Symposium • 51 Anticoagulant rodenticide residue levels in liver samples from a Maine Canada Lynx population

Mariana Acquaviva, Felicia Nutter, and David Needle

Tufts Cummings School of Veterinary Medicine (Acquaviva), Department of Infectious Disease and Global Health (Nutter); University of New Hampshire - New Hampshire Veterinary Diagnostic Laboratory (Needle)

Anticoagulant rodenticides (ARs) are the most commonly used chemical worldwide for lethal rodent control. For predato- ry species that consume prey targeted with ARs, both acute and chronic secondary exposures may occur. Sublethal effects have been documented in wildlife with AR bioaccumulation, which can lead to other causes of death. Canada lynx (Lynx canadensis) occupy areas of Maine where they could have access to exposed prey, however, lynx populations typical- ly prefer insular forests, away from humans, which is assumed to reduce their exposure risk. In fishers,a carnivore with similar habitat preferences, significant AR exposure has been documented. We will evaluate the presence and quantify levels of exposure to first and second generation ARs in Canada lynx from Maine to better understand human-induced threats to species conservation. We hypothesize that 1) the majority of Canada lynx samples tested will contain residues of at least one anticoagulant rodenticide, and 2) there will be more exposure to second generation than to first generation anticoagulant rodenticides. 46 Canada lynx liver samples have been sent to Pennsylvania Animal Diagnostic Laboratory System Toxicology Laboratory to be analyzed for first and second generation ARs. The prevalence by specific compound and by AR generation group will be reported. Potential associations between lynx life history data (sex, age, class) and AR prevalence (by compound and generation group) will be evaluated by using chi-squared tests. We expect that at least 50% of Canada lynx samples will be positive for at least one AR subtype.

Research Grant: from the Cummings School Associate Dean for Research and Graduate Education Student Support: Tufts Cummings School of Veterinary Medicine

Identification and validation of alleles for cardiac arrhythmias and atrial fibrillation with ECG analysis

Em C. Adam, Sian A. Durward-Akhurst, Lauren Hughes, Kendall Blanchard, Jenifer Gold, James R. Mickelson, and Mol- ly E. McCue

College of Veterinary Medicine (Adam), Veterinary Clinical Sciences Department (Durward-Akhurst), Veterinary Popu- lation Medicine Department (Hughes, Blanchard, McCue), Veterinary and Biomedical Sciences Department (Mickelson), University of Minnesota; Veterinary Clinical Sciences Department (Gold), Washington State University.

Cardiac arrhythmias are an important cause of sudden death (SCD). Most horses with arrhythmias have no underlying structural heart disease, making it virtually impossible to detect those that will develop arrhythmias. Similar arrhythmias in human athletes are almost exclusively caused by mutations in ion channel genes. We hypothesized that mutations in ion channel genes are a cause of arrhythmias in racehorses without structural heart disease. We used whole genome sequence of 6 Standardbred racehorses with atrial fibrillation (AFIB) to identify 506 putative arrhythmia-causing mutations based on being: 1) within 5 kb of biologic candidate genes; and 2) rare (< 5% allele frequency) in the equine population. 78 mu- tations (15%) were false positives due to poor reference genome annotation or genotyping errors. Additional evidence of pathogenicity was obtained using PROVEAN, SNPS&GO, and SIFT. In total, 132 mutations (26%) in 77 candidate genes had sufficient evidence to be considered putative arrhythmia-causing mutations.To determine which of these mutations are associated with arrhythmias, we will genotype them in 1,200 racehorses. So far, we have performed ECGs (before, during, and after exercise) on 143 Standardbred and 171 Thoroughbred racehorses. The average heart rates (beats per min- ute) at rest, exercise, and post exercise were 35, 191, and 62 respectively. 6 horses had arrhythmias at rest (3) or a history of AFIB (3). Mutations associated with the presence of arrhythmias will have strong evidence to support them having a functional effect on cardiac polarization. Our long-term goal is to reduce the rate of SCD in racehorses by establishing risk factors that contribute to the development of SCD.

Research Grant: USDA NIFA-AFRI 2017-67015-26296; AQHA; UMN Multistate; CVM Resident and Graduate Student Research Award; AAEP Young Investigator Award; MAF (D20EQ-013); Grayson Jockey Club Foundation Student Support: MAF (D20EQ-606); Boehringer Ingelheim VSS Program; UMN College of Veterinary Medicine

52 • 2021 National Veterinary Scholars Symposium Effect of maternal obesity on placental, fetal, and maternal outcomes in a mouse model of preeclampsia, BPH/5

Daniella M. Adams, Kalie F. Beckers, Viviane L.C. Gomes, Jenny L. Sones

Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA.

Obesity impacts 1/3 of adults. Maternal obesity significantly increases risk of adverse pregnancy outcomes, includ- ing preeclampsia (PE). Late-gestational hypertension, aberrant fetoplacental development, and fetal growth restriction (FGR), hallmarks of PE, are observed spontaneously in BPH/5 mice. Similar to obese preeclamptic women, BPH/5 have increased reproductive white adipose tissue (rWAT). We hypothesized that attenuation of maternal obesity in pregnant BPH/5 mice will improve fetoplacental development by decreasing inflammation and hypoxia at the maternal-fetal in- terface. To test this hypothesis, BPH/5 mice were fed ad libitum (lib) and pair-fed (PF) to C57 ad lib controls beginning at embryonic day (e) 0.5. Implantation sites were collected at e7.5 for real-time PCR analyses (n = 5/group). BPH/5 PF females had reduced rWAT by e7.5 without change to mRNA of IL-15, a pro-inflammatory cytokine elevated in BPH/5 decidua, nor NKp46, a marker of pro-angiogenic decidual natural killer (dNK) cells which are lower in BPH/5 (P > 0.05). Hypoxia-related genes, vascular endothelial growth factor (Vegf) and hypoxia inducible factor (Hif) 1a, were assessed in e7.5 implantation sites. We found a 2-fold increase in Hif1a and Vegf mRNA (P < 0.05) in ad lib BPH/5 versus C57 that was reduced in PF BPH/5 (P < 0.05). Although maternal weight loss in BPH/5 reduced excess hypoxia at the maternal-fe- tal interface, it did not alter dNK cells which are crucial for placentation. Despite this, BPH/5 PF mice have attenuation of FGR. In conclusion, maternal weight loss in BPH/5 beginning at conception may improve placental development in this model. Future investigations are needed to determine the effects on pregnancy outcomes in obese women.

Research Grant: National Institutes of Health Student Support: National Institutes of Health

Longitudinal Assessment of SARs-CoV-2-Specific T Cell Response in Convalescent Orthodox Jewish Adolescents

Leticia Kuri-Cervantes, Betina Pampena, Josetta Adams

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PASchool of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

In previous studies, it has been suggested that SARS-CoV-2-specific memory T cells provide long-term immunity against COVID-19. In this study, we use cytometric analysis to longitudinally investigate the SARS-CoV-2-specific T cell re- sponses in seropositive adolescent males between the ages of 15 and 18 belonging to Orthodox Jewish secondary schools in Baltimore and Chicago. Most of the individuals in this study experienced mild or convalescent COVID-19 during outbreaks in the summer and fall of 2020. Our goal is to determine if mild/asymptomatic infection in adolescents results in detectable SARs CoV-2-specific T cell responses, and determine if there is elevated generalized immune activation of lymphocyte subsets in mild and asymptomatic disease compared to severe disease in adults.

Research Grant: None Student Support: Boehringer-Ingelheim

2021 National Veterinary Scholars Symposium • 53 Antimicrobial resistance in Africa: Pets, wildlife, and humans in danger

Ridwan O. Adesola, Jennifer M. Rudd

Faculty of Veterinary Medicine, University of Ibadan, Nigeria (Ridwan); Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University (Rudd)

The emergence of antimicrobial resistance (AMR) is arguably the most vital threat to pets, wildlife, and human health in Africa and cannot be overlooked. Despite increased reports of antimicrobial resistance in common zoonotic and interspe- cies pathogens, there is a lack of information providing a broad overview of this danger and summarizing recent trends in research related to AMR specifically on the African continent. The main aim of this study is to summarize the emerging threat of AMR in companion animals, livestock, wildlife, and people in African countries. A literature search was carried out through several databases (PubMed, Web of Science, Scopus, Google Scholar) to identify research articles published between 2011 and 2021. Criteria for inclusion focused on AMR in companion animal or livestock from within countries and regions in Africa as well as trends in judicious antimicrobial usage and decision-making among prescribers. A total of 80 articles were identified fitting the criteria for this review. This review extends several bacterial isolates, various anti- microbials, possible mechanisms for AMR spread among animals and humans, interrelationship among the organisms, the public health importance, and future directions of AMR in African countries. A more complete description of cur- rent trends in AMR is paramount to designing new strategic One Health programs in AMR research and judicious usage among domestic animals, wildlife, and people in Africa.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

Relationship between ownership and enrichment in dogs with and without separation anxiety or noise phobia

Cynthia A. Adkisson, Melissa J. Bain

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis, Davis, California

Separation anxiety (SA) and noise phobia (NP) are serious problems in dogs. Veterinarians have limited time to gather full behavior histories. Goals were to determine questions that can most quickly establish a working diagnosis of SA and/or NP in order to treat earlier, as well as to help prevent problems from developing. Hypotheses are: dogs with SA or NP will exercise less, have attended a training class, have been trained with aversive methods, and be more likely to have come from a shelter. Data were collected from patients, 1-10 years old, diagnosed with either SA or NP at UC Davis Behav- ior Service between 1/1/17-5/31/21. Data included: signalment; diagnoses; and other demographic and environmental data. Owners of 1065 healthy patients, 1-10 years of age, of UC Davis Community Practice seen between 1/1/17-5/31/21 were emailed a survey. In addition to patient data already in the EMR, questions were asked about: exercise and training; behavioral questions, partially using a validated behavior survey; and other demographic and environmental data. Prelimi- nary results for dogs from Behavior Service (82 diagnosed with SA, 65 diagnosed with NP, 10 diagnosed with both) were: The average age was 4 years for dogs with SA and 5 years for dogs with NP. Mean weight was 21 kg and 26 kg for dogs with SA and NP, respectively. Future analyses will look at the rest of the data being collected from patients, and statistical analyses will be performed to evaluate differences.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholar funds

54 • 2021 National Veterinary Scholars Symposium Developing and optimizing a flow cytometry-based assay for feline infectious peritonitis virus

Brittany A Allen, Samantha J.M. Evans

Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio

Feline Infectious Peritonitis (FIP) is a deadly viral disease affecting cats that are carriers of the almost ubiquitous feline enteric coronavirus (FECV). Infection with FECV is mostly asymptomatic and transient, but it can mutate into FIP virus (FIPV) causing non-specific, and ultimately fatal clinical signs. Diagnostic testing for FIP is often ambiguous and usual- ly presumptive, with most available tests unable to distinguish between the two different pathotypes (FECV vs. FIPV). A definitive diagnosis is usually made only by biopsy and examination of tissue post-mortem. The aim of this study is to develop and optimize a flow cytometric assay for the detection of FIPV within fluid macrophages. Felis catus whole fetus (fcwf) cells are grown in standard media. Cells are infected with FIPV, along with sham-infected controls. Then, cells are harvested, fixed, permeabilized, and stained with anti-vimentin and anti-feline coronavirus antibodies, and analyzed by flow cytometry. Preliminary results show that flow cytometry detects stained FIPV and vimentin within fcwf cells, and eventually, we expect this within fluid macrophages from feline patients suspected of having FIP. We anticipate that our flow cytometry-based assay for FIP will be more diagnostically reliable (higher specificity) than currently available assays. A definitive antemortem diagnosis is crucial to veterinarians and clients when it comes to making treatment or eu- thanasia decisions surrounding FIP. The next steps for this study include testing fluid samples from suspected FIP patients in a clinical diagnostic trial.

Research Grant: OSU Feline Funds (PI = Evans) Student Support: American Veterinary Medical Foundation Second Opportunity Research Scholarship

SHIP inhibition in canine osteosarcoma: Evaluation of a novel target

Carley B Allen, Anita M Rogic, Brian K Flesner, Angela L McCleary-Wheeler

Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO

Despite advances in the understanding of cancers across species, the treatment paradigm for both human and canine os- teosarcoma (OS) has not changed in decades. SHIP proteins, important phosphatases in the PI3K cell signaling tree, have been shown to promote cell survival in various neoplasms, but their role in canine OS has not been evaluated. Therefore, we sought to determine the in vitro biologic effects of several novel SHIP inhibitors. MTS experiments assessed cell via- bility in three established canine OS lines (Abrams, D-17, and HMPOS). Cells were treated with 3-AC, K118, K149, and K161 at concentrations ranging from 1.25-10.0 mM. To assess cell migration, confluent canine OS cells were wounded using BioTek’s Autoscratch Tool followed by K118 treatment. Wells were imaged for 72 hours post-treatment using the Lionheart-FX. MTS data demonstrated that SHIP inhibitors exhibited a time and concentration dependent decrease in cell viability. The greatest effect for all inhibitors occurred 72 hours post-treatment. K118 was chosen for further evaluation as it demonstrated the highest potency of inhibitors tested. K118 inhibited wound healing and cell migration; its greatest effects were noted at 1.25m M and 2.5 mM concentrations. Preliminary colony formation assays demonstrated significant inhibition of cell growth and colony formation when cells were treated with K118 concentrations of 1.25 mM or greater. Further studies of SHIP inhibitors should investigate the mechanism of cell signaling pathway interference, specifically in the PI3K/mTOR pathway. Clarifying the role of SHIP proteins in OS pathogenesis could promote further in vivo studies, assessing biologic efficacy.

Research Grant: University of Missouri College of Veterinary Medicine Phi Zeta Research Grant. Student Support: MU College of Veterinary Medicine, Department of Veterinary Medicine & Surgery.

2021 National Veterinary Scholars Symposium • 55 Differences in physiologic parameters in sled dogs and shelter dogs undergoing anesthesia

Megan M. Allen, Jane M. Manfredi, Kirk A. Muñoz, Sarah K. Rich, Sarah A. Shull

Small Animal Clinical Services (Allen, Muñoz, Shull), Pathobiology and Diagnostic Investigation (Manfredi), College of Veterinary Medicine, Michigan State University, East Lansing, Michigan; Banfield Pet Hospital (Rich), Loveland, Colorado

Anesthetic monitoring parameters consider differences such as species, size, age, and breed, but little is known about the influences of physical fitness on the established normal ranges.The hypotheses of this study are that conditioned sled dogs will have a 5% lower heart rate (HR), 10% less HR variation, and a 10% lower mean arterial pressure (MAP), during general anesthesia when compared to shelter dogs. Anesthetic reports from sterilization procedures completed by third year veterinary students at one institution during 2018-2019 (n = 166) were evaluated. Sled dogs (n = 62) from one Michigan kennel were compared with shelter dogs (n = 104). Pre-operative parameters studied included body weight, body condition score (BCS), temperature (TEMP), HR and respiratory rate (RR), with TEMP, HR, RR, MAP, hemoglobin oxygen saturation, and ventilation recorded from induction to end of anesthesia. Periods of manual ventilation, apnea, and any complications were noted. Data will be analyzed using descriptive statistics and independent t-tests with significance at P < 0.05. Mean age and BCS of sled and shelter dogs were 3.2 and 2.1 years, and 4.2/9 and 4.4/9, respectively. Sled dogs were Alaskan huskies and shelter dogs were mixed breeds and American Pit Bull Terriers. It is predicted that the cardiovascular parameters of sled dogs will be significantly lower than shelter dogs during general anesthesia. Information gained from this study may be used to determine more appropriate anesthetic guidelines, including a reference range for conditioned dogs, to increase anesthetic safety for canine athletes.

Research Grant: None. Student Support: Boehringer Ingelheim and the Graduate School at Michigan State University.

Sequencing of Infectious Bronchitis Virus

Tiffani B. Allen, Brian J. Jordan

Poultry Diagnostics and Research Center (Jordan), College of Veterinary Medicine, University of Georgia, Athens, GA

Infectious bronchitis virus (IBV) is a gamma coronavirus that infects chickens and causes an economically significant upper respiratory tract disease. When a new variant emerges, it often takes 6-12 months for the virus to be isolated and fully characterized. One major obstacle in identifying new variants is the lack of a diagnostic tests that can detect them, especially when sequence information is unavailable. In Dr. Jordan’s laboratory, a novel IBV variant was detected, and a partial sequence was obtained, but there are many samples in his inventory with a similar variant real-time PCR detection profile. The objective of this project was to re-test a bank of samples to determine how widespread this variant may be, or if other variants were present. 120 cecal tonsil and trachea samples were gathered from 10 farms across the Southeastern US to determine the prevalence of this variant. RNA was purified from each sample, and an RT-PCR was run for each sample using three different primer sets. Any positive RT-PCR reactions were then sequenced to determine the IBV virus type detected. Based on the overall results, it was found that the predominance of the previously detected variant was unable to be determined. Only one sample was positive for IBV via RT-PCR and sequencing and was an Arkansas variant strain of IBV. This discrepancy in detecting IBV by real-time PCR but not being able to obtain sequencing via traditional RT-PCR is common and highlights the difficulty in characterizing potential IBV variants. Additional modifications to this procedure, such as random priming and next generation sequencing, should be made to determine the most efficient meth- od to gather sequence information for future analyses.

Research Grant: None. Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

56 • 2021 National Veterinary Scholars Symposium The effects of liquid storage and cryopreservation on the morphometry of the West Indian manatee spermatozoa

Alyssa Alvarez, Jonathan R. Cowart, Danielle M. Collins, Iskande Larkin

Aquatic Animal Health Program (Alvarez, Cowart, Larkin) Department of Animal Sciences (Collins), University of Florida College of Veterinary Medicine, Gainesville, Florida

Successful reproduction is an essential component in the management and conservation of protected marine mammal spe- cies. Unlike domestic animals, factors related to male fertility are highly under-researched in marine mammals, especially the West Indian manatee. No research has previously been conducted on any sirenian species. Research of this nature allows us to be “proactive” in creating solutions that address key management/conservation issues rather than “reactive” when it is already too late for intervention. This study is significant for the development of semen collection and long-term storage protocols in the West Indian manatee. In doing so, we will examine the effects of (1) different semen extenders, (2) different storage variables (i.e., storage temperature, presence or absence of seminal plasma, and length of storage), and (3) cryopreservation on the morphometry of West Indian manatee sperm. By investigating these specific aims, we can identify the specific conditions that have the least negative impact on the sperm and will result in increased viability of gametes for this species. Semen samples were collected, aliquoted, and subjected to treatments consisting of different semen diluents and the storage parameters listed above. The extenders used were Ham’s F10, Modena, Inra 96, and a homemade Tris egg yolk extender. Each sample was stained with SpermBlue and morphometric data was collected using computer-aided sperm morphology analysis (CASMA) using a Microptic Sperm Class Analyser (SCA) computer-aided sperm analysis (CASA) system, version 6., with the SCA morphology module. Morphometric data is currently being collected.

Research Grant: UF CVM Consolidated Faculty Research Award, Distance Education Program and Florida Fish and Wildlife Conservation Commission Student Support: Boehringer Ingelheim Veterinary Scholar Program, UF CVM Aquatic Animal Health Program

Pathology associated with triploid oyster gametogenesis

Sarah Amblard, Tal Ben-Horin

Department of Clinical Sciences (Amblard, Ben-Horin), College of Veterinary Medicine, North Carolina State University, Raleigh, NC.

Triploids are widely used in commercial fruit, fish, and mollusk production around the world. Having three rather than the normal two sets of haploid chromosomes leads to many beneficial commercial traits, including improved growth and infertility. Triploid oyster production has fueled oyster aquaculture’s global expansion, as triploids provide a consistent, year-round product compared to diploids which are typically commercially unavailable during and after spawning. Nev- ertheless, triploid oysters still undergo gametogenesis, with gonad development being irregular and incomplete. The past decade has seen widespread triploid mortality events timed with peaks in gametogenesis but unclear pathology. Occa- sional erosion of gill epithelium is seen in affected oysters, which suggests an environmental cue. Here we conducted a study investigating the pathology associated with different stages of triploid gametogenesis, hypothesizing that there will be a shift in microbial communities affecting triploid oysters later in gonad development. Our work will inform a recently developed collaborative program developing oyster stocks for sustainable aquaculture production in North Carolina and across the Atlantic coast.

Research Grant: North Carolina Department of Environmental Quality (Contract # CW19141) Student Support: North Carolina State College of Veterinary Medicine Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 57 Cytochrome P450 reaction phenotyping of hydroxyitraconazole metabolism in canine liver

Jennifer Applebaum, Zhong Li, and Jennifer M. Reinhart

Department of Veterinary Clinical Medicine, College of Veterinary Medicine (Applebaum, Reinhart), and the Roy J. Carver Biotechnology Center (Li), University of Illinois at Urbana-Champaign, Urbana, IL

Itraconazole (ITZ) is a valuable systemic antifungal agent and, although it is a safer long-term treatment option, abundant evidence has established adverse effects, particularly hepatotoxicity, in canines. Alterations in ITZ metabolic pathways re- sult in the production of toxic metabolites, which are thought to be linked to hepatotoxicity. In humans and rodents, ITZ is metabolized by the hepatic cytochrome P450 (CYP) 3A4 enzyme to hydroxyitraconazole (OH-ITZ) and then to keto-ITZ. Our laboratory has recently established that CYP3A12, a canine ortholog to CYP3A4, is responsible for converting ITZ to OH-ITZ, but the CYP responsible for converting OH-ITZ to keto-ITZ in canines is unknown. This study aims to identify the CYP(s) responsible for metabolizing OH-ITZ in canines in vitro and we hypothesize that this enzyme is CYP3A12. Initial experiments in canine hepatic microsomes (MICs), which contain aggregated CYPs, established that oxidation of OH-ITZ to keto-ITZ optimally occurred under the following conditions: 1 mg/mL MIC, 1 mM NADPH, and a 30-minute incubation period, with keto-ITZ generation detected using HPLC-MS. Michaelis-Menten reaction kinetics were estab- lished in MICs using OH-ITZ (0-40 mM) and yielded the following parameter values: Vmax = 11.06 pmol keto-ITZ/min/ mg MIC, km = 148.6 mM. Final assays will be performed using Bactosomes, which contain recombinant isolated canine CYPs, to identify the specific CYP(s) responsible for OH-ITZ metabolism.This study will establish a better understanding of ITZ metabolic pathways, which will contribute to the knowledge of ITZ-associated hepatotoxicity. Such advances may permit development of new treatment and preventative strategies in canine patients.

Research Grant: Partially funded by the Companion Animal Memorial Fund, University of Illinois College of Veterinary Medicine Student Support: Office of the Director, NIH, T35 OD011145

Analysis of computed tomography derived laryngotracheal dimensions and correlation with body weight in rabbits

Tessa Arendt, Samantha Loeber, Carrie Schroeder, Tatiana H. Ferreira

School of Veterinary Medicine, University of Wisconsin (Arendt), Department of Surgical Sciences - Diagnostic Imaging (Loeber), Department of Surgical Sciences - Anesthesia (Schroeder, Ferreira), School of Veterinary Medicine, University of Wisconsin - Madison, Madison, WI

Rabbit orotracheal intubation is challenging and typically a blind procedure that is complicated by narrow oropharyngeal anatomy, leading to a high rate of failure. Anecdotally, clinicians have appreciated that rabbits with a larger body size are not necessarily easier to intubate. The aim of this retrospective study was to characterize the relationship between rabbit laryngotracheal dimensions and body weight. Computed tomographic (CT) laryngotracheal luminal diameter and cross-sectional area measurements were made in forty-five rabbits (range = 0.92-5.06 kg, mean6 SD = 2.17 6 0.82) at the rostral thyroid cartilage at the level of the arytenoids, caudal thyroid cartilage/rostral cricoid cartilage, caudal cricoid cartilage/cranial trachea, and trachea at the level of the 5th cervical vertebra. For statistical analysis, rabbits were distrib- uted in three body weight groups: Group 1 = 0.5 - 1.5 kg (n = 9), Group 2 = 1.6 - 2.9 kg (n = 29) and Group 3 = 3 kg and above (n = 7). The following was obtained at the level of the arytenoids: Group 1 mean sagittal diameter (H) 6 SD = 3.76 6 0.91 mm, mean transverse diameter (W) 6 SD = 3.01 6 0.25 mm, and mean cross-sectional area 6 SD = 8.56 6 1.2 mm2. Group 2 mean sagittal diameter (H) 6 SD = 4.48 6 0.96 mm, mean transverse diameter (W) 6 SD = 3.21 6 0.91 mm, and mean cross-sectional area 6 SD = 10.6 6 4.4 mm2. Group 3 mean sagittal diameter (H) 6 SD = 5.11 6 0.27 mm, mean transverse diameter (W) 6 SD = 3.79 6 1.1 mm, and mean cross-sectional area 6 SD = 14.7 6 4.2 mm2. The data for every measurement of airway diameter and body weight revealed a statistically significant, strong positive cor- relation (P < 0.008). This suggests that different methods of orotracheal intubation should be investigated.

Research Grant: None Student Support: Boehringer Ingelheim

58 • 2021 National Veterinary Scholars Symposium Effect of oral gabapentin on directly obtained ambulatory systemic arterial blood pressure in healthy cats

Allison M. Arne, Bianca N. LourenÇo, Melissa De Lombaert, Amanda E. Coleman, Chad W. Schmiedt

Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Situational hypertension, defined as an increase in systemic arterial blood pressure (BP) associated with stressful situa- tions in an otherwise normotensive individual, is common in cats and can make it difficult to diagnose true, pathological systemic hypertension. Gabapentin, an antiepileptic and analgesic, has anxiolytic effects in cats and may mitigate situa- tional hypertension. The objective of this study was to investigate the effect of a single oral dose of gabapentin on directly obtained BP in conscious, undisturbed cats in a home environment. We hypothesized that compared to placebo, gabapen- tin would have no effect on mean systolic blood pressure (SBP) up to four hours post-dose. Six purpose-bred research cats were outfitted with surgically implanted femoral arterial telemetric BP monitors. SBP was measured continuously for 24 hours before and after an oral dose of gabapentin (100 mg/cat) or placebo in a crossover study. For each of 8 consecutive 30-minute intervals following administration of study drug (i.e., up to 4 hours post-dose), average SBP, and change in SBP from baseline was calculated. Mean SBP over the corresponding 4-hour period on the day prior to dosing was considered baseline SBP. Average and change in SBP for each time period were compared between treatment groups using repeated measures ANOVA. No significant difference in absolute or change in SBP from baseline was noted for any time period post-dose. These data indicate that treatment with gabapentin has no significant effect on BP when clear environmental stressors are absent. This information will be used to inform future studies in which the effects of gabapentin on SBP will be measured in cats subjected to simulated veterinary visits.

Research Grant: The Alison Bradbury Endowment for Feline Heath Student Support: Boehringer Ingelheim, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

Investigating relationships between prey preference and infectious disease in southern sea otters

Elizabeth Ashley, Megan Moriarty, Melissa Miller, Joe Tomoleoni, Julie Yee, Jessica Fujii, Richard Grewelle, and Christine Kreuder Johnson

UC Davis School of Veterinary Medicine, Davis, CA (Ashley, Moriarty, Kreuder Johnson); California Department of Fish and Wildlife, Santa Cruz, CA (Miller); US Geological Survey, Santa Cruz, CA (Tomoleoni, Yee); Monterey Bay Aquarium, Monterey, CA (Fujii); Hopkins Marine Station, Stanford University, Pacific Grove, CA (Grewelle)

Southern sea otters (Enhydra lutris nereis) are an endangered keystone and sentinel species found along the California coast. Infectious disease is a major hindrance to sea otter population recovery. Because sea otters exhibit individualized prey preferences, relationships between diet and prevalent causes of death (COD) can be investigated to characterize disease risks. We evaluated the role of diet in two important drivers of otter mortality: acanthocephalan peritonitis (AP) and bacterial infections. We conducted a case-control observational study using 1) gastrointestinal tract (GIT) content data collected at necropsy (CDFW) and 2) live foraging data characterizing diet composition (USGS/MBA) to examine whether the presence of sand/mole crabs or bivalves in the GIT are predictors of AP or bacterial COD, respectively. The multivariate logistic regression model evaluating factors associated with AP included sand/mole crab presence, nutrition- al condition, age class, and sex. Consistent with our hypothesis, sea otters that consumed sand or mole crabs were more likely to have died of AP (P = 0.04). Being a subadult (P = 0.04), being in poor or fair nutritional condition (P < 0.001), and being male (P = 0.04) were also significant predictors of AP. Poor or fair nutritional condition (P < 0.001) and ad- vanced age (P = 0.004) were positively associated with bacterial COD. However, we did not find a significant relationship between bivalve consumption and bacterial COD. Further investigation into specific bacterial pathogens and body systems within this broad COD category may help elucidate dietary and other risk factors of fatal infections.

Research Grant: None Student Support: STAR Fellowship

2021 National Veterinary Scholars Symposium • 59 Surveillance for novel coronaviruses in West African bats

Makda Asrat, Simon J Anthony, Isamara Navarrete Macias, Annie Rein-Weston, Cassie Bonavita

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, CA

The most significant pandemics of the last 50 years have been caused by viral pathogens that originated in wildlife, and several of these zoonotic viruses have been traced back to bat species. Previous work has found that bats are the major wildlife reservoirs of coronaviruses (CoVs) and that there is a significant association between particular lineages of CoVs and different bat species. SARS-like coronaviruses (sarbecoviruses) have been found to be significantly associated with rhinolophid bats (family Rhinolophidae), and recent data supports the hypothesis that sarbecovirus distribution is likely to mirror the distribution of rhinolophid bats. West Africa has high species richness of rhinolophid bats, and the aim of this study is to test the hypothesis that there are undiscovered sarbecoviruses circulating in this region. This study is the first time bats from this West African hotspot have been screened for CoVs. We used broadly reactive consensus polymerase chain reaction (cPCR) to identify known and novel CoVs in previously collected and extracted samples from West African bats. Positive cPCR products were cloned and sequenced using traditional sanger sequencing, and sequences were evalu- ated phylogenetically to identify their taxonomic placement. To date, at least one novel sarbecovirus has been identified in bats from West Africa. Viral surveillance and discovery studies play an important part in pandemic preparedness. Screen- ing for known CoVs allows us to better characterize their geographic range and host-specificity, and the discovery of new CoVs is a critical first step toward functional characterization and determination of their zoonotic potential.

Research Grant: NIH R01 AI149693; Genetic and ecological determinants of recombination in coronaviruses Student Support: Students Training in Advanced Research (STAR) Program

Flexibly induced backlit imaging as a novel diagnostic tool for the diagnosis of chronic enteropathy in cats

Sarah Au Yeung, Paula Giaretta, Richard Levenson, Maria Questa, Sina Marsilio

Departamento de Clinica e Cirurgia (Giaretta), Universidade Federal de Minas Gerias, São Luiz, Brasil; Department of Medicine and Epidemiology (Marsilio, Questa), Department of Pathology and Laboratory Medicine (Levenson); School of Veterinary Medicine and School of Medicine, University of California, Davis, CA

Chronic enteropathies (CE), including inflammatory bowel disease (IBD) and small cell lymphoma (SCL), are common gastrointestinal disorder in older cats. Diagnosis and differentiation require the collection, processing, and histopathologic examination of intestinal biopsy specimens, which is time and resource consuming. Flexibly Induced Backlit Imaging (FIBI) is a novel microscopy method that can be performed on fresh or fixed tissue. Imaging with FIBI can be performed in a non-destructive manner without the necessity of slide preparation, thus providing real time diagnostic information while the tissue remains intact and available for further downstream analysis. This retrospective, pilot study aims to deter- mine the diagnostic concordance between conventional H&E-stained slides and FIBI on formalin-fixed paraffin embedded small intestinal tissue samples from cats with clinical signs of chronic enteropathy. Biopsy specimens from 50 cases were evaluated using the guidelines published by the World Veterinary Small Animal Association (WSAVA) gastrointestinal standardization group. Blocks subsequently underwent superficial deparaffinization, FIBI imaging and histopathologic evaluation using the same guidelines. Histopathologic evaluation of conventional H&E images revealed, 24 cases with IBD and 15 with SCL; 6 cases were ambiguous for either diagnosis. Furthermore, 3 cats showed no active inflammation or neoplasia, 2 cases showed lamina proprial fibrosis, and 1 cat was diagnosed with small intestinal large cell lymphoma. The WSAVA score and diagnostic concordance will be compared between conventional H&E images FIBI.

Research Grant: Center of Companion Animal Health Student Support: Students Training in Advanced Research (STAR) Program

60 • 2021 National Veterinary Scholars Symposium Attitudes towards companion animals in Egypt

Alexandra Awad

Center for Animals and Public Policy, Cummings School of Veterinary Medicine, Tufts University, Grafton, Massachusetts

Internationally and throughout history, humans and animals are intertwined. The relationships between humans and animals are dynamic and can be significant to both species involved. Cultural perceptions often determine the role nonhu- man animals have in a specific society, which can influence the characteristics of the human-animal relationship. Within non-Western countries, there is a gap of knowledge on the relationships people have with pets in the Middle East. In Arab countries, there are many cultural aspects that can influence the human-animal relationship, such as religion andWestern influence. Egypt is one of the most populous Middle Eastern countries but has not been a target of human-animal bond research. Using the Pet Attitude Scale, the attitudes Egyptians have towards companion animals will be explored. The aim of this study is to investigate if these attitudes are influenced by demographics, culture, religion, and/orWestern influence. The hypothesis of this study is that Egyptian attitudes towards companion animals will be more positive than the Mid- dle Eastern countries surveyed to date - Kuwait, Palestine, and Oman. The more positive attitudes are expected based on prior knowledge and experiences in Egypt. In addition to investigating cultural influences, human-animal bond research has shown connections to veterinary medicine, demonstrating that stronger bonds correlate to more veterinary care. By distributing the Pet Attitude Scale to a robust sample in Egypt, a foundation regarding the perceptions towards companion animals can be built. This foundation can be later used to further understand the prevalence of pet ownership, the hu- man-animal bond, and the relationship’s impact on veterinary medicine.

Research Grant: None Student Support: The Lawrence Fund

Investigating the role of Caveolin-1 in cellular response to drug-induced oxidative stress

Dalia R Badamo, Dhirendra P Singh, and Joseph Francis

Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Breast cancer is the second most common cause of cancer related death in women in the United States. Doxorubicin (Dox) is one of the most effective anticancer chemotherapeutic drugs used to treat cancer. Dox induces iron overload in the mitochondria by blocking mitoferrin (MitoFer) and ATP-binding cassette sub-family B member 8 (ABCB8) trans- porters. Iron overload in the mitochondria induces oxidative stress and production of excessive reactive oxygen species (ROS) which results in lipid peroxidation and cell death called ferroptosis. Caveolin-1 (Cav-1) is a key protein of specific lipid rafts, called caveolae rafts, present on the membrane microdomain that has been shown to regulate the process of ferroptosis. The goal of this study is to understand the role of CAV-1 in Dox induced ferroptosis in vitro. To achieve this, CRISPR-Cas9 has been used to develop genetically Cav-1 knock out (Cav-1 KO) murine mammary epithelial 4T1 cells, confirmed by immunoblotting for anticav-1 antibody and immunofluorescence using a Green Fluorescent Protein tag in the donor vector. Concentrations of Dox treatments were optimized using both cell viability assay and ROS production indicated by Electron Paramagnetic Resonance of 4T1 cells upon Dox treatment. Both mRNA qPCR and immunoblotting are in progress to understand the molecular mechanism of ferroptosis upon Dox exposure in both Cav-1 KO and 4T1 WT cells. Lipid peroxidation following Dox treatment in these cell lines will also be quantified to understand the role of Cav-1 in lipid peroxidation upon Dox treatment.

Research Grant: 1P20 GM135000-01A1 NIH 1R01-HL147133-01 Student Support: Arkansas Veterinary Medical Foundation

2021 National Veterinary Scholars Symposium • 61 Probabilistic genotyping in DNA mixtures of canine “bully breeds” using DogFiler

Samantha L. Badgett, Melissa K.R. Scheible, Teresa M. Tiedge, Brittany S. Thomas, Adam J. Birkenheuer, and Kelly A. Meiklejohn

Department of Population Health and Pathobiology (Badgett, Scheible, Tiedge, Meiklejohn) , Department of Clinical Sciences (Birkenheuer, Thomas), College of Veterinary Medicine, North Carolina State University, Raleigh, NC

In forensic casework, animal DNA particularly those from companion animals is often collected as evidence. Complica- tions can arise during DNA analysis when the sample is a mixture of more than one individual and may not be analyzed or be deemed as inconclusive. For example in a dogfighting case, investigators might find blood on the fur of a dog that could include its own blood but also that of its canine attacker. For such mixtures, probabilistic genotyping software could have potential for identifying the number of individual sources and their corresponding genetic profiles.The goal of this study is to assess whether probabilistic genotyping software can be used to separate canine DNA mixtures, such that they could be interpreted in forensic casework. In this study, 80 canine samples from NC State’s Vector Borne Disease labora- tory were obtained from “bully breeds’’ commonly associated with dog fighting cases or breeds used in police work. The Dogfiler panel, designed for canine individualization by UC Davis’s Veterinary Genetics Laboratory, was used to obtain genotypes for each sample. Using this panel, genotype information for 13 of the 15 loci was obtained. Artificial DNA mixtures made from two individuals at varying ratios (1:1, 1:2, 1:3, and 1:4) were also genotyped with DogFiler and were analyzed using probabilistic genotyping software MaSTR (SoftGenetics, State College, PA). We will present results on the challenges of using DogFiler for canine genotyping and preliminary findings on the use of MaSTR for mixture separation.

Research Grant: Seed funds from NC State College of Veterinary Medicine Student Support: NC State Veterinary Scholars Program

Image analysis for monitoring in vitro migration of feline oral squamous cell carcinoma cells

Lindsey Bailey, Erika Pugh, Hailey Wang, and Chelsea Martin

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island.

Feline oral squamous cell carcinoma (FOSCC) is the most common oral malignancy in cats and is highly malignant and invasive. Poor prognosis and local invasion into the jaw bones or tongue are major factors in owners electing for euthana- sia. Cranberries have components that demonstrate anti-migration and anti-invasion properties in human oral squamous cell carcinoma cell lines. The aim of this study is to see if cranberry extract is effective in slowing FOSCC cell migration. A migration assay was performed with 3 FOSCC cell lines - SCCF1, SCCF2, SCCF3 - and with varying concentrations of crude cranberry extract. A migration assay involves making a ‘scratch’, or ‘gap’, in a monolayer of tumour cells in a tissue culture plate and measuring the rate of cells migrating into the defect and closing the gap. The migration assay was photographed daily. This study focuses on the image analysis performed using an opensource program called Fiji (based on ImageJ2). The dimensions of the defect are measured at the onset of the experiment and at 24 and 48 hours of culture. Studying the effect of crude cranberry extract on FOSCC migration will help determine the potential of cranberry extract for the treatment for FOSCC. Furthermore, optimizing the migration assay technique for FOSCC cells will make this can- cer research tool available for other FOSCC studies.

Research Grant: Natural Sciences, Engineering and Research Council of Canada (RGPIN-2019-06898, PI: C.Martin) Student Support: Natural Sciences, Engineering and Research Council of Canada (RGPIN-2019-06898, PI: C.Martin)

62 • 2021 National Veterinary Scholars Symposium Development of highly specific chicken IgY- based immunoassays for the detection of Staphylococcal leukotoxins

Carol L. Baker, Keun S. Seo, Nogi Park, Stephen B. Pruett, Joo Youn W. Park

Department of Comparative Biomedical Sciences, Mississippi State University College of Veterinary Medicine, 240 Wise Center Drive, Mississippi State, Mississippi

Staphylococcus aureus (S. aureus) causes severe infections such as necrotizing , diabetic foot ulcer, and infec- tive endocarditis by producing several redundant virulence factors such as cytotoxins and superantigens. Identification of specific virulence vectors is the key to develop effective therapeutics.Traditionally, mouse or rabbit IgG-based immuno- assays have been used, however, non-specific binding of mouse or rabbit IgG to staphylococcal protein A (SpA) hindered accurate identification and quantification of virulence factors. Chicken ImmunoglobulinY (IgY) has unique fragment crystallizable (Fc) sequences that do not have an affinity to SpA and other mammalian Fc receptors ideal to be used for immunoassays. In this study, chicken IgY specific to staphylococcal leukotoxins were generated and applied for western blot and enzyme-linked immunosorbent assay (ELISA). We demonstrate that chicken IgY successfully detected staphylo- coccal leukotoxins from in vitro culture supernatants and mouse tissue homogenates prepared from subcutaneous S. au- reus infection. ELISA showed high specificity and sensitivity of detecting less than 10 ng of staphylococcal leukotoxins. Chicken IgY-based immunoassays established in this study could be highly reliable, effective, and applicable detection tools to analyze specific virulence factors produced byS. aureus in vitro and in vivo.

Research Grant: "Center for Biomedical Research Excellence on Pathogen-Host Interactions." 1P20GM103646-07 Student Support: None

Molecular identification of arthropod vectors and pathogens using mobile, third-generation sequencing

Julia P. Baker, Cristina M. Blanco, Peter A. Larsen

Department of Veterinary and Biomedical Sciences, University of Minnesota College of Veterinary Medicine, Saint Paul, MN

Changing weather patterns are influencing ecological landscapes globally, leading to changes in the abundance and geo- graphic distribution of insect vectors of veterinary importance. As vector ranges expand, they are typically accompanied by pathogens that can negatively impact naive host populations, thus contributing to disease emergence. To keep pace with changing landscapes, biosurveillance methods must be nimble; providing meaningful data in a short period of time with limited a priori information. Third-generation nanopore sequencing platforms and other mobile, genomic technolo- gies are perfectly poised to allow for real-time, field-based surveillance, information that can be leveraged to detect and confront emerging vector-borne disease outbreaks. Here, we used portable nanopore sequencing and RT-PCR methods to detect molecular evidence of arthropod-borne pathogens of veterinary importance from native genomic samples; specifi- cally focusing on the surveillance of biting midges (genus Culicoides), to monitor for epizootic hemorrhagic disease virus (EHDV), an emerging pathogen of particular concern in Minnesota. CDC light traps were deployed in areas adjacent to ungulate housing and waste-collection zones where Culicoides spp. are most abundant. Insect trapping, sample prepa- ration and data analysis is ongoing. We anticipate our results will inform vector-borne disease surveillance initiatives throughout the state and upper Midwest. Our preliminary nanopore sequencing results for mosquito surveillance have al- ready identified several known mosquito vector species andDirofilaria immitis, the causative agent of canine heartworm.

Research Grant: Larsen Lab University of Minnesota College of Veterinary Medicine Student Support: DVM/PhD Program at the University of Minnesota College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 63 Impact of tumor heterogeneity on the stromal landscape in a mouse model of pancreatic ductal adenocarcinoma

Devon Bankler-Jukes, Meghan Kopp, Richard L. Barrett, Dr. Ellen Pure

Department of Biomedical Sciences (Bankler-Jukes, Kopp, Barrett, Pure) and Penn Vet Cancer Center (Bankler-Jukes, Barrett, Pure), University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

Malignant cells accumulate genetic and epigenetic alterations that lead to heterogeneity in their molecular, cellular, and clinical features. These differences also drive heterogeneity in the tumor microenvironment (TME) which in turn, can shape the evolution of the cancer cells. Extracellular matrix (ECM), stromal cells, immune cells, are some of the TME components which affect the progression of disease and clinical outcomes.Although heterogeneity and the nature of the TME determine the response to various therapies, the mechanisms involved in driving stromal heterogeneity and acquired resistance to treatment are poorly understood. Desmoplasia, involving a fibroproliferative response and dynamic remod- eling of ECM, is typical of many human tumors. My focus is to create a model to evaluate how tumor cells drive the stromagenic response underlying desmoplasia. Using a single parental pancreatic cell line derived from an autochthonous PDA isolated from a KPC (Kras/p53/cre) mouse, we take advantage of the natural heterogeneity of the parental line by selecting for high/low stromagenic clones based on ECM (collagen, fibronectin, ) and cancer-associated fibroblasts (CAFS) markers (FAP,a SMA, Ly6c, vimentin). Having clones that drive stromal variability from a single pa- rental cell line will allow us to identify differentially expressed genes that promote/restrain the desmoplastic response.We can also evaluate how tumor cells drive stromagenesis and how those differences influence other components of theTME such as, immune cell infiltration and function. Future studies will hopefully reveal which candidate genes provide novel therapeutic targets for solid tumors and improve current immunotherapies.

Research Grant: Unknown Student Support: NIH T35 OD010919

Development and validation of a novel canine visual acuity measurement protocol and procedure

Dustin Banooni, Ceylan Ersoz, Siddharth Suresh, Emily J. Ward, Freya Mowat

School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI (Banooni, Ersoz, Mowat)Department of Psychology, University of Wisconsin-Madison, Madison WI (Suresh, Ward)Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI (Mowat)

There are currently no standardized or validated methods of measuring canine visual acuity (the ability to detect detail). If visual acuity were measurable, we could develop a more comprehensive understanding of dog vision in health and disease. The aim of this study was to develop a method of accurately measuring canine visual acuity that is minimally invasive, inexpensive, and usable for research. We used a two-alternative forced choice task to measure visual acuity. On each trial, the dog was stationed 1.5 meters in front of two computer monitors, and a target stimulus of black and white vertical bars appeared on one of the monitors. The dog was rewarded for approaching the monitor displaying the target. The spatial frequency of the target, (i.e. bar width in cycles/degree) changed based on the dog’s performance, according to a QUEST staircasing method. This approximates the visual acuity threshold by finding the spatial frequency at which the dog could no longer reliably detect the target. Prior to this threshold testing, each dog was required to achieve an overall accuracy of 75% on 20 trials that used above-threshold targets. To date, 7 dogs have completed training at home and in the laboratory. Only 1 dog has reached 75% training accuracy and undergone threshold testing. In that dog, the QUEST stair- case estimated the visual acuity threshold as 1.14-1.17 cycles/degree. Although many dogs require a substantial amount of training to learn the task, initial results suggest dog visual acuity appears lower than the human standard of 30 cycles/ degree. Future directions include using eye tracking to account for the short dog attention-span and to limit the need for extensive training prior to threshold testing.

Research Grant: UW-Madison Companion Animal Fund (Mowat, Ward). Mowat faculty startup funds. Student Support: 2021 Boehringer Ingelheim Veterinary Summer Scholars Program

64 • 2021 National Veterinary Scholars Symposium Messenger RNA therapy to induce expression of bovine cathelicidins to combat respiratory disease pathogens

Cassandra Barber, Santiago Cornejo, Daryll Vanover, Hannah Peck, Philip J. Santangelo, and Amelia R. Woolums

Department of Pathobiology and Population Medicine, Mississippi State University, Starkville, MS (Barber, Cornejo, Woolums); Wallace H Coulter Department of Biomedical Engineering, Emory University School of Medicine and Georgia Institute of Technology, Atlanta, GA (Vanover, Peck, Santangelo)

Respiratory disease resulting from infection by viruses, bacteria, or a combination of both frequently causes sickness and death in animals and humans. In cattle, coronavirus and are common contributors; related patho- gens lead to respiratory disease in humans and other species. Various methods to prevent bovine respiratory disease have either been inadequately effective or have led to antimicrobial resistance. Better methods of prevention could improve animal and human health. A novel preventive approach is to activate production of innate antimicrobial molecules by messenger RNA (mRNA) transfection of the respiratory epithelium, to induce rapid protection against respiratory disease. Cathelicidins are a group of innate immune mediators produced by white blood cells and epithelial cells with direct an- timicrobial, inflammatory, and chemotactic effects. Our overall objective is to transfect bovine epithelial cells with syn- thetic mRNA encoding bovine cathelicidin 2 and modified bovine cathelicidin 5 (Syn 1 of Sahoo et al.), and to measure the antibacterial and antiviral effects of the expressed cathelicidins againstP. multocida and bovine coronavirus (BCoV). Transfection of bovine cells with cathelicidin mRNA resulted in peak expression at 24 hours as indicated by the linked reporter molecule NanoLuciferase. Ongoing work will define the conditions for optimal antimicrobial effect.This research will form the basis for future research to confirm efficacy in vivo trials.

Research Grant: Foundation for Food and Agriculture Research Veterinary Student Research Fellowship and USDA Formula Funds Student Support: Foundation for Food and Agriculture Research Veterinary Student Research Fellowship

Search of acaricidal bacteria for the control of the lone star tick (Amblyomma americanum)

Hannah M. Barber, L. Paulina Maldonado-Ruiz, Yoonseong Park

Kansas State University College of Veterinary Medicine (Barber) and Department of Entomology (Barber, Maldonado-Ruiz, and Park), Kansas State University, Manhattan, KS.

The lone star tick (Ambylomma americanum) is an important vector of disease for animals and humans in North America. With its increasing distribution throughout the Midwest and eastern United States, new control measures are needed to decrease the risk of tick-borne illnesses. Our previous work has shown that ticks voluntarily drink water when dehydrated in the field. In this study, we aim to test the oral toxicities of 30 different spore forming bacteria;Bacillus and other related strains (Paenibacillus and Brevibacillus) on adult lone star ticks to find a perspective candidate for tick management.The 30 differentBacillus and related strains were selected based on their toxicities to other invertebrates described in previous studies and were obtained from the Bacillus Genetic Stock Center. , found to have oral toxicity to the lone star tick in our previous study, was used as a positive control. The bacterial strains were pooled in groups of 4-5 and female ticks were allowed to voluntarily feed. Ticks were fed on the pooled bacteria with equal inoculum sizes of each, then observed for mortality for 6 days. Bacterial colonization of the midgut was tested by plating the homogenized tissue. Identifying bacteria that can colonize the midgut and are pathogenic to ticks will lead to the potential field applica- tion of acaricidal bacteria for management.

Research Grant: None Student Support: Elanco

2021 National Veterinary Scholars Symposium • 65 Characterizing the roles of the Toxoplasma gondii calcium binding proteins, TgCalnuc and CaM2

Ruth Pentlarge Barrow, Abigail Calixto, and Silvia N.J. Moreno

College of Veterinary Medicine (Barrow) and Center for Tropical and Emerging Global Diseases (Calixto, Moreno), University of Georgia, Athens, GA

Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cells in warm blooded organisms. One third of the world is infected with T. gondii which causes abortions and encephalitis in immunocompromised individu- als. The parasite’s ability to disseminate to all organs and cause disease is directly linked to its lytic cycle consisting of invasion, replication, and egress from host cells. Because calcium (Ca2+) signaling is vital to stimulation of the lytic cycle, we need to understand Ca2+ signaling in the parasite to understand how it causes disease. Calcium binding proteins (CBPs) are the first proteins to bind Ca2+ entering the cytosol from the external environment or intracellular stores, thus acting as key transducers of numerous cell signaling pathways. This study aimed to understand the roles of the CBPs TgCalnuc and CaM2 in the lytic cycle, which regulate Ca2+ storage/release in the ER and Golgi and therefore effect the lytic cycle Ca2+ signaling. ΔTgCalnuc and ΔCaM2 and complemented mutants were generated using CRISPR-Cas9 and shown to be de- fective for completion of the lytic cycle. To further characterize the phenotypes of the mutants with regards to the lytic cy- cle, we transfected the KO and complemented mutants with a red fluorescent protein, tdTomato, and a genetically encoded Ca2+ indicator, GCaMP6-mScarlet, which allows for visualization of Ca2+ signals in live parasites. Growth and replication assays were completed to discern the roles of TgCalnuc and TgCaM2 in the lytic cycle. With a better understanding of the CPBs that regulate the Ca2+ signaling necessary for the parasite to infect host cells, it is possible to better understand the mechanism by which Toxoplasma causes disease.

Research Grant: NIH Grants AI154931 and AI128356 Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

Bladders and biofilms: chronic urinary tract infections in dogs

Mary Catherine Beard, Elizabeth A. Swanson, Orion Rivers

Department of Clinical Sciences, Mississippi State University College of Veterinary Medicine, Mississippi State, MS (Beard, Swanson); Institute for Imaging and Analytical Technologies (Rivers)

Chronic and antibiotic-resistant urinary tract infections (UTIs) are a widespread complication in human and canine patients and can lead to life-threatening bacteremia if left untreated. Certain factors are known to increase the risk of chronic UTIs, such as long-term catheterization and diabetes mellitus. Enterococcus faecalis is a multi-drug resistant and biofilm-forming pathogen commonly isolated in these patients with chronic UTIs. Current research lacks understanding in how the urothelium in chronic UTI patients is altered such that biofilm colonization is possible.The objective of this study focused on identifying epithelial morphology of bladder tissue exposed to catheter trauma and/or diabetic-level urine glucose concentrations. It also aimed to determine whether E. faecalis biofilms grownin vitro were stimulated by high glucose concentrations. Biofilms were grown using a constant-flow bioreactor and measured using confocal laser scanning microscopy. Epithelial morphology was evaluated using scanning and transmission electron microscopy. It was found that catheter damage and/or high glucose concentrations altered the membrane morphology of cells, and significantly disrupted the epithelial barrier. It was also noted that the thickness of E. faecalis biofilms grown in glucose-enriched media was less than that of biofilms grown in standard media. Overall the findings demonstrated that catheterization and diabetic-level urine glucose compromise the protective urothelial barrier, however high glucose concentrations may not be the sole cause of biofilm stimulation in diabetic patients.

Research Grant: The Centers of Biomedical Research Excellence in Pathogen Host Interactions Grant P20GM103646 Student Support: National Institutes of Health Grant 5T35OD010432

66 • 2021 National Veterinary Scholars Symposium O-GlcNAc-Ca2+-calmodulin kinase activity on K-channel isoform within atrial and ventricular cardiomyocytes

Estefania D. Benavides, Emily Flam, Zoltan P. Arany

School of Veterinary Medicine, University of Pennsylvania, and Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

The molecular processes that occur during pathological events may differ between species with a heart, even though one may serve as a model in research. Type I diabetic humans have been presented to develop either premature contractions of the atria or ventricles; these patients are also at risk for developing atrial fibrillations. Similarly, depending on the breed and age, atrial fibrillation can be detrimental to canines, though no significant correlations to type I diabetes have been documented. Insulin-resistant cardiomyocytes are known to activate the hexosamine biosynthesis pathway, leading to increased levels of O-GlcNAc. This modification has been associated with calmodulin kinase II hyperactivity. The ex- pression of ion channels such as sodium and potassium channels, essential for maintaining electrophysiological integrity of cardiac tissue, are influenced by calmodulin kinase. In vitro, potassium channel isoforms been down regulated; this conclusion led to support the prolonged Q-T interval. A separate study also noted a decrease in potassium channel expres- sion in vivo, though only aimed for detecting in the left ventricle. This study aims to determine the extent diabetic-induced O-GlcNAcylated calmodulin kinase II has on potassium channel isoform expression between atrial and ventricular cardio- myocytes in mice. Western blotting and quantitative polymerase chain-reaction procedures will be utilized to measure po- tassium channel populations alongside the presence of O-GlcNAc-calmodulin kinase II. The results may aid in extending the knowledge of arrhythmias in mouse models to determine the translational benefits mice may provide for both human and veterinary cardiovascular medicine.

Research Grant: None Student Support: NIH Training Grant T35 OD010919

Correlation between sepsis scores and arterial blood pressures and their variability in anesthetized foals

Elizabeth Benton-Levith, Chiara E Hampton, Colin Mitchell, Xiaocun Sun, Anderson daCunha, Patricia Queiroz-Williams, Mark Mitchell

Dpt. of Veterinary Clinical Science (C. Mitchell, Queiroz-Williams, M. Mitchell), Louisiana State University, Baton Rouge, LA; Dpt. of Large Animal Clinical Sciences (Hampton, Sun), University of Tennessee College of Veterinary Medicine, Knoxville, TN; Animal Health Institute (DaCunha), Western University, Glendale, AZ.

Sepsis is a leading cause of morbidity and mortality in foals. Blood pressure variability indexes (BPVI) have been cor- related with severity of sepsis in humans. The goal of this retrospective study with data gathered from electronic anes- thetic records was to investigate the correlation between pre-operative sepsis scores, arterial pressures (SAP, DAP, MAP), and their respective dynamic BPVI in septic and non-septic foals anesthetized for surgical procedures at a tertiary referral center between January 2017 and May 2021. The correlation between sepsis scores and the first invasive arterial pressure measurement for MAP (MAP1), SAP, DAP, and MAP averaged for the duration of the anesthetic episode, along with calculated BPVI (standard deviations of arterial pressures, arterial pressure nadirs, differences between maximum and minimum arterial pressures, and their coefficients of variation) was investigated via Spearman’s correlation coefficient. Significance was set at P < 0.05. Data from 79 foals (n = 10 newborn, 40 neonate, 38 juvenile; n = 21 septic) were includ- ed in the analysis. Median age at the time of procedure was 11 days (range 0-108). Incidence of hypotension for MAP1 and averaged MAP was 43% and 4.6%, respectively. Higher pre-operative sepsis scores were correlated with lower av- eraged SAP and an increased number of interventions used to treat hypotension. No differences in the investigated BPVI were found. Foals with higher sepsis scores displayed lower systolic arterial pressures throughout the anesthetic event and required more complex blood pressure support strategies, which, in this cohort of patients, were effective in providing a drastic reduction in the incidence of hypotension and blood pressure variability.

Research Grant: None. Student Support: Boehringer Ingelheim; Louisiana State University School of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 67 Validation of a primary thyroid cell line to study feline hyperthyroidism

Alexandra E. Bergen, Chris J. Martyniuk, Chris L. Souders II, Alex E. Gallagher, Richard C. Hill, Joseph H. Bisesi, Deirdre H. Love

Department of Physiological Sciences (Bergen, Martyniuk, Souders,), Small Animal Clinical Sciences (Gallagher, Hill), College of Veterinary Medicine, Department of Environmental and Global Health (Bisesi, Love), College of Public Health and Health Professions, University of Florida, Gainesville, FL

Hyperthyroidism is the most common feline endocrine disorder. Therapeutic interventions in cats include methimazole, radioactive iodine treatment, surgical thyroidectomy, and dietary iodine restriction. The lack of validated cell assays to test new compounds for their ability to modulate production of thyroid hormones (TH) from cat thyrocytes impedes the discovery of new therapeutic interventions. The objective of this study is to optimize primary cell cultures from thyroid tissue of euthyroid and hyperthyroid cats to assess compounds for their potential to modulate TH. As a first step, we suc- cessfully cultured euthyroid feline thyrocytes and aim to expose cells to methimazole, a highly prescribed pharmaceutical for treating hyperthyroidism in cats. Cell cytotoxicity data was collected to identify doses for exposure. Based upon two assays, Cell Titer Glo and Cell Tox Green, there was no evidence for cytotoxicity up to 2,000 ng/mL methimazole over 72 hours. Based on these preliminary investigations, we will expose cat thyrocytes to 20 ng/mL and 2,000 ng/mL methima- zole to study its effect on TH production in vitro. We aim to characterize the expression profile of the thyroid system (key enzymes, transporters, and receptors in the TH production pathway) and thus far have optimized expression assays for thyroid peroxidase, thyroid hormone receptor alpha, and thyroid hormone receptor beta. Understanding baseline respons- es in a euthyroid cell culture to TH modulators will lay the foundation for testing the efficacy of therapeutic agents and dietary factors that can treat feline hyperthyroidism.

Research Grant: Unknown Student Support: Morris Animal Foundation and Florida Veterinary Scholars Program at UFCVM

Incidence of gallbladder sludge in cats and correlation with routine laboratory parameters

Dena Berlin, Todd Carter, and Daniel Foy

Department of Specialty Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Identification of gallbladder sludge in cats is an uncommon ultrasonographic finding. Still, the presence of sludge has been described as significant, but limited research has evaluated the implication of this finding in cats.The aim of this study was to compare the populations of cats with and without gallbladder sludge noted via ultrasonography and to explore potential associations between the presence of sludge and sex, breed, and clinicopathologic variables. Our retrospective study examined cats having an abdominal ultrasound performed at the Midwestern University Companion Animal Clin- ic from January 2015 through May 2021. Five hundred six cats were divided into 2 groups: no evidence of gallbladder sludge (430 cats) and evidence of gallbladder sludge (76 cats). Signalment, weight, clinical concern, ultrasonographic findings, and clinicopathologic variables (complete blood count, chemistry, and urine specific gravity) were collected us- ing the electronic medical records system. Data analysis found no difference between groups based on age, weight, breed, or sex. Similarly, no significant difference in the incidence of sludge was noted when stratifying cats by age (< 10 years versus > 10 years). When evaluating laboratory variables in cats with a mild amount of sludge compared to those with a moderate amount of sludge, cats with moderate gallbladder sludge had significantly higher GGT (P = 0.037) and choles- terol (P = 0.042). In summary, 15.0% of cats had gallbladder sludge noted during ultrasound. No association between the presence of gallbladder sludge and descriptive or laboratory parameters was found; however, cats with moderate gallblad- der sludge had evidence of possible cholestasis based on chemistry values.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

68 • 2021 National Veterinary Scholars Symposium Influence of chondrodystrophy on intervertebral disc mineralization and vertebral geometry in the dog

Catarina A. Bianchi, Denis J. Marcellin-Little, Tanya C. Garcia, Danika L. Bannasch

From the Veterinary Orthopedic Research Laboratory (Bianchi, Marcellin-Little, Garcia) and the Department of Population Health & Reproduction (Bannasch), School of Veterinary Medicine, University of California, Davis

Chondrodystrophy in dogs results from the presence of one or more copies of the fibroblast growth factor 4 FGF4( ) retro- gene on chromosome 12 (CFA12). Chondrodystrophy yields shortened limbs and abnormal calcification of intervertebral discs, contributing to intervertebral disc disease (IVDD). The Nova Scotia Duck Tolling Retriever (NSDTR) has been used to study FGF4 on CFA12 because NSDTRs may have 0 (controls), 1, or 2 or more copies of FGF4 on CFA12 while appearing to be free of other mutations that influences skeletal development, such asFGF4 on CFA18 (chondrodysplasia). CT scans of the spine were acquired for 22 NSDTR dogs with 0 (n = 7), 1 (n = 8), or 2+ FGF4 copies (n = 7) on CFA12. CT scans were imported into medical imaging visualization software (Horos) and analysis software (Mimics and 3-Matic, Materialise, Plymouth, MI). Investigators were blinded regarding the dogs’ genetic status. Intervertebral disc mineraliza- tion (location, geometry, size) and the geometry (size, curvature, vertebral canal geometry) of the third cervical (C3), thir- teenth thoracic (T13), and first lumbar vertebrae (L1) were analyzed. For C3, vertebral canal height and the vertebral canal height / width ratio were larger in dogs with 2 FGF4 copies compared to control dogs (P = 0.048 and P = 0.033, respec- tively). No other differences in vertebral geometry were identified between dog groups. Intervertebral disc mineralization is being analyzed. Clear geometric differences in the size, curvature, or vertebral canal geometry of a cervical, thoracic, and lumbar vertebra were not identified among 22 NSDTRs with and without chondrodystrophy. Chondrodystrophy does not appear to have a clear impact on vertebral geometry in dogs.

Research Grant: Funded in part by the grant from the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis (UCDSVM) Student Support: Funded in part by the Students Training in Advanced Research (STAR) Program, UCDSVM

Does closed glove exchange result in bacterial contamination of the surgeon’s hands?

Kathryn E. Biehl, Kelley Thieman Mankin

Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Closed glove exchange (CGE) is common practice in small animal surgery. The incidence of hand contamination occur- ring while performing this technique has never been assessed in the veterinary literature. Hand asepsis is of paramount importance because of the risk of glove perforations during a surgical procedure. This study aimed to determine if per- forming CGE resulted in a greater number of colony forming units (CFU) on hand culture when compared to baseline bacterial contamination of the surgical team’s hands. The hands of participants in 65 surgical procedures were sampled, resulting in 200 different sampling events.Pre-CGE, or baseline, contamination was found on 17 dominant hands and 13 non-dominant hands. After performing CGE, contamination was found on 14 and 15 dominant and non-dominant hands, respectively. No statistical difference was detected between the number of CFUs detected on a surgeon’s hands pre CGE and the number of CFUs detected on a surgeon’s hands post- CGE. 12 different bacterial species were detected, the most common bacterial species detected was Staphylococcus (63%). Our findings indicate that CGE does not statistically in- crease bacterial hand contamination over baseline levels.

Research Grant: Internal funding through Texas A&M University Small Animal Clinical Sciences Student Support: Boehringer Ingelheim VSP, Texas A&M College of Veterinary Medicine & Biomedical Sciences

2021 National Veterinary Scholars Symposium • 69 The use of mefenoxam to treat cutaneous and gastrointestinal pythiosis in dogs: a retrospective study

Phillip C. Billings, Stuart A. Walton, Domenico Santoro

Department of Small Animal Clinical Sciences, University of Florida College of Veterinary Medicine, Gainesville, FL

Pythium insidiosum is an emerging aquatic oomycete pathogen of dogs, horses, and cats in tropical and subtropical cli- mates all over the world. The microorganism is most commonly found in stagnant, warm bodies of water; however, it has been documented more recently in temperate regions. This pseudo-fungus causes pythiosis which most often results in gastrointestinal or dermatological disease in dogs. P. insidiosum is considered to be a water mold because its cellular char- acteristics lie between that of a fungus and algae. Therefore, it is particularly challenging to manage since it lacks chitin and ergosterol: the cell wall components that most antifungal drugs target. Current treatment methodologies include ag- gressive surgical removal, including appendage amputation if applicable, followed by adjunctive medical treatment with a combination of antifungal and antibiotics aiming to prevent infection resurgence. Prognosis is guarded to poor with a less than 20% recovery rate as a result of late detection and poorly effective treatments.An agricultural antifungal known as mefenoxam has demonstrated the ability to inhibit P. insidiosum proliferation in vitro and has promising results when used orally in a number of individual canine case reports. Since large scale literature on the use of mefenoxam is sparse, this retrospective evaluation displays the efficacy of mefenoxam in the treatment of canine gastrointestinal and dermatological pythiosis over a larger sample size at the University of Florida Small Animal Hospital. Data collection and analysis is still ongoing. We hypothesize that oral administration of mefenoxam will improve the survivability of both the dermatological and gastrointestinal form of pythiosis in dogs.

Research Grant: Unknown Student Support: Florida Veterinary Scholars Program, UF College of Veterinary Medicine

Dog and Adolescent Interactions Amid Animal Assisted Interventions

Ryan L. Bixby, Megan K. Mueller, Seana D. Guyer, Katie E. Dabney

Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA

Animal-assisted interventions (AAIs) with therapy dogs are increasingly used as a therapeutic treatment strategy for adolescents experiencing anxiety, however additional research is needed on the nature of human-dog interactions in this setting. When adolescents experience social anxiety in a therapeutic setting, proactive action by the animal has the poten- tial to reduce their anxiety, as well as create a positive feedback loop with sustained interactions that increases the AAI’s efficacy. However, therapy dogs experiencing stress may display stress-reducing or fearful behaviors, which could be interpreted by a socially anxious youth as personally directed at them. The individual agency of the animal has the poten- tial to have drastic effects on the outcome of an AAI, therefore further exploration of dog behaviors is needed. This study explores the behavior of therapy dogs in an AAI setting with adolescents experiencing a laboratory-based social stressor. The experiment was recorded and videos are being coded in Noldus Observer XT 14 software. Researchers will use an ethogram to code social, physical, and verbal contact between therapy dogs and human participants. The dogs’ behaviors are coded based on affiliative and stress-linked behaviors, while the human participants are coded in terms of actions involving the therapy dog. The coded behavioral data will be analyzed with human psychophysiology data to determine common behaviors between dogs and physiological correlations with specific behaviors. Gaining a better understanding of how the behaviors of therapy dogs affect adolescents amid animal-assisted interactions may allow for better design of interventions for the well-being of both the animals and people involved.

Research Grant: NIH R03 HD103300-01A1 Student Support: Cummings School of Veterinary Medicine at Tufts University

70 • 2021 National Veterinary Scholars Symposium Nanopore-based adaptive sequencing for mosquito-borne disease surveillance

Cristina M. Blanco, Julia P. Baker, Evan Kipp, Laramie Lindsey, Peter Larsen

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN

Single-molecule nanopore sequencing is an emerging powerful tool for real time sequencing based biosurveillance and pathogen discovery. Recent software advancements have made it possible to selectively enrich for viral and bacterial communities without the need for host depletion protocols in an approach known as adaptive sequencing. This project uti- lized nanopore-based adaptive sequencing for surveillance of mosquito-borne pathogens in the Minneapolis and St. Paul areas of Minnesota. Mosquitos were collected with CDC light and gravid traps, from urban and forested ecosystems, and identified using morphological characteristics and adaptive sequencing for molecular identification. DNA and RNA were sequenced for pathogen and strain identification. An initial nanopore sequencing run was conducted using genomic DNA from 8 barcoded mosquitoes and generated over 3 billion base pairs in approximately 48 hours. Preliminary results indi- cate potential matches to mosquito species including Culex pipiens, Aedes aegypti, and Culiseta alaskaensis. Dirofilaria immitis (heartworm) was also potentially detected in the samples, and additional phylogenetic analyses will be performed to confirm these results. Future steps include additional mosquito collection, blood meal analysis, and RNA analysis for pathogen discovery.

Research Grant: Unknown Student Support: Research reported in this publication was supported by the NIH training grant T35OD011118

Supporting the human-animal bond among homeless and housing insecure pet owners in Philadelphia, PA

Zelda Blowers, Lauren Powell, Chelsea Reinhard, Brittany Watson

Department of Shelter Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

The rate of pet ownership for those experiencing homelessness is estimated to be between 5-25%. Previous studies have described the benefits and liabilities of the human-animal bond (HAB) among homeless pet owners as well as the attach- ment dimension of the relationship. There is limited quantitative information describing commitment within the HAB and further investigation of the impact of pets on the experience of homelessness throughout the US is warranted. This study aims to characterize the HAB between pet owners experiencing homelessness or housing insecurity and their companion animals. Research questions include: (1) What do levels of commitment to pets look like in this population? (2) How does owning a pet impact the owner’s experience of homelessness or housing insecurity? Following IRB approval, mixed method surveys will be distributed at local housing support services and shelters in Philadelphia, PA. Quantitatively, this study will explore the commitment dimension of the HAB utilizing the Miller-Rada Commitment to Pets Scale adapted by Rauktis et al. (2021). Qualitatively, the study aims to describe the ways pet ownership impacts respondents’ experience of homelessness or housing insecurity. It is hypothesized that participants will report high levels of commitment to their pet as previous studies have described high levels of attachment in this population of pet owners, attachment is correlated to commitment and because income is not a significant predictor of commitment.The impact of pet ownership will likely follow previous studies which include both positives, like improved emotional and physical health, as well as limitations such as barriers to support services.

Research Grant: None Student Support: NIH T35 Training Grant OD010919, Rosenthal Shelter Medicine Fellowship Program

2021 National Veterinary Scholars Symposium • 71 Developing a thromboelastography assay in Elasmobranchs

Kayla L. Bonadie, Laura K. Ruterbories, Craig A. Harms, Alex M. Lynch

College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Thromboelastography (TEG) is a viscoelastic hemostatic assay that evaluates clot initiation time, kinetics, and strength, as well the extent of fibrinolysis, in whole blood or plasma. TEG has been thoroughly studied in mammals, but has been used less extensively in non-mammalian species such as elasmobranchs. Elasmobranchs, a subclass containing sharks, rays, and skates, are often found in aquarium collections, with wild populations currently declining. Non-traumatic coagulopa- thies often go unnoticed in exhibit aquarium animals until advanced stages of disease have developed. It is therefore chal- lenging to provide definitive or palliative treatment to affected animals.The goal of this study therefore was to establish a protocol for TEG in elasmobranchs that may improve detection of hemostatic defects and expand therapeutic options. Pooled samples of citrated plasma and brain from wild caught and aquarium elasmobranchs were stored at -808C until day of use and recalcified with 0.2 M CaCl2. The samples were equilibrated to room temperature for 30 minutes, rested for 30 minutes and then used for analysis. TEG was performed using a variety of clotting initiators, specifically RapidTEG (tis- sue factor with kaolin), kaolin, Reptilase, and elasmobranch derived thromboplastin. Initial tests yielded no quantifiable TEG reactions with any of the initiators investigated. TEG analyses may have been negatively impacted by the naturally high plasma calcium concentration of elasmobranchs, or the brain-derived thromboplastin may have been inactivated in preparation due to low urea concentration. Further studies investigating the influence of temperature, calcium concentra- tion and fresh whole blood versus citrated plasma are warranted.

Research Grant: Support Fund for Aquatic Animal Medicine (NC Veterinary Medical Foundation account) Student Support: NC State CVM Veterinary Scholars Program

Retrospective evaluation of whole-body CT findings and prognostic outcomes in canine trauma patients

Daniel A. Bonce, Thomas Denegamage, Federico Vilaplana Grosso

Department of Small Animal Clinical Science (Bonce, Vilaplana Grosso) and Department of Large Animal Clinical Science (Denegamage), College of Veterinary Medicine, University of Florida, Gainesville, FL

Motor vehicle accidents and bite wounds from dog fights are the most common causes of trauma affecting dogs, and both commonly lead to polytrauma. Polytrauma is defined as a patient with severe injuries in at least two or more areas of the body. Injuries in polytrauma patients can be life-threatening, and therefore prompt recognition and treatment are paramount for a successful outcome. Several studies have detailed the prevalence of common injuries reported following different types of trauma, however, limited information has been documented on the prognostic outcome and survivability associated with types of trauma and their resulting whole-body computed tomographic (WBCT) findings. In this study, we performed a retrospective search of medical records for all canine patients admitted into the Emergency and Critical Care service for trauma at the University of Florida Small Animal Hospital. Inclusion criteria required canine presentation of trauma, WBCT evaluation, and known final disposition. We categorized types of trauma as either blunt or penetrating trauma. Lesions reported on WBCT were categorized by the affected body regions. Finally, multivariable logistic regres- sion was used to assess the association between types of trauma combined with the affected body area and outcome. Data is currently in collection. We hypothesize that blunt-type trauma will result in an increased number of body areas injured and a worse prognosis, with blunt-spinal injuries resulting in the highest mortality. These results should be used to better assess the prognosis of patients with injuries related to trauma, assist in developing an appropriate diagnostic and treat- ment plan, and inform owners of potential outcomes.

Research Grant: None Student Support: University of Florida College of Veterinary Medicine, Department of Small Animal Clinical

72 • 2021 National Veterinary Scholars Symposium Accuracy of volume or weight measurement of cooked homemade diets for dogs

Grace L. Boothby, Jacqueline M. Parr, Melanie R. Parham, and Joseph W. Bartges

Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA

Cooked homemade dog diets have increased in popularity as owners become more involved in their pets’ lives. Measure- ment of ingredients during preparation of diets must be accurate to ensure the diet will be complete and balanced. Prior research has demonstrated that repeatedly measuring dry dog food with a measuring cup results in significant inaccuracy. Significant over- and under-estimation of dry dog foods also occurs with other measuring devices such as dry food mea- suring cups, graduated liquid measuring cups, and commercial food scoops. Thus, measuring ingredients using volumetric devices may be inaccurate resulting in incomplete and/or unbalanced diets. Cooked homemade diets for dogs formulated by a Board-Certified Veterinary Nutritionist typically consist of a source of protein, starch, a fiber, fat, and a canine-spe- cific vitamin and mineral supplement. This necessitates accurate measurements of a variety of ingredients. The purpose of this study was to determine the accuracy of preparing cooked homemade diets for dogs measuring ingredients by either volume (dry-food measuring cups and spoons) or weight (digital gram scale and syringes). Twenty-one participants were randomized to one of the two methods and instructed on homemade dog food preparation. Diet samples were sent to a third-party laboratory and underwent complete proximate analysis with minerals. Metabolizable energy was calculated for each sample. Data from both groups were compared to the anticipated nutrient profile to determine which method resulted in the most accurate homemade cooked diet preparation.

Research Grant: Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia Student Support: Boehringer Ingelheim, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

Lack of efficacy of routine and enhanced hygiene practices in removing rotavirus A from rubber piglet mats

Sarah R. Botkin, Benjamin W. Blair, James Lowe

Applied Epidemiology Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL

Porcine rotavirus, a pathogen typically impacting neonatal pigs, is becoming increasingly important in commercial swine breeding herds. Conventional control methods have resulted in limited impacts on rotavirus associated disease. For this study, rubber mats, typical of those found within farrowing crates for piglet comfort, were contaminated with a modified live porcine rotavirus vaccine. A slurry of two times strength rotavirus vaccine, 5 grams of feces and 5 ml of water was spread over a 103.2 cm2 inoculation area (IA). Mats were reinoculated every 48 hours, a total of three inoculations. Mats were kept under plastic sheets to create a warm, humid environment.48 hours after the last inoculation, the mats were sub- jected to decontamination methods, 1: High-pressure washing alone(HP), 2: HP followed by chemical disinfection with accelerated hydrogen peroxide at a 1:16 dilution with 5 minutes of contact time(DW), 3: HP followed by detergent applied at low pressure(WS) and 4: WS followed by chemical disinfectant(DWS). 16 times, a total of 64 treatments. A toothbrush was used to systematically scrub it across the IA and placed into a 50ml tube containing 15mL of D/E neutralizing broth. Samples were sent to the ISU VDL for routine multiplex Rotavirus rtPCR. All treatments had RNA detected; HP:11/16, DW:9/16, WS:6/16, and DWS:7/16. The proportion of samples with rotavirus RNA following treatment was not different between treatment groups. This data suggest that conventional and enhanced cleaning methods were ineffective in lower- ing the prevalence rotavirus A from mats. This demonstrates that rubber mats can serve as an environmental reservoir for rotavirus in clinically diseased herds despite proper cleaning practices.

Research Grant: Foundation for Food and Agriculture Research Student Support: University of Illinois

2021 National Veterinary Scholars Symposium • 73 The impact of netarsudil on endothelial regeneration in a murine model of Fuch’s endothelial corneal dystrophy

Madeline C. Bradford, Sangwan Park, Sara M. Thomasy

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA

Fuch’s endothelial corneal dystrophy (FECD) in humans is a degenerative disease of the cornea that progresses to blind- ness if left untreated. Up to 4% of the U.S. population over 40 years of age is affected by FECD and it is the most com- mon indication for corneal transplantation. Netarsudil is a topical ROCK inhibitor FDA approved for treating glaucoma. In corneal injury models using normal animals, ROCK inhibition has been shown to stimulate corneal endothelial cell (CEC) proliferation and migration. However, studies are lacking in animals that mimic FECD. This study aims to evaluate the effects of the topical ROCK inhibitor netarsudil, following cryoinjury in mice deficient inTAZ, encoded by Wwtr1. These mice recapitulate the clinical phenotype of late-onset FECD, including softening of Descemet’s membrane, guttae formation, and decreased CEC density. Corneal cryoinjury was performed on Wwtr1+/- mice 6-9 months of age. Then, the mice were treated twice daily with topical netarsudil or vehicle control for 14 days. Data is being collected at days 1, 3, 7, and 14 post-cryoinjury. Central corneal thickness and CEC density will be measured at each timepoint to assess injury response and healing over time. Corneas collected from mice euthanized at day 3 and day 14 will be assessed for CEC proliferation and migration over the wound center using EdU cell proliferation assay and Alizarin red stain, respectively. Analysis of cornea structure and CEC regeneration by these methods will provide insight into the effects of netarsudil on corneal endothelial healing in TAZ deficient mice.

Research Grant: NIH R01EY01634 (SMT) and P30EY12576 Student Support: UC Davis School of Veterinary Medicine STAR Program; NIH T35 Training Grant 5T35OD010956-22

Development of antigen based diagnostic ELISA for Mycoplasma hyopneumoniae

Rachel Bradley, Venkatramana D. Krishna, Maria Pieters, Jian-Ping Wang, Maxim C-J. Cheeran

Department of Veterinary Population Medicine (Bradley, Krishna, Pieters, Cheeran, College of Veterinary Medicine, and Department of Electrical and Computer Engineering (Wang), College of Science and Engineering, University of Minnesota, St. Paul, Minnesota

Mycoplasma hyopneumoniae (M.hyop) is one of the most prevalent respiratory bacterial pathogens that causes enzootic pneumonia, which leaves pigs vulnerable to other respiratory pathogens. Timely detection of disease is critical for treat- ment, control, and preventing spread. Current diagnostic testing such as culture and PCR are time consuming, require specialized laboratory facilities, and skilled personnel. Using monoclonal antibodies specific to M.hyop, an ELISA was optimized and evaluated for antigen detection using various clinical sample matrices. The optimized ELISA was tested with serial dilutions of M.hyop to determine dynamic range and limit of detection (LOD) for the pathogen. Analytical specificity was determined using various bacterial and viral respiratory pathogens. Finally, the assay will be evaluated using M.hyop spiked clinical samples and clinical samples from experimentally infected pigs. Current results show that the optimum concentration of capture and detection antibodies is 1 mg/mL. The dynamic range for the optimized assay was 226.57 ng/mL -7,250 ng/mL of M.hyop lysate, and the LOD is 226.57 ng/mL. The ELISA did not cross-react with any other respiratory viral or bacterial pathogens or commensal M. flocculare. Testing spiked and experimentally infected samples is currently underway. Once optimized for clinical sample matrices, the monoclonal antibodies will be attached to magnetic nanoparticles (MNP) to detect antigens using a magnetic particle spectroscopy (MPS) platform. MPS detects alterations in the harmonics of antibody-functionalized MNPs when bound to mycoplasma antigen. MPS technology will give producers access to quick, reliable, point of care diagnostics that can be applied in the field.

Research Grant: USDA- National Institute of Food and Agriculture Award Number 2020-67021-31956, and Project No: MN62-123 (SAES, University of Minnesota College of Veterinary Medicine Signature Program) Student Support: Swine Disease Eradication Center

74 • 2021 National Veterinary Scholars Symposium Impact of military chemical simulants on wicking of canine hair

Morgan Brathwaite, Steven D. Holladay, Brian France, William Bell, and Robert M. Gogal Jr.

Department of Biomedical Sciences (Brathwaite, Holladay, Gogal), College of Veterinary Medicine, University of Georgia, TDA Research (France, Bell), Wheat Ridge, CO.

Military working dogs (MWDs) have been an active part of the US military since the Revolutionary War. Currently, these working dogs, assigned to every branch of the US military, are frequently deployed worldwide into hazardous military field environments placing them at risk to chemical exposure (i.e. hair6 skin). Presently, little is known on how canine hair, as a potential natural barrier, interacts with military-based environmental chemicals. The present study examined the wicking of select chemical simulants on canine hair across multiple exposure timepoints. Back and thigh skin samples were collected from a canine donor dog that had been euthanized due to health-related reasons. The skin with hair was frozen (-808C) and then freeze-dried, after which it was cut into 1cm2 plugs for examination of chemical wicking. Hair plugs (N = 3/chemical/hair region/timepoint) were dipped in simulant chemicals (i.e. methyl salicylate, 2-chloroethylphe- nylsulfide and Glo Germ) for 2-second duration at a 0.5 cm depth. Hair was sampled at 30 sec, 5 and 10 min for a total of 27 hair plugs/hair region/simulant. Preliminary results, with these 3 chemical simulants, suggest that canine hair in 1 cm2 clusters show little simulant chemical wicking activity up to 10 min. These results suggest that clipping of the outer-most 0.5 cm of hair may largely remove simulant chemicals from an exposed dog. This also raise questions about the rapid use of shampoos and water on dogs exposed to immobile chemicals on their hair tips, in that these may serve as a conduit of the immobile chemicals to the skin. Future studies will focus on most effective procedures to remove the simulant chemi- cals from the hair, while preventing enhanced exposure to the skin.

Research Grant: DOD W911NF-20-P-0022 Student Support: DOD W911NF-20-P-0022

Genome-wide association study of degenerative suspensory ligament desmitis in the Peruvian horse

Kiley Brauer, Mehdi Momen, Sabrina H. Brounts, and Peter Muir

Department of Surgical Sciences, University of Wisconsin-Madison School of Veterinary Medicine, Madison, Wisconsin

Spontaneous rupture of ligaments and tendons as a consequence of tissue degeneration or damage is a common problem shared across species. In humans, degeneration of the Achilles tendon and the rotator cuff often results in injury, while in dogs, rupture of the cruciate ligaments in the stifle is very common. Degenerative suspensory ligament desmitis (DSLD) is a devastating disease in horses that is predisposed to the Peruvian Horse, Akhal-Teke, and Warmblood-type breeds. Degenerative tendon/ligament rupture typically leads to early retirement from athletic activity, has negative economic impacts, and in the case of DSLD often results in euthanasia of affected horses. Currently, there remains a major gap in the understanding of genetic contribution to spontaneous rupture of tendon/ligament in companion animals and humans. Therefore, we are undertaking a genome-wide association study (GWAS) combined with signature of selection analysis (SOS) of a large data set of single nucleotide polymorphisms (SNPs) from DSLD case and control Peruvian Horses. Pre- liminary work analyzing pedigrees of study Peruvian Horses suggests DSLD is a complex disease with a polygenic mode of inheritance and moderate heritability of ~0.246. We anticipate association analysis will discover strong DSLD can- didate genomic loci. Identification of associated DSLD SNPs and candidate genes will enable development of a genetic screening test to assess risk of DSLD in Peruvian Horses used for breeding as well as allow early treatment for affected horses. Furthermore, gene mutations that influence risk of DSLD in the Peruvian Horse represent important candidate genes for human and canine spontaneous tendon/ligament rupture.

Research Grant: NIH 5R21AR073301-02 Student Support: NIH T35OD011078-11

2021 National Veterinary Scholars Symposium • 75 Are beef calves placed in a herd with familiar cohorts more resilient to stress than unfamiliar calves?

Caleb M. Brezina, Grant A. Dewell, Renee D. Dewell, Rebecca L. Parsons, Anna K. Johnson, Derek B. Haley, Suzanne T. Millman

Veterinary Diagnostic & Production Animal Medicine (CMB, GAD, RLP, STM), Center for Food Security & Public Health (RDD), Department of Animal Science (AKJ), Department of Biomedical Sciences (STM), Iowa State University, Ames, IA; Department of Population Medicine (DBH), University of Guelph, Guelph, ON

Beef calves commonly experience stressors including weaning, transportation, social mixing, and castration. The objective of this experiment was to identify if social buffering among familiar (F) calves confers stress resilience relative to calves unfamiliar (U) with their pen-mates. The F calves were defined as those from the same source farm and U calves were sourced from different farms. We hypothesized that U calves would experience greater stress than F calves during social mixing (D0), castration (D+14) and wound healing (D+28) phases. Fifty-four calves from 14 source farms were random- ly assigned to F or U treatments and placed into 9 pens, each comprised of 6 calves (3F, 3U). All calves were processed weekly for 4 consecutive weeks. Stress outcomes included body weight gain, chute exit speed, plasma and hair cortisol, and wound healing score. Mortality, morbidity, treatments, and feed intake data were collected from farm records; a CBC panel was performed on D28. Preliminary examination suggests F calves had higher average body weight gain (F:9.97% vs. U:3.95%), lower average daily gain (F:2.19 lbs. vs. U:2.34 lbs.), fewer medical treatments (F:15% vs. U:39%), and lower D28 neutrophil count (F:3.14 x103/ mL vs. U:3.97 x103/ mL). These preliminary results provide some early support that maintaining social buffering among familiar calves confers stress resilience and may improve calf welfare.

Research Grant: USDA NIFA Animal Health and Production & Animal Products: Animal Well-Being Program [grant no. 2019-67015-29572/project accession no. 1019068] from the USDA National Institute of Food and Agriculture Student Support: Iowa State University College of Veterinary Medicine; Boehringer Ingelheim Animal Health

Anticarcinogenic effects of whole blueberry extract in MDA-MB-231 cell line

Rebecca M. Broussard Banker, Niya M. Lewis, Rashmi Pathak, Dhirendra P. Singh, Joseph Francis

Comparative Biomedical Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA (Banker, Pathak, Singh, Francis), Environmental Toxicology, Southern University and A&M College, Baton Rouge, LA (Lewis)

Breast cancer is the leading cause of cancer-related deaths in females globally. Immune evasion by cancer cells allows tumor progression and presents a major challenge in cancer therapy. Natural products like blueberries are well known to possess a variety of anti-carcinogenic properties. However, evidence supporting the effects of blueberries on immune eva- sion in cancer cells is lacking. Among the different immune checkpoints, PD-L1 (programmed cell death ligand 1) over- expression on cancer cells is linked to immune evasion making it an essential target in many malignancies such as breast cancer. The present study investigated the immunomodulatory effects of blueberries in addition to exploring its effects on cell viability and migration in MDA-MB-231 (triple-negative breast cancer) cell line. The survival of MCF-10A (non-tu- morigenic) and MDA-MB-231 (cancerous) cells after treatment with whole blueberry extract (BB) at various concentra- tions (0, 0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/mL) was determined by MTT assay, which indicated that BB exhibited higher toxicity towards MDA-MB-231 compared to MCF-10A cells. Further, BB induced morphological changes in MDA- MB-231cells when observed under the microscope. BB also inhibited MDA-MB-231 cell migration in a dose-dependent manner as shown by wound healing assay. Immunoblotting assay demonstrated that BB significantly reduced the expres- sion of PD-L1 levels. Overall, our findings suggest that BB diminished viability, induced morphological change, inhibited cell migration, and reduced PD-L1 expression in MDA-MB-231 cells. Further research should explore the potential use of blueberries as an additive to standard therapy to optimize future cancer treatment options.

Research Grant: NIH 1P20 GM135000-01A1, NIH 1R01-HL147133-01 Student Support: NIH Biomedical Research Experience for Veterinary Students, LSU SVM

76 • 2021 National Veterinary Scholars Symposium Evaluation of urine for Dirofilaria immitis antigen detection in dogs

Andrew C. Brown, Meriam N. Saleh, J. Mack Fudge, Guilherme G. Verocai, Mary B. Nabity

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX (Brown, Saleh, Verocai, Nabity); Hill Country Animal League, 924 N. Main St, Boerne, TX, 78006 (Fudge)

Infection by the canine heartworm, Dirofilaria immitis, causes a potentially fatal, multisystemic disease in dogs. Diagnosis of D. immitis relies on serologic antigen detection coupled with microfilariae identification. It has been shown that -im mune-complex dissociation (ICD) of serum/plasma by heat treatment increases detection of infection, especially in dogs from endemic areas and with inconsistent heartworm prevention. The aim of this study was to assess the use of urine for heartworm antigen detection in dogs using a commercial ELISA (DiroCHEK ). Matching serum/plasma and urine of 24 experimentally infected dogs at different time points post-infection were tested to assess the feasibility of antigen detec- tion in urine. Matching serum and urine samples of 56 dogs admitted to a spay/neuter clinic with unknown infection status were also tested. In the experimental samples, antigenemia was detected in 91.7% of samples both pre- and post-ICD, while antigenuria was detected in 75% and 70.8% of dogs pre- and post-ICD, respectively. In the clinic samples, antigen- emia was detected in 5.4% of dogs pre-ICD and 7.1% post-ICD, while antigenuria was detected in 14.3% and 5.4% of dogs pre- and post-ICD, respectively. In experimental samples, sensitivity (sn), specificity (sp), and positive predictive value (PPV) of antigenuria were high (82%, 100%, 100%, respectively). In clinic samples, sn, sp, and negative predictive value (NPV) were high (100%, 92%, 100%, respectively). In contrast, NPV in experimental samples (33%) and PPV in clinic samples (50%) were low. Overall kappa agreement between antigenemia and antigenuria was moderate. These data suggest that urine might be a useful biofluid for heartworm antigen detection in dogs.

Research Grant: None Student Support: Texas Veterinary Medical Foundation, Texas A&M College of Veterinary Medicine & Biomed Sciences

Interrogating MARCKS inhibition as a strategy to alter bovine neutrophil responses to S. typhimurium

Chalise F. Brown, Haleigh E. Conley, M. Katie Sheats

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Salmonellosis is a disease of major importance in both livestock and humans. Salmonella infection in cattle causes eco- nomic loss due to animal death and human food supply contamination. Neutrophils play a key role in Salmonella enteritis, causing inflammation that ultimately enablesSalmonella to colonize the gut. With growing concerns regarding antibiotic resistance, particularly in animal agriculture, non-antimicrobial interventions/treatments for are needed. One proposed method for treatment is to target the host response by reducing the inflammation that supportsSalmonella colo- nization with the gut. Previous research in our lab has shown that MARCKS protein plays an essential role in neutrophil inflammatory functions, including respiratory burst and adhesion.Therefore, we investigated whether MARCKS protein inhibition would be a strategy to alter bovine neutrophil responses to S. typhimurium. We hypothesized that treatment of isolated bovine neutrophils with a MARCKS-specific inhibitor peptide, known as MANS, would attenuate Salmonella-in- duced respiratory burst and adhesion. Whole blood was collected from healthy, lactating Holstein cows for neutrophil isolation (IACUC #20-115). Neutrophils were stimulated with late exponential phase S. Typhimurium strain HA420 (MOI 50:1). Plate-based fluorescence assays using dihydrorhodamine (DHR) were used to measure the production of reactive oxygen species (ROS). Static plate-based assays with calcein-labeled neutrophils were used to measure adhesion. Prelim- inary results with MANS peptide treatment show a trend for a concentration dependent attenuation of respiratory burst in Salmonella-stimulated bovine neutrophils.

Research Grant: USDA Student Support: NC State Veterinary Scholars Program, FFAR Veterinary Student Research Fellowship

2021 National Veterinary Scholars Symposium • 77 Analysis of commonly encountered toxins in avian patients presenting to a wildlife rehabilitation service

Kristen N. Brown, Fiep de Bie, and Dr. Lara Cusack

Zoo, Exotics, and Wildlife Service, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island.

Countless numbers of raptors are introduced to rehabilitation clinics annually, many with signs of toxicity. Some of the most common and lethal toxins include lead, anticoagulant rodenticides, organophosphates, and pentobarbital. A retro- spective study of 129 raptors presented to the Atlantic Veterinary College’s Zoo, Exotics, and Wildlife Service (ZEW) from 2015-2020 was performed to assess the exposure of these toxins in the Atlantic Canada region. Patients were cate- gorized as Predated (injury consistent with predation), Suspect (history consistent with toxin exposure), Probable (clini- cal signs of toxicity), and Confirmed (positive testing for toxins). Data from ZEW intake and AVC Pathology found that 52.71% (n = 68/129) of all raptor intakes were Probable or Suspect and 33.33% (n = 43/129) were Predated. Clinical signs were more commonly associated with lead (16.28%, n = 21/129), followed by anticoagulant rodenticide (9.3%, n = 12/129). Due to budgetary limitations, samples from only five raptors (7.3%, n = 5/68) that presented as Probable or Suspect for toxicity were sent for confirmation. Of these, 60% were Confirmed (n = 2 lead, n = 1 pentobarbital). In addition to the financial constraints often associated with wildlife rehabilitation, there are limitations in antemortem toxin testing. Additionally, clinical signs are often nonspecific. For these reasons, it is recommended that toxicity be considered a differential diagnosis for raptors presenting to wildlife rehabilitation centers and that history and clinical signs be used in conjunction with testing, when available, to direct clinical decision making. Additional testing of Suspect and Probable cases may allow improved recognition of antemortem signs and patient care.

Research Grant: None Student Support: Veterinary Summer Research Award, Sir James Dunn Welfare Center

Selenium as a Potential Antiviral for PRRSV Infection

Matthew H. Browning, Brittany M. Pecoraro, Alba Frias-De-Diego, Elisa Crisci

College of Veterinary Medicine, Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina, USA

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a pathogen that heavily impacts the porcine industry through high rates of morbidity, mortality, and dramatic economic losses. Currently, PRRSV is only partially managed and controlled through vaccination strategies. Selenium (Se) is a micronutrient and feed additive used in swine that is essential to selenoprotein formation. Selenoproteins help protect against oxidative stress and have immunostimulating properties. These properties have led to investigations on how selenium supplementation can impact immune functions during viral infections. The purpose of this research is to investigate the antiviral properties of Selenium to determine its potential as a treatment during PRRSV infection. Different selenium-based compounds have been investigated; however, for this study, L-Selenomethionine was tested at three different concentrations: 0.3, 0.03, 0.003 ppm (parts per million).The highest con- centration legally permitted in the swine industry is 0.3 ppm due to its potential toxicity. MA 104 cell line (African Green Monkey kidneys) was used for PRRSV infection, since it is the standard cell line for PRRSV studies. The compound was also used with PRRS viruses of different virulence and with a modified live virus vaccine strain.The results obtained showed a decreased viral load in cells treated with L-Selenomethionine in comparison to untreated cells in all tested viral strains. In vitro data indicate that L-Selenomethionine has the potential to be used as an antiviral for PRRSV infection, but additional experiments are needed to fully confirm its effectiveness, particularly in lung macrophages, which are the target of the virus, and in in vivo studies.

Research Grant: PI Startup Funds Student Support: NC State Veterinary Scholars Program

78 • 2021 National Veterinary Scholars Symposium Spectroscopic Characterization of Collagen and Biomechanical Changes in Alcohol- Induced Osteoporosis in Mice

Allison Brunner, Gombojav Ariunbold, Lauren Priddy, Haifeng Wang, Michael Jaffe

Department of Veterinary Clinical Science (Brunner, Jaffe), Department of Physics & Astronomy (Ariunbold), Department of Agriculture & Biological Engineering (Priddy), Department of Industrial & Systems Engineering (Wang), Mississippi State University College of Veterinary Medicine, Mississippi State, MS

Osteoporosis as a disease characterized by decreased bone mass and deterioration of bone microarchitecture of which more than 75 million people worldwide suffer. Secondary osteoporosis is predisposed by risk factors including alcohol abuse. There is little that is known about the role of alcoholism on the collagen content and mineral composition of the bone and its biomechanical properties. The aim of this pilot study is to determine the difference in collagen content of the bone of genetically similar but phenotypically different BXD mice. Raman spectroscopy was used to identify the collagen in the bone, and the Keyence system allowed us to quantify surface roughness of the BXD mice bones. The results are still being collected and data analyzation is an ongoing process. It is suspected that there are collagen content differences between the BXD mice. The data collected from this study is preliminary data to be used in distinguishing biomechanical, histopathological, and spectroscopic properties of a variety of collagen-containing tissue types in a mouse model of osteo- porosis secondary to chronic alcohol abuse for future studies.

Research Grant: Mississippi State University College of Veterinary Medicine Student Support: Mississippi State University College of Veterinary Medicine

Effects of E. coli Heat-labile enterotoxins on immune cell-cell communication mediated by exosomes

Andreya L. Bryson, Toufic Nashar

Tuskegee University College of Veterinary Medicine, Tuskegee, Alabama

Enterotoxigenic Escherichia coli (ETEC) is the cause of diarrhea in human and animals in many parts of the globe, result- ing in fatalities if left untreated. ETEC secretes a heat-labile enterotoxin (LT) that binds to GM1 receptor on many cells including gut cells causing an increase in cAMP level leading to electrolyte imbalance and the resulting diarrhea. LT is also a strong immunogen. Extracellular vesicles (EV’s) include micro vesicles, exosomes, and apoptotic bodies, are lipid bound particles that are naturally secreted to the extracellular space from many cells. Exosomes shuttle proteins, nucleic acid, and lipids between cells, they act as an important mode of intercellular communication. we examined effects of tox- ins on EVs from immune cells to clarify the role of ETEC and their toxins played in disease. LT and its non-toxic subunit LTB were incubated with A20 B cells, supernatants were collected, exosomes purified and labelled, and their transfer to recipient B cells examined. A structurally and functionally related toxin to LTB, cholera toxin B (CTB) was incubated with B cells or loaded into exosomes from dendritic cells. Transfer in exosomes to recipient B cells was examined. Un- expectedly, LT and LTB significantly reduced transfer of exosomes to recipient B cells and their overall protein content. CTB incubation with B cells, and CTB in exosomes were transferred to recipient B cells. Overall, our results demonstrate a significant inhibitory effect of LT/LTB on secretion and delivery of exosomes to recipient B cells, and CTB is transferred through exosomes. We suggest that LT interruption of cell-cell communication may help ETEC inducing diarrhea in the host before an active immune response.

Research Grant: NIH #2T35OD010432, RCMI Corefacility#U54MD007585 Student Support: NIH #2T35OD010432

2021 National Veterinary Scholars Symposium • 79 Hypertension is associated with glucocorticoid excess and insulin resistance in aged SHRSP rats

Ireyon Monique Buchanna, Dr. Anne Dorrance, Dr. Theresa Lansdell

Department of Pharmacology and Toxicology (Dr. Dorrance and Dr. Lansdell), College of Veterinary Medicine, Michigan State University, East Lansing, MI.

Objective: Insulin resistance is the most significant risk factor for dementia development, aside from advanced age. Fur- thermore, hypertension plays a role in the development of cognitive decline due to decreased cerebral perfusion; we hy- pothesized that hypertension causes insulin resistance.Methods:6-month-old spontaneously hypertensive/stroke-prone rats (SHRSP)(7 male, 8 female) were compared to age-matched normotensive Sprague Dawley rats (6 male, 8 female). Blood pressure was measured using a tail-cuff blood pressure system (CODA-6 Kent Scientific). Steroids were extracted from fecal pellets, and corticosterone was measured using an ELISA (Arbor Assays). Fasting glucose (Freestyle lite portable glucose meter) and insulin (ELISA assay)were measured and used to calculate insulin resistance index using the homeo- static model of insulin resistance. Glucose clearance will be measured using the intraperitoneal glucose tolerance test (IP-GTT). Results: Both male and female SHRSP had higher mean arterial pressure than their normotensive controls (P < 0.0001). Male and female SHRSP had higher fecal corticosterone levels than their controls (males, P < 0.0001; females, P = 0.0017). However, male SHRSP produced significantly more corticosterone than females (P < 0.0001).There was no difference in fasting blood glucose between the male hypertensive and normotensive rats. However, female SHRSP had higher fasting blood glucose than controls (P = 0.0045). Conclusions: In SHRSP rats, hypertension is associated with glucocorticoid excess and insulin resistance,two factors that often contribute to vascular damage and intensify the devel- opment of dementia.

Research Grant: National Institutes of Health Grant R01-HL-137694-01 (AMD) Student Support: NIH Grant R25HL103156, BRUSH Summer Research Program

History of eccentric contractions in Duchenne muscular dystrophy

Katherine E. Bukovec and Robert W. Grange

Department of Human Nutrition, Foods, and Exercise, College of Agriculture and Life Sciences, Virginia Tech, Blacks- burg, VA (Bukovec, Grange)

This review describes the history of ex vivo eccentric protocols including the foundation of research used to support the protocol’s development and its applications in Duchenne muscular dystrophy (DMD) research. DMD is a fatal neuromus- cular disease characterized by progressive muscle weakness. DMD is caused by a genetic mutation resulting in the lack of the protein dystrophin. Dystrophin’s functions are believed to be both mechanical and signaling because of its localization and association with the dystrophin-associated glycoprotein complex (DGC) at the sarcolemmal membrane. Because of dystrophin’s structural function, methods to assess muscle’s mechanical properties have frequently been used in this area of research. It is now well accepted that unaccustomed eccentric contractions (muscles that are lengthened while activat- ed) damage muscle more than concentric (muscles that are shortened while activated) or isometric (muscles that maintain length while activated) contractions. Early twentieth-century research reported observations of severe delayed muscle soreness in human subjects following rhythmic contractions. Later research in the mid-twentieth century further charac- terized the type of exercise that caused more severe muscle soreness in humans as negative (eccentric) work. This under- standing led to the development of eccentric contractile protocols to assess muscle susceptibility to damage. While the origins of ex vivo eccentric protocols lay in observations of human soreness following exercise there has been a returned emphasis on the eccentric components of physiologic movements such as during gait, walking downhill, and descending stairs.

Research Grant: None Student Support: T35OD011887D

80 • 2021 National Veterinary Scholars Symposium Postnatal development of vocal movements in a Peromyscus mouse model

Daegan Bulloch, Tobias Riede

Department of Physiology, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Mammals, including humans, produce a voice through coordinated breathing and laryngeal and oral movements. We investigated the postnatal development of those movements in the California mouse (Peromyscus californicus). Audio and video recordings of spontaneous vocal behavior in young and old animals were made. A frame-by-frame video analysis tracked mouth movements by measuring the distance between the upper and lower lip (“mouth gape”). Acoustic signals were analyzed through spectrograms and tracings of call fundamental frequency (F0). We tested the hypotheses that every call is associated with a stereotypic mouth movement. In 318 calls produced by 22 pups, we found that 93% of calls were associated with mouth movements. In contrast, all 12 calls from two adult mice were associated with a prominent mouth movement. Calls that were associated with a mouth movement demonstrated high coordination, as call onset was within 332 ms from the beginning of mouth opening. The relationship between “mouth gape” and F0 (49 calls from 4 pups; R2 = 0.0578; P = 0.003) suggests that mouth movement does not track F0. The experiments we report here provide two new findings that contribute to our understanding of how California mice use their oral cavity to modulate their calls.We show that a majority of calls are accompanied by previously undescribed changes in upper vocal tract filter configuration -in volving movements of the mandible affecting oral cavity geometry. Furthermore, California mice demonstrate age-depen- dent changes in how mouth movements are synchronously employed and coordinated with laryngeal sound production. In future work, we will increase samples from adult animals and explore the acoustic consequences of mouth movements.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Host-pathogen interactions during wound repair in zebrafish

Ariela Burk, Anna Huttenlocher

Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, WI.

The skin microbiome is a vast assortment of microorganisms that normally exist symbiotically, but have the potential to influence patient health and immune response to injury and infection. It’s previously been shown that commensal skin bacteria can inhibit the proliferation of , however, innate immune response to different types of bac- terial pathogens is less well characterized. Thermal injury induces tissue damage which triggers a rapid innate immune response that affects host defense mechanisms. These injuries are frequently confounded by secondary infections where the effect of pathogenic versus commensal bacteria on host innate immune response is not well understood. During innate immune response, the mechanisms that drive damaging versus protective leukocyte inflammation also remain unclear. The aim of this study is to better understand the impact that commensal skin bacteria have on innate immune response during infectious processes and wound healing. A transgenic zebrafish model expressing fluorescently tagged neutrophils and macrophages was used in conjunction with widefield microscopy to quantify immune cell recruitment to the sites of ther- mal injury with exposure to either pathogenic bacteria (Pseudomonas aeruginosa) or commensal bacteria (Staphylococcus epidermidis). The results showed that exposure to P. aeruginosa after burning resulted in significantly more neutrophil re- cruitment to the site of injury at 2 and 6 hours compared to the uninfected group, while there was a trend towards reduced neutrophil recruitment with exposure to S. epidermidis. Additionally, exposure to both bacteria concurrently resulted in less neutrophil recruitment than P. aeruginosa alone.

Research Grant: NIH R35 GM118027, NIH R01 AI134749 Student Support: NIH T35 OD011078

2021 National Veterinary Scholars Symposium • 81 The effects of multimodal pain management during disbudding on health and performance in neonatal goat kids

Erin M. Burke, Whitney A. Knauer, Emily A. Barrell, Tanzania M. Menn, Hannah Bakke, and Michaela M. Thomas

College of Veterinary Medicine (CVM), University of Minnesota, St. Paul, Minnesota

Current standard of care in the dairy goat industry is to disbud neonatal goat kids without pain control, creating a welfare issue. The objective of this study was to investigate the effect of pain management during cautery disbudding on health and performance of neonatal goat kids in the 6 wks following the procedure. A total of 560 mixed breed goat kids (5-10 days old) from a commercial goat dairy were weighed and evaluated using the UMN Goat Kid Health Scoring System (GKHSS). Mentation, body posture, respiratory secretions, abdominal fill, fecal staining, and rectal temperature were as- sessed on a 0-2 scale. Fifty kids that either scored 2 in any category or had a total health score $4 were excluded. The re- maining 510 kids were randomly allocated to either control (CON; n = 254; no pain management) or treatment (TRT; n = 256; average (range) of 1.7 mg/kg (1.0-3.4 mg/kg) oral meloxicam, 4.1 mg/kg (2.5-8.2 mg/kg) SQ sodium bicarb buffered lidocaine, 0.05 mg/kg (0.03-0.12 mg/kg) IM xylazine) by ear tag. All kids underwent cautery disbudding by two trained technicians, and weekly weights and health scores were collected for the subsequent 6 wks. Farm treatments and mortal- ity events were collected from dairy records. At enrollment the average body weight was 4.4 6 1kg, with no difference between treatment groups (P = 0.34). TRT kids received meloxicam, xylazine, and a lidocaine nerve block an average of 67, 30, and 16 minutes prior to disbudding, respectively. Follow up data collection is ongoing. Future analysis will include the comparison of weights, health scores, and pre-weaned morbidity and mortality events between treatment and control groups during the 6 wk follow-up period.

Research Grant: This work is/was supported by the USDA National Institute of Food and Agriculture, Animal Health project number 1017915 through funds granted to the CVM at the University of Minnesota Student Support: CVM, Ruminant Center. Supported by the NIH training grant T35OD011118

Comparison between qPCR and ddPCR for quantifying AMR genes in Salmonella enterica in swine fecal samples

Daniella A Burleson, Claudia O Trujillo, Javier Vinasco, Morgan H Scott, Keri N Norman

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Antimicrobial resistance (AMR) is one of the most serious public health threats of the century and quantitative PCR (qPCR) is utilized to determine the quantity of AMR genes in a sample; however, it is difficult to associate these genes to specific bacterial species. Droplet digital PCR (ddPCR) is a third generation PCR technology that is more accurate at quantifying genes compared to qPCR without the need for standard curves. DdPCR has not yet been applied in an industry setting, thus, we hypothesize that ddPCR technology will detect and quantify antimicrobial resistance genes more accu- rately than qPCR; more importantly, ddPCR will allow for the association of AMR genes in non-typhoidal Salmonella enterica from agricultural animal fecal samples. Data from qPCR of invA and qnrB genes will be compared to data from ddPCR multiplexing of invA and qnrB genes. For the invA qPCR protocol, 20uL reactions were mixed containing 1.0uL of 1.5uM forward and reverse primer, 0.4uL of 2.0uM probe, 3.0uL of DNA, 4.6uL of water, and 10.0uL of Brilliant III Mas- ter Mix. For the qnrB qPCR protocol, 20uL reactions were mixed containing 1.0uL of 2.5uM forward and reverse primer, 1.0uL of 2.5uM probe, 5uL of water, 2.0uL of DNA, and 10uL of Brilliant III. At the time of drafting the abstract, ddPCR protocol is being optimized and data analysis is still underway; however, we anticipate that ddPCR will more accurately and efficiently quantifyinvA and qnrB genes in Salmonella enterica from agricultural animal fecal samples. Overall, the consistency and reliability of ddPCR as compared to traditional PCR methods will facilitate the identification ofAMR genes in Salmonella and potentially other foodborne pathogens of concern to public health.

Research Grant: Texas A&M University, College of Veterinary Medicine & Biomedical Sciences, Veterinary Medical Scientist Research Training Program Student Support: Foundation for Food & Agriculture Research Fellowship, Texas A&M College of Veterinary Medicine

82 • 2021 National Veterinary Scholars Symposium Retrospective analysis of Avitrol toxicity in rock pigeons (Columbia livia) presenting to a wildlife service

Joseph W. Buzzell, Fiep de Bie, Lara M. Cusack

Zoo, Exotics, and Wildlife Service, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island

The pest mediation substance 4-aminopyridine (trade name “Avitrol”), is marketed as a humane and safe frightening agent that causes pain free behavioral changes with little to no mortality. Despite this labelling, the Atlantic Veterinary College’s Zoo, Exotics, and Wildlife Service (ZEW) sees an average of 21 rock pigeons (Columbia livia) (pigeon) patients annually (7.45% of total patient intakes, n = 125/1677) with suspected Avitrol poisoning. A retrospective study on pigeon intakes between 2015-2020 (n = 209) was conducted to identify clinical signs and history suggestive of Avitrol poisoning. Cas- es were defined as Confirmed (positive Avitrol test of crop sample), Probable (clinical signs of toxicosis), and Suspect (history consistent with toxicosis). Using ZEW intake and AVC Pathology data, estimates of Probable (n = 50/209) and Suspect (n = 75/209) cases and case outcomes were compiled. Of all pigeon intakes, 58.37% (n = 122/209) resulted in a fatal outcome (died in care, dead on arrival, or euthanasia). A total of 60.23% of pigeon intakes were classified as Probable or Suspect (n = 125/209), of which 56% (n = 70/125) resulted in mortality. Samples from 11 pigeons were submitted for Avitrol testing (5.2%, n = 11/209, of total pigeon intakes) and 100% were Confirmed. Of pigeon fatalities, 57.38% (n = 70/122) were deemed related (Confirmed, Probable, Suspect) to Avitrol. The results of this study suggest that Avitrol as- sociated morbidity and mortality may be higher than claimed by the manufacturer. As there is no antemortem Avitrol test and clinical signs are non-specific, it is suggested that future research includeAvitrol testing of all pigeon fatalities as this will improve assessment of Avitrol toxicosis in pigeons.

Research Grant: Atlantic Veterinary College Summer Research and Leadership Program Student Support: Veterinary Student Research Award, Sir James Dunn Welfare Center

Seroreactivity of tick-immunized calves to five recombinantD. andersoni proteins

Kyle J. Cacciatore, Sammuel Shahzad, Kyle Hoffman, Stefan H. Keller, Bridgette Rogers, Roger W. Stich

Department of Pathobiology (Cacciatore, Shahzad, Hoffman, Stich, Rogers, Keller), College ofVeterinary Medicine, University of Missouri, Columbia, MO

Ticks are blood-feeding ectoparasites known to spread diseases to people and other vertebrate hosts. Ticks are traditionally controlled with chemical acaricides, but resistance to these products is a concern. There is a need to develop new ways to control tick vectors. Previous studies have shown that immunization of cattle with D. andersoni MG or SG tissues im- pacted tick performance, with tick salivary gland tissues greatly reducing feeding performance of female D. andersoni. In order to identify protein antigens reactive with protective immune sera, 2-D electrophoresis and Western blot analysis localized uniquely seroreactive regions for shotgun protein sequencing with a Bruker TIMS QTOF mass spectrometer. Bioinformatic analysis of 258 sequences led to five proteins for preliminary evaluation of this forward-proteomics ap- proach. The objective of this study was to overexpress and test recombinant forms of these five proteins for seroreactivity to SG-immunized and MG-immunized bovine sera. Coding sequences for these five proteins were inserted into pET28 transformed into Escherichia coli strain BL21 DE3. Overexpression was optimized for each clone which included inducer concentration (IPTG), induction time and induction temperature. Western blot analysis of the overexpressed recombinant proteins is underway to identify a candidate(s) that is uniquely reactive with immunoglobulins from protected cattle. This work is expected to provide proof of concept for adaptation of this bovine model system for development of a novel strat- egy to mitigate tick-borne diseases of cattle, domestic animals and people.

Research Grant: USDA National Institute of Food and Agriculture Award 17-67015-26630 Student Support: Stipend for Kyle Cacciatore is supported by an endowment established by IDEXX-BioAnalytics

2021 National Veterinary Scholars Symposium • 83 Glial cell inhibitor fluoroacetate suppresses enteric glial activity marker GFAP in ischemia-injured jejunum

Madison Caldwell, Laurianne Van Landeghem, Anthony Blikslager, Amanda Ziegler

Department of Clinical Sciences (Caldwell, Blikslager, Ziegler), Department of Molecular Biomedical Sciences (Van Landeghem), NC State University College of Veterinary Medicine, Raleigh, NC.

Ischemic injury to the intestinal epithelial barrier is associated with clinically relevant diseases such as neonatal necrotiz- ing enterocolitis in which mortality rates remain overwhelmingly high. The enteric glial network regulates function and repair of this epithelial barrier via paracrine signaling. In our pig ischemia-repair model, 30 minutes of ischemia leads to changes in the enteric glial network illustrated by an increase in the glial activity marker glial fibrillary acidic protein (GFAP). Also using this model, the Krebs cycle blocker fluoroacetate (FA), to which glia are purported to be preferentially sensitive, results in an inhibition of epithelial restitution. We hypothesized that treatment with FA leads to alterations in the enteric glial network mirrored by changes in expression of GFAP. To determine this, ischemia-injured jejunal tissue of juvenile Yorkshire cross pigs which had been exposed to 500mM FA during ex vivo recovery were stained for GFAP using immunofluorescence. GFAP intensity in submucosal ganglia was quantified by corrected total cell fluorescence. Results show that treatment with FA significantly decreased GFAP expression (p = 0.0215) in tissues exposed to 30 minutes of ischemia (432621 6 544777 RFU) as compared to controls (863363 6 876665 RFU). Suppression of ischemia-driven increases in GFAP in submucosal enteric glia by FA suggests that the enteric glial network is altered in tissues which had failed epithelial restitution. This may indicate an integral role for the enteric glial network in acute epithelial restitution after ischemic injury.

Research Grant: NIH K01 OD 028207, NIH P30 DK 034987, NIH-NICHD R01 HD095876, USDA-NIFA VMCG-0065 Student Support: NIH T35 Training Grant 5 T35 OD 11070-12

Prognostic significance of CD204+ tumor-associated macrophages in canine cutaneous melanocytic neoplasms

Jessica Callisher, Sylvia Ferguson

Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Melanocytic neoplasia is common in dogs and occurs in the oral mucosa and skin. Tumor-associated macrophages (TAMs) are known to promote angiogenesis and cancer cell proliferation in various canine tumor types, such as oral melanomas, mammary carcinomas, and lung adenocarcinomas. To date, the presence or role of TAMs in the oncogenesis of canine cutaneous melanocytic tumors has not been investigated. TAMs can be subdivided into M1 and M2 phenotypes; M2 are considered most critical for tumor progression. Immunohistochemical expression of CD204, a polarized M2-mac- rophage receptor, is used to identify these M2-TAMs. High densities of CD204+ TAMs have been significantly correlated with negative prognostic factors, like tumor stage, lymphovascular invasion/metastasis, and relapse. The objective of the current study was to determine the presence and quantity of CD204+ TAMs in 10 canine cutaneous melanocytic neo- plasms and investigate their correlation with known histologic indicators of malignancy. We hypothesized that CD204+ TAM quantities would positively correlate with these known negative prognostic indicators and be a useful prognostic factor. CD204+ TAMs were quantified in all 10 cutaneous melanocytic neoplasms, in addition to determining mitotic counts and nuclear pleomorphism. Statistical analyses did not identify a significant correlation between CD204+TAM quantity and neoplastic mitotic counts or nuclear pleomorphism. These preliminary results indicate that CD204+ TAM quantities are not a reliable prognostic factor in cutaneous melanocytic neoplasms; however, given the low number of cases evaluated, continued prospective recruitment of cases is warranted.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

84 • 2021 National Veterinary Scholars Symposium Pharmacokinetics and efficacy of high and low dose meloxicam in mice

Brinley Cannon, Layne Freeman, Sarah Tan, Jeffrey Kim, Lon Kendall

College of Veterinary Medicine, Texas A&M University (Cannon), Colorado State University (Cannon, Freeman, Tan, Kim, Kendall)

Despite the mouse being the most commonly used animal in research, analgesic protocols are not well established. Thus, an effective pain management and dosing parameter are needed to ensure the wellbeing of laboratory mice. Meloxicam is a non-steroidal anti-inflammatory drug (NSAID) widely used in veterinary medicine to relieve surgical pain and inflam- mation. In this study, we evaluated both the duration of action and efficacy of 10 mg/kg and 20 mg/kg meloxicam in mice through pre-surgical administration and pharmacokinetic analysis. To assess the efficacy of these dosages, an experimental ovariectomy via ventral laparotomy was performed on female CD1 mice. Mice were administered either meloxicam at 10 mg/kg (n = 2) or meloxicam at 20mg/kg (n = 4) preoperative and were re-administered at 12, 24, and 36 h after surgery. Postoperative pain was accessed through behavioral analysis measured by nest building, rearing, orbital tightness, wound licking, grooming, arched posture, and activity 3, 6, 12, 24, and 48 h postop. Through pharmacokinetic analysis, the 10 mg/kg meloxicam’s half-life, time to peak plasma concentration and maximum concentration were found to be 5 h, 1 h, and 28.5 mg/mL respectively. For 20 mg/kg meloxicam these values were found to be 4 h, 30 min, and 54.9 mg/mL. Two mice treated with meloxicam (20 mg/kg) demonstrated profound behavioral effects of pain, resulting in unexpected death and euthanasia. Necropsy of both mice revealed gastrointestinal perforation indicating an adverse reaction to the high dose meloxicam. Overall, this suggests that 20 mg/kg meloxicam is not a suitable dose of analgesic for murine studies due to its ability to cause NSAID toxicity.

Research Grant: None Student Support: ASLAP Foundation

Canine osteosarcoma, a spontaneous large animal model for solid tumor CAR T cell therapy

Jennifer W. Cao, Jessica Lake, Lyndah Chow, Jade Kurihara, Renata Impastato, Dylan Ammons, Ashley Yingst, Michael Verneris, Steven Dow

Department of Microbiology, Immunology and Pathology (Cao, Ammons, Dow) and Department of Clinical Sciences (Chow, Kurihara, Impastato), College of Veterinary Medicine, Colorado State University, Fort Collins, CO; Department of Hematology/Oncology/BMT(Lake, Yingst, Verneris) University of Colorado, Aurora, CO

Chimeric Antigen Receptor (CAR) T cell therapy allows for the targeting of a specific surface antigen to generate an adap- tive immune response. CD19 and CD20 CAR T cells have shown miraculous clinical success in treating advanced and relapse B cell malignancies. Application of CAR T cell therapy to solid tumors have not shown the same clinical success, despite the identification of promising tumor antigens and successful preclinical data.The standard preclinical animal model utilizes the NOD SCID-gamma mouse strain that is severely immune deficient, which allows for human tumor xe- nografts and infusion of human CAR T cells. However, lack of myeloid lineage cells does not model the interaction of the tumor microenvironment (TME) on cellular antitumoral immunity, which is proposed as a major factor in the gap in trans- lation from benchtop to clinical success. Tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) that reside within the TME through cell signaling and secreted cytokines dampen antitumoral cellular response. Osteosarcoma (OS) is an aggressive solid tumor that occurs in high prevalence in both adolescent children and large breed dogs. The checkpoint molecule B7-H3 has been identified in human (OS) as a marker correlated with poor prognosis, increased metastasis and decreased tumor infiltrating lymphocytes.This study aims to develop canine OS as a solid tumor model for pediatric OS to evaluate the role of TAMs and MDSCs on CAR T cell efficacy targeting B7-H3. We found that B7-H3 was over expressed in primary canine OS while normal liver and spleen had low to no expression of B7-H3. Level of B7-H3 correlated to canine B7-H3 CAR T cells activation as measured by secreted interferon gamma.

Research Grant: V foundation Grant Student Support: Unknown

2021 National Veterinary Scholars Symposium • 85 Clostridioides difficile in horses hospitalized with colic: nosocomial pathogen or innocent bystander?

Kyle Cappo, Laurel Redding, Louise Southwood, Eman Anis, Donna Kelly, and Amanda Barnard

Department of Clinical Studies (Redding, Southwood), Department of Pathobiology (Anis, Barnard), College of Veterinary Medicine (Cappo), University of Pennsylvania, Philadelphia, PA

Clostridioides difficile is a bacterial pathogen that can cause acute enterocolitis and has up to a 42% mortality rate in hors- es. Antibiotic treatment and the subsequent dysbiosis can increase the risk of C. difficile associated diarrhea. Other data suggests that hospitalization outside of antimicrobial administration can be a risk factor for C. difficileinfection. There has yet to be definitive genetic link betweenC. difficile from the hospital environment and the isolates from hospitalized horses. This study takes fecal isolates from patients undergoing colic treatment and records their medical history such as signalment, diagnosis, and medications. The isolates are collected upon admission and discharge and are being tested with multiplex and duplex qPCR that selects for the genes coding A & B toxins. Environmental samples were collected from several surfaces and floors from the hospital, and then tested with 16s qPCR due to its high sensitivity. Upon any positive results, the environmental samples will also be tested with the same multiplex and duplex qPCR. Fecal isolates and envi- ronmental positive results will be cultured, and the cultured C. difficile is genetically sequenced. All samples are collected and currently undergoing testing. This data will provide information about C. difficile presence in horses upon admission and discharge, the genetic relatedness from pathogenic C. difficile horse isolates and the environmental isolates, and assess which factors could contribute to initial contraction of C. difficile.

Research Grant: NIH T35 Grant Student Support: NIH T35 OD010919

CollagenV Haploinsufficiency Results in Delayed Healing and Altered Wound Matrix Post-Injury in Murine Tendons

Jaclyn A Carlson, Mei Sun, Sheila M Adams, Stephanie N Weiss, David E Birk, Louis J Soslowsky

McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA, (Carlson, Weiss, Soslowsky) and Morsani College of Medicine, University of South Florida, Tampa, FL (Sun, Adams, Birk)

Patients with Classic Ehlers-Danlos syndrome (cEDS) suffer from tendon/ligament fragility and abnormal wound healing. Collagen V (ColV) haploinsufficiency leads to abnormal tissue development and altered collagen assembly, and mechan- ical loading of the mouse patellar tendon shows a delay in healing and alterations in mechanical properties post-injury (PI). Therefore, the objective of this study was to determine the effect of ColV deficiency in female mice on wound matrix formation and resultant structure-function relationships when mechanical load is applied PI. We hypothesized that ColV deficiency will have effects PI, resulting in increased fibril diameter and cellularity, decreased mechanical properties and leading to a delayed healing response when compared to WT tendons. Col5a1 expression was significantly increased in WT tendons at 1- and 3w PI compared to uninjured controls, with no significant changes inCol5a1 expression seen PI in Col5a1+/- tendons. TEM analysis showed Col5a1+/- fibrils PI were larger and more broadly distributed than WT fibrils. Further, mechanical testing showed Col5a1+/- tendons continuing to realign longer than WT tendons. Lastly, Col5a1+/- ten- dons had a significant increase in cellularity persisting to 6w PI when compared to uninjured tendons, that was not seen in WT tendons. Additionally, increased cellularity in Col5a1+/- tendons alters the matrix alignment and architecture. This study indicates that the lack of an early increase in Col5a1 expression PI in Col5a1+/- tendons influences matrix architec- ture, alignment, and cellularity throughout tendon healing, demonstrating altered and delayed healing compared to WT tendons.

Research Grant: This study was supported by NIH/NIAMS AR065995 and the Penn Center for Musculoskeletal Disorders (AR069619) Student Support: Unknown

86 • 2021 National Veterinary Scholars Symposium The effects of intravenous opioids on leukocyte oxidative burst and phagocytosis in dogs

Imani M. Carswell, Heather Perkins, Jared A. Jaffey, and Anderson da Cunha

Department of Specialty Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Methadone is a schedule II synthetic opioid that is commonly used for sedation or pain management in canines. Opioids have been shown in various species to alter immune function. Canine studies performed in vitro and ex vivo have had conflicting results regarding the immunomodulatory effects of opioids. However, methadone has not been evaluated in any canine studies. This lack of information, combined with the common use of methadone in dogs, necessitates a bet- ter understanding of methadone’s immunomodulatory effects. Therefore, the objective of our study was to determine if intravenous methadone administration in dogs affects granulocyte/monocyte oxidative burst or phagocytic capacities.Ten dogs were randomized to receive either methadone or placebo (0.9% NaCl), followed by the opposite intervention 7 days later. Each intervention was administered intravenously once every 6 hours for 24 hours (4 total doses). Phlebotomy was performed for immunologic testing at baseline (before intervention) and then 10 min, 6 hours, and 24 hours after interven- tion administration. Granulocyte/monocyte phagocytic function and oxidative burst was analyzed using flow cytometry.

Research Grant: Midwestern University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program

Demographics of community cats presented to a veterinary clinic over a ten-year span

Abigail Cashel, Emily McCobb

Center for Animals and Public Policy, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA.

Overpopulation of unowned, free-roaming community cats detrimentally impacts the environment, animal welfare, and public health, sparking a need for human intervention. Trap-Neuter-Return (TNR), a method where community cats are trapped, sterilized, and returned to the trapping site, currently serves as the most humane and effective method of popula- tion control. However, most studies view TNR through the lens of community cat colonies, failing to consider impacts on clinic operations. Before a standardized model of United States community cat population demographics can be devel- oped from empirical evidence, a primary model of characteristics of cats presenting for TNR throughout the northeastern United States is essential. A primary model would allow TNR programs in this region to more efficiently allocate limit- ed resources to address overpopulation demands. This exploratory study examined patient-level clinical parameters in community cats presented to the Tufts Luke and Lily Lerner Spay/Neuter Clinic in North Grafton, MA between 2009 and 2019. Existing data from over 4000 cats was extracted from the clinic’s electronic database to analyze trends in factors that could affect the time, expertise, and medical supplies needed forTNR. These parameters included sex, estimated age, prior sterilization, rates of euthanasia on intake, cryptorchidism, adoption placements, intraoperative complications, prenatal litter size, and the seasonality of estrus, lactation, and pregnancy. Geographical origin of trapping sites was also traced over time. The hypothesis was that these values would display variation throughout the ten-year period and that they would differ from previously reported findings from other United States regions.

Research Grant: Research Grant: Cummings School of Veterinary Medicine at Tufts University Student Support: Student Support: Tufts Summer Research Training Program

2021 National Veterinary Scholars Symposium • 87 Autologous biologic therapies for treatment of full thickness cutaneous wounds in horses

Nicole Cassale, Nora Springer, Charan Ganta, Dylan Lutter

Department of Clinical Sciences and Anatomy and Physiology (Cassale, Lutter), Department of Diagnostic Medicine/ Pathobiology (Springer, Ganta), College of Veterinary Medicine, Kansas State University

Full thickness cutaneous wounds are a leading cause of morbidity in horses in the United States. Horses have delayed in- flammatory responses, especially on the distal limb, leading to improper wound healing. Previous studies have shown that autologous biologic therapies such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are inexpensive treatments that may be critical to wound healing and preventing chronic wounds. We hypothesized that autologous mesenchymal stem cells (aMSC) in combination with PRF and aspirate would enhance wound healing in horses compared to untreat- ed wounds. In this randomized controlled experimental study, twelve full thickness cutaneous wounds, 2.5 cm x 2.5 cm, were created on six horses (six distal limb wounds and six gluteal wounds). Wounds were randomly treated with PRP, PRF, aMSC, PRF+aspirate, PRF+aMSC+aspirate, and an untreated control. Wounds were then sampled via 6 mm punch biopsy day 0, 7, 14, 21, and 28 and the tissue was prepared for histopathology evaluation and RT-qPCR gene expression of HPRT1, COL1A1, COL3A1, and TGF-b, a housekeeping gene and three major genes involved in the healing process, respectively. Histopathologic evaluation of the wounds showed no significant difference in epithelialization, fibrosis, or- in flammation between control and all treatment groups. Although histology results did not support the hypothesis that autol- ogous biologic therapies would improve wound healing, the results from RT-qPCR gene expression analysis are pending.

Research Grant: Grant Support: Kansas State University College of Veterinary Medicine Mentored Clinical, Applied, or Translational (MCAT) Research Award Student Support: Student Support: Midwest Institute for Comparative Stem Cell Biology

Antibiotic resistance in dairy cattle: a foe, or no? A survey-based study of dairy farmers

Eleni Casseri, Sebastian Llanos Soto, Ece Bulut, Michelle Wemette, Alison Stout, Amelia Greiner Safi, Robert Lynch, Paolo Moroni, Renata Ivanek

Department of Population Medicine and Diagnostic Sciences (Casseri, Soto, Bulut, Wemette, Stout, Safi, Moroni, Ivanek) College of Veterinary Medicine, Cornell University, Ithaca NY; Department of Communication (Safi) and Department of Animal Science (Lynch) College of Agriculture and Life Sciences, Cornell University, Ithaca, NY

Antibiotic use is an important component in herd protocols to treat common bacterial diseases and keep up the welfare standards on dairy farms. However, antibiotic misuse and/or overuse by farmers might promote the emergence of resistant pathogens, an issue that can increase the cost and decrease the effectiveness of treatments.We conducted a cross-sectional web-based questionnaire study to evaluate the attitudes towards antibiotic resistance and the antibiotic use habits of dairy farmers and managers in the northeastern United States (US). A total of 118 responses were subjected to statistical anal- ysis of factors associated with the levels of concern about antimicrobial resistance and interest in adopting new farming practices. Preliminary findings show that nearly half of the dairy farmers are undecided or disagree with the statement “Antibiotic resistance due to antibiotic use in may negatively impact dairy cattle”. In contrast, the majority of farmers do not treat with antibiotics at the first sign of mastitis and have either fully or partially implemented or in- tend to implement culture-based mastitis treatment on their farm. The results suggest that judicious use of antibiotics is a widespread practice of dairy farmers, despite the majority being un-concerned about antibiotic resistance as a threat to cattle. No significant associations were found between various demographic groups within the study population and farm- er survey responses. The results highlight the need to further investigate the northeastern US farmer motivations for their appropriate antibiotic use and adopting culture-based testing, as no clear motivating factor was identified, to help promote judicious antibiotic use in livestock.

Research Grant: USDA-NIFA Hatch grant # 1014331 Student Support: NIH T35 OD010941, Cornell University College of Veterinary Medicine

88 • 2021 National Veterinary Scholars Symposium Prenatal Oxycodone Exposure and Pup Vocalizations

Jillian F. Celatka, Kerri E. Budge, Sara B. Isgate, Katie R. Fleming, Fair M. Vassoler, Elizabeth M. Byrnes

Department of Comparative Biology (Celatka, Budge. Isgate, Fleming, Vassoler, Byrnes), Cummings School of Veterinary Medicine, Tufts University, Grafton, MA.

The use of prescription opioids has increased over the past decade, resulting in a rise of infants exposed in utero, many of whom experience neonatal opioid withdrawal syndrome (NOWS). There is a gap in our understanding of what factors influences NOWS severity and what symptoms are associated with long-term outcomes. Previous findings report both increased ultrasonic vocalizations (USVs) in pups and disrupted maternal behavior in rat dams who self-administer (SA) oxycodone throughout pregnancy. The current study uses a cross-fostering design to determine whether effects on USV quantity or quality are due to direct fetal exposure to oxycodone or to changes in maternal behavior due to withdrawal in the dam. Female Sprague-Dawley rats were surgically implanted with a jugular catheter and trained to self-administer oxycodone (0.1 mg/kg/infusion) for 3 weeks during daily operant conditioning sessions prior to mating. Once females were pregnant, they had daily access to oxycodone for 6h/day. Oxycodone access ended on the day of parturition to induce postpartum withdrawal. On postnatal day 1 (PND1) litters were culled to 4 males and 4 females and cross-fostered with litters from time-mated drug naive donor rats. Pup USVs and maternal behaviors were studied on PND3, 6, 9, and 12. Initial data suggest that oxycodone SA dams have impaired maternal retrieval. Additional analyses will determine whether these maternal effects impact pup USVs and the extent that the amount of oxycodone self-administered during pregnancy correlates with postnatal outcomes independent of maternal effects.These data will be used to dissociate direct effects of oxycodone on pup neurodevelopment from those associated with altered maternal care.

Research Grant: National Institute on Drug Abuse R01 DA049531 Student Support: Cummings School of Veterinary Medicine Office of Research and Graduate Education

In vitro cardioprotective effects of epigallocatechin-3-gallate

Alexandra Cerrato, Dhirendra Singh, Dalia Badamo, Rashmi Pathak, Joseph Francis

Comparative Biological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Myocardial infarction (MI) occurs when blood flow to the heart ceases, leading to necrosis and eventual death of the cardiac muscle. Reperfusion of the heart is the first line of treatment to bring blood flow back to the once deprived cardiac muscle, but also causes additional damage as well. Immediate and long-lasting damage to the heart is caused by reactive oxygen species (ROS) and iron overload within the cardiac cells leading to lipid peroxidation and more ROS production. Epigallocatechin 3-gallate (EGCG) is a known antioxidant in green tea with iron chelating properties. The objective of the study was to determine the cardioprotective effects of EGCG from the long-term damage caused by hemin.We hypothe- sized that EGCG will provide an antioxidant effect as well as chelate iron, thus protecting the cardiac cells from additional cellular damage. To test this hypothesis, we used H9C2 myoblast cardiac cells from the rat species Rattus norvegicus and treated them with hemin and EGCG in various combinations. The treated H9C2 myoblast cardiac cells were processed via western blot to determine ROS-related protein production, as well as with electron paramagnetic resonance (EPR) to quantify ROS production, and with an iron affinity assay to determine EGCG’s iron chelating ability. In this study, we demonstrated that epigallocatechin 3-gallate (EGCG) is a scavenger of ROS as well as an iron chelator, ameliorating the iron induced ROS damage in H9C2 cardiac myoblasts.

Research Grant: NIH 1P20 GM135000-01A1 and NIH 1R01-HL147133-01 Student Support: NIH Biomedical Research Experience for Veterinary Students Grant 5T35OD011151-18

2021 National Veterinary Scholars Symposium • 89 Platelet-endothelial associations are dependent on CD40/CD40L interactions in the context of EcoHIV

Alyssa L. Chalmin, Yeji Cho, Claire E. Lyons, Kelly A. Metcalf Pate

Division of Comparative Medicine (Chalmin, Cho, Lyons, Metcalf Pate), Massachusetts Institute of Technology, Cambridge, MA. University of Pennsylvania School of Veterinary Medicine (Chalmin), Philadelphia, PA.

Currently, 38 million people globally are living with HIV infection and latent viral reservoirs create a barrier to establish- ing a cure. EcoHIV is a chimeric HIV that uses mouse-specific receptors to infect mouse cells using the same mechanisms as found in human HIV pathophysiology. HIV forms a latent reservoir in the brain very early in infection and SIV-infected macaque models of HIV have shown the formation of platelet-endothelial associations (PEAs) in the microvasculature of the brain. Previous experiments have suggested that platelets play a protective role against increased vascular permeability in the brain through PEAs. HIV infection is associated with moderate thrombocytopenia which may decrease the abili- ty of platelets to form these protective associations. PEAs in HIV are predicted to be mediated by interactions between CD40 receptor on endothelial cells and CD40 ligand on platelets, mirroring what is found in cerebral malaria. We utilized CD40-knockout mice to determine whether CD40 was essential for PEA formation in the context of EcoHIV infection. Brain sections from EcoHIV-infected and mock inoculated CD40-knockout and wild-type C57BL/6J mice were stained with anti-CD41 antibody to identify platelets associated with blood vessels and are currently being analyzed. We predict that mice lacking the CD40 receptor will show decreased PEA formation in brain tissue compared to wild-type mice.

Research Grant: NIH R03 OD026691 Student Support: None

The prevalence of endoparasites of the Cuban treefrog and Greenhouse frog in Florida

Ellis Chase, Kelsey Lykins, Robert Ossiboff, Steven Johnson, Terry Farrell, Nina Thompson, Heather Walden

Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL (Chase, Lykins, Ossiboff, Thompson, Walden), Wildlife Ecology and Conservation, University of Florida, Gainesville, FL (Johnson), Biology Department, Stetson University, Deland, FL (Farrell)

Many human pathogens are a result of zoonotic origin, having been spread from organisms encountered in everyday life. One such organism is Angiostrongylus cantonensis, also known as rat lungworm (RLW). RLW can infect humans and mi- grate to the brain, causing eosinophilic meningitis. A major definitive host of RLW is rats,Rattus rattus, and snails play a significant role in its transmission as intermediate hosts. Previous research has determined RLW to be present throughout Florida within rats and snails; however, few studies have been conducted on which animals are possible paratenic hosts for this parasite. The aim of this study is to discover the prevalence of RLW within the non-native Cuban treefrog, Osteo- pilus septentrionalis, and greenhouse frog, Eleutherodactylus planirostris. These frogs share habitats with snails in areas previously shown to have the highest prevalence of A. cantonensis, which are also highly populated by humans. In this study, 200 live Cuban treefrogs (N = 100) and greenhouse frogs (N = 100) were collected throughout Florida in the coun- ties of Alachua, Hillsborough, Orange, Miami-Dade, St. Johns, and Volusia. These frogs were combined with a collection of previously euthanized and preserved Cuban treefrogs (N = 94) from Alachua County. Necropsies of each frog were performed and morphological identification of parasites, along with PCR of parasites and tissues, used to determine the prevalence of RLW, and other parasite species. It was hypothesized RLW would be found in up to 50% of frogs sampled, with the highest infection rates in south and central Florida. Additional parasites have been recovered, some considered invasive and threatening to native wildlife species. This research is still ongoing.

Research Grant: University of Florida College of Veterinary Medicine and University of Florida Unique Opportunity Grant Student Support: University of Florida College of Veterinary Medicine

90 • 2021 National Veterinary Scholars Symposium Quantitative MRI measures of the biochemical and biomechanical properties of canine intervertebral discs

Kayla L. Chase, Erick Buko, Alexandra R. Armstrong, Susan A. Arnold, Elizabeth W. Bradley, Arin M. Ellingson, Casey P. Johnson

Department of Veterinary Clinical Sciences (Chase, Buko, Armstrong, Arnold, Johnson), College of Veterinary Medicine, University of Minnesota, Saint Paul, MN; Department of Orthopedic Surgery (Bradley) and Department of Rehabilitation Medicine (Ellingson), Medical School, University of Minnesota, Minneapolis, MN

Intervertebral disc degeneration (IVDD) is a debilitating condition affecting the canine spine, often resulting in , paraparesis/paraplegia, and even death. Clinically, T1- and T2-weighted magnetic resonance imaging (MRI) is the leading modality to diagnose disc herniation and dictate surgical treatment. However, these conventional MRI methods have limited ability to assess the health of non-herniated discs and their risk of future herniation. The goal of our project is to establish noninvasive, quantitative MRI measures of the biochemical and biomechanical properties of canine interver- tebral discs to assess their health. We hypothesize that quantitative MRI measures (T2*, T2, T1ρ, and ADC) in the inter- vertebral disc will provide noninvasive, correlative measures of key disc physical properties including glycosaminoglycan (GAG), collagen, and water content and disc pressure. Vertebral column segments (T10 to S1) from donated dog bodies will be assessed ex vivo. In this study, we report on our initial experience and current procedures to evaluate our initial specimens. Our first specimen exhibited a range of quantitative MRI values across the discs at 3T MRI. Biochemical anal- ysis will assess the GAG, collagen, and water content of four separate locations on each disc (nucleus pulposus and later- al, dorsal, and ventral annulus fibrosus) spatially matched to the quantitative MRI maps.We will also obtain histological sections of each disc. We are continuing to develop procedures for biomechanical assessment of the discs given challenges posed by their small size. This work is an initial step toward the validation of new, noninvasive imaging biomarkers to advance clinical management and treatment of canine IVDD.

Research Grant: College of Veterinary Medicine Signature Program Grant; Medtronic; and NIH P41 EB027061 Student Support: NIH Training Grant T35 OD011118

A retrospective study of absence seizures in canines

Emma Chawner, Masayasu Ukai, William Sears, Fiona James

Department of Clinical Studies (Chawner, James), Health Sciences Centre (Ukai), Department of Population Medicine (Sears), Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Seizures are the most common neurological condition diagnosed in dogs. Absence seizures are a type of generalized onset seizure that may include twitches (myoclonus). Absence seizures can only distinguished from focal seizures based on brain activity seen on a diagnostic test known as electroencephalography (EEG). Agreement on their identification by veterinarians is minimal based on visual assessment alone. With unknown prevalence in canines a retrospective study evaluated the distribution of seizure types presented to the Ontario Veterinary College Companion Animal Hospital over four years (May 2017-April 2021). A total of 528 cases were included via a medical record search for dogs with epilepsy and/or seizures presented to the neurology or emergency services. Cases were manually categorized into seizure types based on reported clinical signs. Each year, 53%-63% of seizure cases were described as generalized tonic clonic sei- zures (GTCS), 9%-15% GTCS with additional atypical events and 29%-35% suspected focal or absence seizures without GTCS. EEG confirmed absence seizures in 12 of 44 EEGs, 5 cases having a history of GTCS and 7 without prior GTCS event. This preliminary study suggests absence seizures may be relatively common as one third of seizure cases in a refer- ral population may present with clinical signs not indicative of GTCS. Prospective studies using EEG analysis are merited to further evaluate the prevalence of absence seizures in dogs. Acknowledging the impact of these seizures will improve awareness, increase diagnostics, and support potential future exploration for effective treatments.

Research Grant: Ontario Veterinary College Pet Trust Fund (#054488) Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 91 Determining cardiomyopathies and cardiac disease via Vertebral Heart Score in a large population of rabbits

Katherine R Chen, Emily Hsieh, Jin Yu, Sari R Kanfer, Curtis Eng

Exotic Animal Veterinary Care Center, Pasadena, CA

Rabbits are one of the most popular pets owned in the United States. As we improve their care and the average lifespan increases, we are seeing increased cases of cardiac disease. The vertebral heart scoring (VHS) system is a technique which compares cardiac dimension and body length measurements on a radiograph. These numbers can then effectually be used to derive a score of normal heart size or cardiomegaly. While VHS has been widely applied to many species to measure heart size, domestic rabbits have minimal representation in this type of research. This study is to investigate whether there is a statistical difference in the VHS of healthy, clinically normal, rabbits compared to the VHS of rabbits with non-de- script cardiac disease. To assess the measure of cardiac disease in rabbits, the VHS values were gathered on a lateral and ventrodorsal radiograph from each patient. Additionally, we measured the cardiac and lung silhouette size, age, weight, body condition score, sex and breed. Our findings reveal a statistically significant difference VHSin of healthy, clinically normal rabbits compared to the VHS of rabbits with non-descript cardiac disease. Similarly, the cardiac silhouette size of healthy, clinically normal, rabbits was smaller compared to that of cardiac disease rabbits. Finally, our study suggests that a ventrodorsal radiograph may be the best and most accurate to derive a vertebral heart score in rabbits. In its entirety, the findings derived from the data we have gathered will be able to assist a practitioner in patient diagnosis and treatment. In addition, with such a large data set, we will also open up more doors for future cardiological research.

Research Grant: None Student Support: WesternU of Health Sciences, College of Veterinary Medicine Research Advisory Committee

ThrombinatorTM thrombin fibrin sealant retains BioCartilage in congruent, high-load cartilage defects

Jacqueline Chevalier, Marta Cercone, Lisa A. Fortier

Department of Clinical Science, College of Veterinary Medicine, Cornell University, Ithaca NY

Treatment of articular cartilage defects with biologic scaffold adjuncts has promising clinical outcomes. It is unknown if a fibrin sealant is necessary to retain the scaffold, particularly in highly congruent articulations.We hypothesized that a BioCartilage graft requires a fibrin sealant for retention in a non-load bearing articular surface, but is unnecessary in congruent, load bearing regions. Proximal (congruent, high-load) and distal (low-load) 10mm diameter, full-thickness cartilage defects were made in the lateral trochlear ridge of each femur of 6 horses. Defects were filled with BioCartilage preparation, and limb defects were left unsealed (control) vs sealed with platelet poor plasma and Thrombinator throm- bin fibrin clot. After 3 weeks, horses were euthanized and scored grossly for defect coverage and surrounding cartilage fibrillations with India ink. Percent-fill was measured with microcomputed tomographym ( CT). Outcome measures were compared within the same horse (high vs low-load; control vs sealed) using paired t-tests. There was no difference in India ink scoring between high and low-load (P = 0.4, n = 3 control; P = 0.7, n = 4 sealed) or between control and sealed (P = 0.5, n = 9 defect pairs). Cartilage coverage of the defect revealed no difference between high and low-load (P = 0.4, n = 3 control; P = 0.6, n = 4 sealed) or between control and sealed (P = 0.2, n = 9 defect pairs). mCT showed within the sealed defects, there was significantly more fill in high compared to low-load defects (P = 0.02, n = 4 limbs). Within high-load, there was a trend towards increased fill in sealed compared to control (P = 0.09, n = 4 horses). In conclusion, a fibrin seal retains BioCartilage in high-load areas but is not required in low-load areas.

Research Grant: Supported by Arthrex Inc. Student Support: Cornell College of Veterinary Medicine AVMA/AVMF 2nd Opportunity Research Scholarship

92 • 2021 National Veterinary Scholars Symposium Gestational sleep apnea induces autism-relevant behavioral and neuronal aberrations in the offspring

Jacqueline Chi, Amanda M. Vanderplow, Tracy L. Baker, Jyoti J. Watters, and Michael E. Cahill

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, WI

Sleep disordered breathing (SDB) is characterized by recurring breathing cessations during sleep and causes intermittent oxygen deprivation (hypoxia). By the third trimester, obstructive sleep apnea, the most common form of SDB, is prevalent in 10-32% of all pregnant women. Complications known to result from gestational SDB are known risk factors for the de- velopment of neurodevelopmental disorders, in particular autism spectrum disorder (ASD). ASD is a neurodevelopmental disorder characterized by cognitive and social impairments, affecting 1 in 59 children in the USA.While the exact cause of ASD remains unclear, studies implicate an interplay between genetic and environmental risk factors. The present study investigates the potential involvement of maternal SDB during pregnancy as a risk for the development of ASD. We posit that gestational SDB results in the dysregulation of dendritic spines, the sites of most excitatory connections in the brain, thereby predisposing offspring to develop behavioral phenotypes relevant toASD. To investigate the consequences of gestational SDB in offspring, we subjected pregnant rat dams to chronic intermittent hypoxia or normoxia (GIH and GNX, respectively). Previous findings indicate that male GIH offspring exhibit several behavioral deficits in conjunction with an increase in dendritic spine density in the medial prefrontal cortex. In order to further delineate the breadth of the behav- ioral and mechanistic aberrations in GIH offspring, ongoing studies are aimed at further defining the behavioral deficits via a reversal learning test, by assessing dendritic spine morphology and density in the hippocampal CA1 region, and by investigating specific synaptic signaling pathway alterations.

Research Grant: Brain & Behavior Research Foundation and the National Institute of Mental Health Student Support: Boehringer Ingelheim and NIH T35 OD011078

Optimizing gene editing approaches in Doberman Pinscher fibroblasts by targeting PDK4 and DCM

Cristina F. Chinchayan, Chris L. Souders, Amara Estrada, Christina Pacak, Chris Vulpe, Chris Martyniuk

Department of Physiological Sciences, University of Florida College of Veterinary Medicine, Gainesville, Florida (Chinchayan, Souders, Martyniuk, Vulpe), Small Animal Clinical Sciences, UF CVM (Estrada), University of Minnesota, Minneapolis, Minnesota (Pacak)

Dilated cardiomyopathy (DCM) is a cardiac condition observed in humans and large breed canines. Risk factors including age, diet and genetic mutations are linked to the condition. This study focuses on pyruvate dehydrogenase kinase (PDK), a mitochondrial protein involved in cardiac metabolism, and the 16-base pair deletion in Pyruvate Dehydrogenase Kinase 4 (PDK4). Previous studies performed on dermal fibroblast have observed homozygous (PDK4del/del) and heterozygous (PDK4wt/del) candidates with impaired pyruvate dehydrogenase complex activity in glucose starved conditions, leading to inadequate energy production and cardiac dysfunction. Current treatment options include pharmacological interven- tion yielding low survival rates with no permanent remedy; therefore, we are pursuing gene editing as a viable treatment option. The objectives of this study were to verify gene mutations through gene sequencing of Doberman Pinscher fibro- blast cell lines carrying mutations. We also aimed to characterize the metabolic differences between genotypes using the XFe24Flux Analyzer and conducting a glycolytic stress test. We hypothesize the mutant cells will differ phenotypically for oxidative phosphorylation and by editing these genes, metabolic function should improve. Data comparing genotypes are currently under way and guide RNAs for gene targeting via CRISPR editing are being developed. This study contribution is important to the development of CRISPR techniques and future therapies to resolve DCM in Doberman Pinschers.

Research Grant: Morris Animal Foundation (MAF), and the UF College of Veterinary Medicine, University of Florida. Student Support: Boehringer Ingelheim Veterinary Scholar Program, Florida Veterinary Scholar Program UFCVM

2021 National Veterinary Scholars Symposium • 93 Differential effects of acetaminophen-induced cytotoxicity on fibrinogen expression by mouse hepatocytes

Alexandra Chiusano, Dafna Groeneveld, Lauren G. Poole, Holly M. Cline-Fedewa, Zimu C. Wei, Anna-Katherine Fournier, James P. Luyendyk

College of Veterinary Medicine, Michigan State Uni., E Lansing, MI. (Chiusano), Department of Pathobiology and Diagnostic Investigation, Michigan State Uni., E Lansing, MI. (Groeneveld, Poole, Cline-Fedewa, Wei, Fournier, Luyendyk), Department of Pharmacology and Toxicology, Michigan State Uni., E Lansing, MI. (Luyendyk)

Acetaminophen (APAP) is an over-the-counter medication for which overdose is a leading cause of acute liver injury and liver failure. Prior studies have shown that the blood clotting protein, fibrinogen, accumulates in theAPAP-injured liver and exerts hepatoprotective functions. The origin of fibrinogen in the APAP-injured liver is not fully understood. Fibrin- ogen circulates in plasma and this is often presumed as a primary source of fibrinogen deposits in the injured liver. Acute phase fibrinogen gene induction is also evident in the APAP-injured liver, but its contribution to fibrinogen synthesis in the context of APAP cytotoxicity is unknown. We tested the hypothesis that APAP treatment increases fibrinogen expres- sion in cultured liver parenchymal cells (i.e., hepatocytes). Primary mouse hepatocytes were treated with APAP (2.5 or 5 mM) or vehicle for 3 or 6 hours. In preliminary studies, APAP treatment induced cytotoxicity at each time point, indicated by release of alanine aminotransferase (ALT) into the culture medium. Interestingly, selective induction of the mRNA encoding the fibrinogen Aa chain, and not Bb or g chains, was evident and both concentrations and times in APAP-treated hepatocytes. The concentration of fibrinogen in the culture supernatant from vehicle-treated cells increased over time, in- dicating secretion of fibrinogen. Notably, compared to vehicle-treated hepatocytes, APAP treatment attenuated fibrinogen secretion into the culture medium, most evident 6 hours after APAP treatment. The results reveal an apparent disconnect between fibrinogen secretion and de novo fibrinogen induction in the early phase ofAPAP cytotoxicity in cultured hepato- cytes.

Research Grant: NIH Grant 5R01DK105099-04 Student Support: NIH Grant 5T35OD016477-19

Creation and validation of Mycobacterium tuberculosis granuloma recognition software

Diana Choi, Gillian Beamer

Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts

Acid Fast stained bacilli are a key diagnostic visual feature observed in the lung tissue of Diversity Outbred (DO) mice in- fected with Mycobacterium tuberculosis. Previous studies have exploited the artificial intelligence (AI) potentials to detect classic features of TB granuloma such as regions of neutrophil clusters or macrophage foci. However, the pathogenesis of tuberculosis incites a complex host response beyond the well-recognized cells in M. tuberculosis infection. Currently, no published automated algorithms can detect individual, singular cells within TB granuloma, such as individual bacilli or individual nuclei. Here, we used whole slide lung images from M. tuberculosis infected Diversity Outbred mice to train, validate, and performance test an algorithm to automatically detect and classify each of the individual mycobacteria, normal nuclei, and pyknotic nuclei. The performance of the proposed machine-learning model was evaluated using two main outcome measures. In the first layer, M. tuberculosis infected lung tissue was detected following the elimination of the non-tissue background. In the second layer, correct detection of potential regions, and correct classification of potential regions into mycobacteria, normal nuclei, and pyknotic nuclei were evaluated. We used 8 lung images from 8 different DO mice for training and validation, and we used new lung images from different DO mice for performance testing. Overall, our results provide a means of converting visual data into measurable, quantitative information suitable for statistical anal- ysis. Furthermore, statistical data of the number, location, and distribution of these individual cells within TB granuloma improves our understanding of tuberculosis.

Research Grant: Unknown Student Support: Boehringer Ingelheim Veterinary Scholars Program

94 • 2021 National Veterinary Scholars Symposium Dental and temporomandibular joint pathology of the Island Fox (Urocyon littoralis)

Anneka L.M. Christie, Michelle X. Lee, Siobhan S. Rickert, Frank J. M. Verstraete

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California

The dental pathology of the Island Fox (Urocyon littoralis) includes the presence and incidence of dental diseases in the examined museum specimens. By understanding U. littoralis dental pathology we can infer their oral health; granting insight into diet, environmental, genetic, and behavioral factors, and how these affect theU. littoralis population and its critical role in the Channel Islands ecosystem. Additionally, the study will contribute to ongoing research on North Amer- ican carnivores’ dental health and its role in species survival. Research on the dental and temporomandibular joint (TMJ) pathology will provide helpful insight into possible environmental pressures, and behavioral or genetic factors affecting U. littoralis. The hypothesis of this study is that U. littoralis will have similar pathology to the Grey Fox and Kit Fox. Due to the species isolation and lack of genetic diversity, we hypothesize an increased likelihood of congenital abnormalities. This study relies upon predefined criteria and protocol established by previous studies to macroscopically classify congen- ital, developmental, and acquired dental and TMJ abnormalities. These criteria will ensure a thorough examination and create consistency across species for future comparison. Data catalogued will include date, collection location, age, sex, subspecies, and examination findings. The sample size will include 346 specimens as determined by the Santa Barbara Museum of Natural History’s collection. The magnitude of the association between the presence or absence of specific forms of dental pathology and individual characteristics measured on a nominal or ordinal scale will be quantified using prevalence odds ratios and 95% confidence intervals.

Research Grant: None Student Support: Students Training in Advanced Research Program - School of Veterinary Medicine of the UC Davis

Evaluating the oncolytic potential of HSV-1 mutants (VC2, VC2GMCSF; VC2PGDH) using murine breast cancer model

Samantha D Clark, Konstantin G Kousoulas, Rafiq U Nabi

Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Rationale and objectives: Cancers have an exceptional ability to escape from immune surveillance and metastasize from an initial site. Conventional cancer therapies are costly with several limitations such as high toxicity, lack of specificity, and inability to induce long-lasting immunity. Consequently, immunotherapies such as oncolytic viruses (OVs) are very promising. OVs can be engineered to infect and selectively kill cancer cells and have the possibility to overcome conven- tional limitations. Most available oncolytic viruses are genetically modified to enhance tumor tropism and reduced viru- lence for non-neoplastic host cells. Due to its large number of receptors and large genome size, Herpes Simplex Virus-1 (HSV-1) can be used as a promising OV. Here we are proposing to evaluate and compare the efficacy of three mutant HSV-1 developed in Dr. Kousoulas’ s lab. Methods: In this study, we will utilize murine 4T1 breast cancer model and the mutant HSV-1 (VC2) with deletions in envelope proteins gK (31-68) and UL20 (4-22) that prevent it from entering neu- ronal axons. In addition, two other viruses were constructed replacing the glycoprotein C (gC) with GMCSF and PGDH. Intratumoral injections will be given after the establishment of the tumor and metastasis in the lung will be quantified to evaluate the efficacy.Expected results: We expect to observe significantly less lung metastasis in VC2 treated animals compared to PBS treated controls. In addition, we expect to see a robust T cell infiltration in the tumor of VC2 treated animals. GM-CSF is a critical growth factor for dendritic cells (DC) and overexpression of PGDH was reported to reduce PD-1 expression. Addition of these factors may increase the efficacy of VC2.

Research Grant: Unknown Student Support: NIH unknown grant number

2021 National Veterinary Scholars Symposium • 95 Comparison of microstructural values of equine proximal sesamoid bones at different CT resolutions

Abigail Clifford, Jesus Hermida, Timothy Lescun

Department of Veterinary Clinical Sciences, Purdue College of Veterinary Medicine, Purdue University, West Lafayette, IN

Proximal sesamoid bone (PSB) fracture is the leading cause of equine musculoskeletal injury leading to humane euthana- sia on racetracks in the USA. Studies have shown that exercise and training cause changes in the microstructure of equine bones. It has also been shown that microstructural values are different in the contralateral PSBs from horses with fractured PSBs compared to control horses and may be predictive of fracture. Unfortunately, these values are collected from micro CT images which currently cannot be obtained in a live horse. The recent development of equine standing CT equipment bypasses the need for horses to be under anesthesia for CT imaging and could enable routine screening of racehorses. The goal of this study is to determine if clinical CT scans give microstructural values of PSBs comparable to the gold standard micro CT imaging. We hypothesized that the current clinical CT resolution is too low to accurately identify bone micro- structure, but that useful correlations exist between clinical and micro CT images. We quantitatively compared the mi- crostructure of fractured and intact PSBs at different scan resolutions. Forelimb PSBs were collected from 20 racehorses that were euthanized on Indiana racetracks, 10 for PSB fractures and 10 for non-musculoskeletal reasons. The PSBs were imaged at a resolution of 625 mm (clinical CT) and 144, 90, and 65 mm (micro CT). Bone volume fraction, trabecular thickness, and degree of anisotropy values were collected and the statistical significance of differences between resolu- tions was determined. The ability to screen horses and identify microstructural changes in PSBs prior to fracture could prevent catastrophic injuries and improve equine welfare.

Research Grant: Purdue College of Veterinary Medicine Competitive Equine Research Funds, American College of Veterinary Surgeons Zoetis Dual Training Research Grant Student Support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

Evaluation of accuracy of point-of-care analyzers in primates to enhance monitoring in decentralized settings

Cassidy M. Coats, Scott H. Oppler Jr., Melanie L. Graham

College of Veterinary Medicine, University of Minnesota, Saint Paul, MN (Coats, Oppler), Department of Surgery, University of Minnesota, Saint Paul, MN (Graham, Oppler), Veterinary Population Medicine Department, University of Minnesota, Saint Paul, MN (Graham)

Point-of-care testing (POCT) is a patient-centric technology designed to bring conventional reference laboratory tests to clinical and research settings to facilitate rapid results for disease screening or therapeutic monitoring. In both settings, the decision to pursue lab testing may rely on accessibility or time constraints; POCT has the potential to bridge this gap to conventional testing by using minimal sample to provide real-time diagnostics to facilitate superior medical care and reduce burden on research animals. Concern about quality of POCT is a major barrier to acceptance in preclinical re- search, especially in disease modeling. The use of consistent, validated diagnostic tools is essential to accurately assess disease status and therapeutic outcomes, make informed treatment decisions, and prevent inaccurate results that impair study reproducibility and translatability. POCT of creatinine is a simple example owing to the central role of creatinine in monitoring disease and therapies that affect kidney function, such as kidney injury, diabetes, transplantation, or nephro- toxic pharmacotherapies. In this study, serum chemistry was analyzed in parallel using POCT and in the central laboratory to evaluate agreement between conventional reference standard and POCT methodology. Specimens from 139 rhesus and cynomolgus macaques representing a range of pathological values were used for assessment of accuracy using correlation agreement and repeatability. Validation of diagnostic tools is imperative to make the best educated and individualized medical decisions, optimize welfare, and accurately evaluate therapeutic outcomes in both the preclinical research and clinical settings.

Research Grant: NIH U01AI120130, NIH 6R42DK109853-02, JDRF 2-SRA-2019-761-S-B, JDRF 3-SRA-2016-259 Student Support: NIH Award T35OD011118

96 • 2021 National Veterinary Scholars Symposium Chemotherapeutic efficacy of carfilzomib in canine glioma cells in-vitro

Gena Cody, Travis Laver, Robert Gogal

Department of Small Animal Medicine and Surgery (Laver) and Department of Biomedical Sciences (Gogal), College of Veterinary Medicine, University of Georgia, Athens, Georgia

Glioma is the second most common primary brain tumor in dogs. These tumors are frequently intra-axial (located with- in the brain parenchyma) where surgical excision is not often a viable treatment option. Non-surgical treatment options include radiation and/or chemotherapy, but chemotherapy options are very limited. Carfilzomib is a second-generation proteasome inhibitor that irreversibly binds to the 20S proteasome that has been shown to reduce cell viability and migra- tion in human glioblastoma (a form of high grade glioma) cell lines. The current study aims to evaluate the cytotoxicity of carfilzomib in two canine glioma cell lines (J3T and SDT3G). Cells were grown in 96-well plates and treated with increas- ing concentrations of carfilzomib (0-1000 nM) for 24-72 hours. Cell viability was assessed using a commercially-avail- able luminescence-based ATP assay. The results show that both canine glioma cell lines are sensitive to carfilzomib in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) ranging between 152-230 nM for J3T and 141-205 nM for SDT3G. Overall, carfilzomib was shown to have cytotoxic effects on canine glioma cells. Further stud- ies are needed, but this study supports the premise that carfilzomib could be considered as a chemotherapeutic option in canine glioma.

Research Grant: This work was funded by the Georgia Veterinary Scholars Program and a generous donation from the Casey Project, sponsored by Carolynn Cooper and Pratap Mukharji Student Support: Georgia Veterinary Scholars Program

Cardiac extracellular matrix remodeling in horses with cardiac pathology

Jennifer R. Colangelo, Francisco A. Uzal, Joshua T. Morgan, Jessica M. Morgan

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California - Davis, Davis, CA

Cardiac remodeling and its potential role in arrhythmogenesis contributing to sudden death is poorly understood in the horse. Histologic changes and changes in myocardial extracellular matrix are associated with heart failure and arrhythmo- genesis in other species. This study aims to characterize changes to the myocardium in horses with cardiac diseases. It was hypothesized that horses that die of congestive heart failure and race-horses that experience exercise associated sudden death have evidence of remodeling in the myocardium compared to horses that died without evidence of cardiac disease. Evaluation of cardiac remodeling included histologic evaluation of the myocardium and specific staining for fibrosis and expression of matricellular proteins Tenascin-C and Osteopontin. Remodeling was evaluated in histologic sections of myocardium from horses with heart failure (6) and age matched controls (6). Fibrosis was evaluated with Mason’s Trichome staining. Matricellular protein expression was evaluated with immunohistochemistry. Slides were evaluated both subjectively and objectively with automated image analysis (Matlab). Horses with cardiac disease showed increased regions of fibrosis and differential expression of matricellular proteinTenascin-C. These findings support the hypothesis that myocardial remodeling is associated with equine cardiac disease. Increased understanding of the role of remodeling in arrhythmogenesis make this a potential therapeutic target to reduce the incidence of sudden cardiac death in horses.

Research Grant: This project was supported by the Center for Equine Health with funds provided by the State of California satellite wagering fund and contributions by private donors Student Support: Center for Equine Health Funds & Students Training in Advanced Research Program, UC Davis SVM

2021 National Veterinary Scholars Symposium • 97 Investigating Bovine Anaplasmosis via analysis of ticks from Ohio livestock

DaZane L. Cole, Justin Kieffer, Jeff LaKritz, Risa R. Pesapane

Department of Veterinary Preventative Medicine (Cole, Pesapane), College of Veterinary Medicine, The Ohio State University, Columbus, Ohio; School of Environment and Natural Resources (Pesapane), College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Columbus, Ohio

Currently no livestock-specific tick studies in Ohio are published in the literature, despite recurring outbreaks of bovine anaplasmosis in the state and the presence of five tick species of veterinary concern.The presence of bovine anaplasmosis in the United States is attributed to an obligate intra-erythrocytic rickettsial bacteria, Anaplasma marginale, which can be transmitted in three ways, including: mechanically via biting insects or blood-contaminated instruments (i.e fomites), biologically via ticks, and vertically from mother to calf. Bovine anaplasmosis manifests in cattle as reduced milk produc- tion, fever, weight loss, abortion, and many times, death. While there are numerous genotypes of A. marginale present in the U.S, it is not currently known which genotypes are circulating in Ohio. In addition to this, Ohio is facing invasion by an exotic tick which targets livestock-the Asian longhorned tick (Haemaphysalis longicornis). Asian longhorned ticks can also transmit Theileria orientalis which can cause bovine theileriosis. The aim of this investigation is to determine if the ticks which infect Ohio livestock carry the pathogens Anaplasma marginale or Theileria orientalis, and to begin to char- acterize the species and genotypes of the pathogens present in cattle herds.

Research Grant: USDA Animal Health and Disease Research Student Support: NIH T35 Training Grant T35OD010977

Genetic analysis of feline cannabinoid receptors—a pilot study

Jasmine Coleman, Kamoltip Thungrat, Dawn Boothe

Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL

The endocannabinoid system (ECS) is a highly conserved biological system found in all mammals that is involved in a variety of physiological activities especially neurologic health. The ECS is comprised of two major receptors - cannabi- noid receptor 1 and 2 (CBR1 and CBR2). It has been theorized that certain chronic neurological conditions are a result of a malfunction in ECS signaling, such as epilepsy disorders. These deficiencies may be a result of genetic mutations to one or both CBRs. Genetic analysis of CBR1 and CBR2 has been performed in humans and dogs but to a much lesser extent in cats. The goal of this study is to genetically characterize the feline CBR1 and CBR2 and to determine the presence of mutations in cats with the epilepsy disorder, Tay-Sachs Disease (TSD). We expect that there are mutations present in the CBR1 and CBR2 genes in the cats with TSD which could contribute to the presence of disease. In this study, tissue samples were taken from 5 cats - 2 with TSD and 3 controls - for genomic DNA extraction and sequencing of the CBR1 and CBR2 genes using the primer walking method with 123 design primers to determine the presence of mutations on the CBR1 gene. Primers were designed using the reference sequence NC_018727.3 for the feline CBR1 gene from the NCBI database. 75% of CBR1 nucleotides have been analyzed and we found one single nucleotide polymorphism (SNP) in exon region 1 of 1 at the location (81,795,652 - 81,820,403) and two SNPs in intron region 1 of 2 at location (81,801,194 - 81,820,244) in all 5 cats. More research must be done to determine if the SNPs found result in a change to the protein structure of the CBR1 receptor. Sequencing of the CBR2 gene must also be completed.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

98 • 2021 National Veterinary Scholars Symposium Nuclear factor-kB inducing kinase (NIK) regulates macrophage metabolism to support GBM growth and invasion

Olivia M. Combs, Justin N. Keeney, Raquel M. Sitcheran

Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, College Station, TX

Glioblastoma (GBM) is a lethal cancer with a 6.7% 5-year survival rate. Despite the development of new efficacious can- cer therapies, such as immunomodulators, GBM patient survival remains poor. Tumor associated macrophages (TAMs) suppress immune responses in the tumor microenvironment (TME) and contribute to the strong resistance of GBMs to im- munotherapy and other treatments. We have previously established that NF-kB-inducing kinase (NIK) is a critical driver of GBM growth and invasion through NF-kB-independent regulation of mitochondrial dynamics and metabolism. Prelim- inary data demonstrate that NIK also promotes mitochondrial oxidative metabolism in bone-derived macrophages, which is a characteristic feature of immunosuppressive M2-like TAMs. Moreover, compared to wild type (WT) macrophages, NIK knockout (KO) macrophages exhibit an M1-like phenotype, with an increased induction of glycolysis and inflamma- tory gene expression. Based on these data, we hypothesize that NIK inhibition in the TME will impair GBM growth and invasion due to de-regulated TAM metabolism and impaired immunosuppression. To test this hypothesis, we evaluated the effect of WT or NIK KO macrophages on GBM proliferation and invasion using co-culture assays. Preliminary findings demonstrate that compared with WT macrophages, co-culture with NIK KO macrophages reduced GBM proliferation, measured by BrdU staining. We anticipate that the migration and invasion of GBM cells will also be diminished when co-cultured with NIK KO macrophages that fail to acquire an oxidative, immunosuppressive TAM phenotype. These findings will help establish the therapeutic potential of targeting NIK-dependent immunometabolic functions in GBM to improve patient outcomes.

Research Grant: NIH R01 NS082554, Texas A&M College of Medicine Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Development & standardization of cardiac diagnostic parameters of the bearded dragon (Pogona vitticeps)

Jessica Conner, Bart Huber, Curtis Eng

Zoo, Exotics & Wildlife Department (Conner, Eng), College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA; Animal Medical Center of Corona (Huber), Corona, CA

Heart disease in reptiles has not been well described & clinical signs are fairly non-specific (lethargy, weakness, etc.), and could very well be diagnostically under-represented. As reptiles live longer in captivity it is probable that the prevalence of heart disease will continue to rise with age. With bearded dragons being one of the most popular lizards owned by pet owners, cardiovascular pathologies like congestive heart failure & aneurysms have been previously reported in this spe- cies. One of the many diagnostic tools in detecting cardiovascular disease is measurement of heart size. While the verte- bral heart score is well known and quite established in cats and dogs, there are no such standards for diagnostic cardiac evaluation in reptiles. This retrospective study aims to define and standardize normal heart parameters in a small popula- tion of bearded dragons. Using past cases that required radiology and computed tomography, the long axis of the heart was measured at its longest section; the short axis was then measured at the tallest section in a perpendicular segment from the long axis. Additionally, the widest sections of the heart-from left to right-were measured and recorded. The measurements of the long and short axis of the heart will be compared to various vertebrae in hopes of creating a reptilian vertebral heart score equivalent. For further comparison, the same measurements were recorded from bearded dragons previously diag- nosed with CHF, cardiomegaly or other cardiac pathologies. Descriptive data analysis provided through this study will begin the process of creating a set of normal or standard heart parameters for future clinical cardiac diagnoses as well as contribute to reptile cardiology research.

Research Grant: None. Student Support: University Research Department, Western University of Health Sciences

2021 National Veterinary Scholars Symposium • 99 Antiretroviral therapy effects on Feline Immunodeficiency Virus salivary viral load

Megan Conry, Sue VandeWoude, Jeffrey Kim, Mary Nehring, and Elisa Behzadi

Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

Feline Immunodeficiency Virus (FIV) is the feline analogue of Human Immunodeficiency Virus and is associated with gingivostomatitis and chronic gingivitis in infected cats. We are testing the effect of a novel combination antiretroviral therapy (cART) on salivary viral load of cats infected with FIV subtype C. We hypothesized that cART would result in a marked decrease in salivary viral load which ultimately relates to severity of oral disease. Saliva was collected from cats with FIV that had been treated with cART or a placebo control (n = 6/group). DNA and RNA extractions were performed using Qiagen DNeasy and QIAamp RNA extraction kits, respectively. We optimized the DNA extraction protocol using an ethanol precipitation step. Viral load will be assessed by using digital droplet PCR, which has been shown to be more precise and sensitive than real time PCR. We will use FIV-C specific primers and probes. We will be quantifying levels of viral DNA and complementary DNA in saliva along with housekeeping gene CCR5. This will allow us to assess the ratio of infected cells to total cells in saliva. It will also allow us to validate the nucleic acid integrity and control for error by acting as an internal reference gene. This study will determine the efficacy of the cART to alter transmission dynamics of FIV between cats and modulate development of gingival lesions commonly associated with FIV.

Research Grant: NIH Grant 1R21DE029733-01A1 Student Support: Boehringer Ingelheim Veterinary Scholar

Characterization of microglia in canine brain metastatic melanoma

Blaire A. Consales, Ryan G. Toedebusch, and Christine M. Toedebusch

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA

Microglia, the resident immune cells of the brain, have been shown to facilitate brain metastases in human metastatic mel- anoma (MM). While brain metastasis occurs at similar rates in canine MM, the microglial response in canine MM has not been evaluated. Thus, we hypothesized that 1) canine brain metastatic foci would have increased microglial density rela- tive to normal canine brain and 2) melanoma-associated microglia would exhibit a pro-tumorigenic phenotype. Archived formalin-fixed paraffin-embedded tissue was used for immunofluorescence and confocal microscopy. On initial review, we observed that the microglial density in brain metastatic foci was distinctly heterogenous. Therefore, we evaluated the following groups: normal canine brain (control; n = 4), metastatic melanoma without brain involvement (nonCNS-MM; n = 4), brain metastatic melanoma- high microglial density (CNS-MM (high); n = 4), and brain metastatic melanoma- low microglial density (CNS-MM (low), n = 4). We found that microglia density in CNS-MM (low) (3.2%) was decreased relative to normal brain (6.4%; P = 0.0412), nonCNS-MM (6.8%; P = 0.0155), and CNS-MM (high) (9.2%; P = 0.0001). While not significant, microglial density was increased 1.5-fold in CNS-MM (high) relative to normal brain (P = 0.0778) and nonCNS-MM (P = 0.168). Compared to normal brain (24.4%), microglial expression of pro-tumorigenic arginase-1 (Arg-1) was significantly lower in CNS-MM (high) (3.24%; P = 0.0016) and CNS-MM (low) (0.6%; P = 0.0003). This data indicates a heterogenous microglial response in canine brain MM with variable cellular density and down-regulation of arginase-1 expression. This work is ongoing to further elucidate the microglial phenotype in canine brain MM.

Research Grant: None. Student Support: UC Davis School of Veterinary Medicine STAR Program NIH T35 Training Grant 5T35OD010956-22

100 • 2021 National Veterinary Scholars Symposium Serotonin production and signaling in mouse trophoblast giant cells

Taylor Cook, Cheryl Rosenfeld, Jessica Kinkade, Shrikesh Sachdev, R. Michael Roberts

Department of Biomedical Sciences (Cook, Rosenfeld), Bond Life Sciences Center (Kinkade, Sachdev) Department of Animal Sciences (Roberts), College of Veterinary Medicine (Cook), University of Missouri, Columbia, MO

Selective Serotonin Reuptake Inhibitors (SSRIs) are increasingly used to treat depression in pregnant women. More than thirteen percent of pregnant women suffer from depression and are prescribed SSRIs. Serotonin guides many aspects of early fetal brain development, and it is being increasingly recognized that serotonin can exert effects on other fetal organs, such as the placenta. Robust evidence suggests that the placenta supplies most of the serotonin that orchestrates the initial stage of fetal brain development. However, controversy remains as to whether the placenta actually synthesizes this com- pound or accrues maternal serotonin through the serotonin transporter (Slc6a4/SERT). The objective of this study is thus to determine the degree which the placenta synthesizes itself vs accumulates maternal serotonin. We hypothesize that the placenta of the mouse produces serotonin, and the parietal trophoblast giant cells are the primary source for this produc- tion. For the procedure, mouse trophoblast stem cells are converted to trophoblast giant cells. Gene expression studies with cDNA generated from undifferentiated and differentiated cells is currently ongoing to determine whether these cells have the genetic machinery needed to produce serotonin. As part of these studies, we are examining genes associated with serotonin synthesis, transport, signaling, and metabolism. These findings will indicate the degree to which serotonin is produced by mouse trophoblast giant cells, and will thereby help to determine what effect, if any, SSRI drugs consumed by the mother may have on the placenta, and in turn the fetus. This is an area that is critical for pregnant women and their babies but has received little attention to date.

Research Grant: The work is supported by NIH grants 1R01ES025547 (to CSR) and 1R01HD094937 (to RMR) Student Support: Stipend for Taylor Cook is supported by a grant from Boerhinger Ingelheim

The impact of maternal dietary deficiencies in folic acid and choline on offspring neurological health

Sarah Coonrod, Nafisa M. Jadavji

College of Veterinary Medicine(Coonrod, Jadavji), Department of Biomedical Sciences, College of Graduate Studies (Jadavji), Midwestern University, Glendale, AZ

Stroke is an unmet medical need within human medicine and is associated with significant mortality and morbidity. Nu- trition is a modifiable risk factor for ischemic stroke. There is an opportunity for advancing therapeutic intervention by completing additional research aimed at understanding the mechanistic impact of nutrition on ischemic stroke. Dietary de- ficiencies of metabolites of one-carbon (1C) metabolism have been associated with an increased risk for stroke. Important 1C metabolites include folic acid and choline, which play a vital role in early-life neurodevelopment. Maternal dietary de- ficiencies in these vitamins and nutrients have been reported to impact stroke outcome in 2-month-old offspring; however, the mechanisms through which this occurs remains unknown. The aim of this study is to investigate the impact of mater- nal dietary deficiencies in folic acid or choline on neuronal variability and skilled motor function after stroke.This project will use hypoxia as an in vitro model of stroke. Female mice will be placed on a control, folic acid-deficient or choline-de- ficient diet for 4 weeks prior to mating. Primary neurons will be isolated from embryonic tissue and exposed to hypoxia; neuronal viability will be assessed after treatment. In vivo, after ischemic stroke to the sensorimotor cortex, skilled motor function will be assessed in 2-month-old adult male and female offspring to determine stroke outcome.These results will provide insight into the role of 1C metabolism on stroke recovery.

Research Grant: Research Grant: American Heart Association 20AIREA35050015 Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

2021 National Veterinary Scholars Symposium • 101 Optimizing surveillance of flaviviruses in free-ranging non-human primates

Olivia Cords, JoAnn Yee, Peter Nham, Tierra Smiley Evans, Pranav Pandit

UC Davis School of Veterinary Medicine (Cords), Primate Assay Laboratory Core, California National Primate Research Center (Nham, Yee), One Health Institute (Evans, Pandit), University of California, Davis, California

Flaviviruses are transmitted by arthropod vectors and have caused numerous epidemics, infecting up to 400 million peo- ple annually. Free-ranging non-human primates (NHP) are an important potential source of emerging infectious agents, including flaviviruses. Although some species are natural reservoirs for flaviviruses with epidemic potential, such as dengue, Yellow fever, and Zika, there are substantial gaps in our understanding of flaviviruses in NHP. This is in part due to the logistical challenges involved in sampling free-ranging NHP as well as a lack of understanding of how to target surveillance. This project evaluated the utility of a non-invasive oral sampling technique, involving distributing a rope for primates to chew on and the collection of oral fluid from the rope for the detection of virus-specific antibodies.A Luminex multiplex bead-based assay was used to detect antibodies specific to 7 flaviviruses from oral samples and compared to serum, the traditional sample type collected from free-ranging macaques from Southeast Asia. Oral samples tested for NHP IgG, IgM, and IgA were found to have concordant results with serum samples. Additionally, this project examined the ecological and biological traits of primate species with confirmed flaviviruses to better understand which species to prioritize for future surveillance. Our results suggest that traits including body mass, habitat type, and population status are predictive of flavivirus positivity. These findings will help maximize the effectiveness of future surveillance efforts, with potential implications for understanding flavivirus infections in primates and their role in spillover to humans.

Research Grant: NIH T32 Medical Scientist Training Program Grant 2020-2021 (NIH Grant T32GM136559) Student Support: UC Davis School of Veterinary Medicine, Students Training in Advanced Research Grant

Establishing serum biomarkers of Mycobacterium tuberculosis infection in Asian elephants

Laura L. Coughlin, Carlos R. Sanchez, Margot I. Monti, Felicia B. Nutter, and Gillian L. Beamer

Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA (Coughlin, Nutter, Beamer); Veterinary Medical Center, Oregon Zoo, Portland, OR (Sanchez, Monti)

Mycobacterium tuberculosis infection is a threat to elephant health and conservation and human public health worldwide. Cases of tuberculosis (TB), the disease due to M. tuberculosis in those susceptible, have occurred on multiple continents and affected both elephants living in captivity and wild elephants.Approximately 10% of captive elephants in North America, 23% of captive Asian elephants in Nepal, and 6-9% of free-ranging African elephants in Kruger National Park have tested positive for exposure to M. tuberculosis by serological testing for antibodies. Humans are the natural host for M. tuberculosis and main source of transmission to elephants. Infection can then spread from elephants to other elephants, to other mammals, and possibly back to humans. This project examines five biomarkers (TNF-a, IFN- g, IL-10, CXCL1, and MMP8) to determine if they are significantly elevated in the serum of elephants infected with M. tuberculosis com- pared to healthy controls. All five biomarkers are known to be involved in immune responses toM. tuberculosis infection in elephants or other similarly affected species. We will use commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits to determine the concentrations of these biomarkers in serum samples from elephants that have tested positive for M. tuberculosis infection and healthy controls. Results of the study are forthcoming. Identification of serum biomarkers of M. tuberculosis infection in elephants may improve diagnostic testing capabilities, allowing for earlier de- tection of TB positive elephants and reduced risk of spread to other species.

Research Grant: Tufts Elephant Conservation Alliance (TECA) Elephant Conservation Fellowship Student Support: Cummings School of Veterinary Medicine at Tufts University

102 • 2021 National Veterinary Scholars Symposium Screening and characterization of coronaviruses in veterinary patients

Sarah Cover, Holly McQueary, Joe Flint, Kaitlyn McGarvey, Tina Yu, Alexandra Presher, Nicole Andre, Gary Whittaker, Michael Stanhope, and Laura Goodman

Royal Veterinary College, London UK (Cover), Cornell University College of Veterinary Medicine, Ithaca NY (McQueary, Flint, McGarvey, Yu, Presher, Andre, Whittaker, Stanhope, Goodman)

In recent years, three separate wildlife spillover events have caused outbreaks of novel coronaviruses in humans (SARS, MERS, SARS-CoV-2). Focus of coronavirus discovery efforts have centered on bats. Importantly, coronaviruses are highly diverse and appear commonly in companion animals. The genetics of certain of these, such as equine coronavi- rus and canine enteric coronavirus, have not been researched extensively. This research attempts to both discover novel coronavirus species in companion animals and to use whole genome sequencing (WGS) to better characterize known coronaviruses. Nucleic acid samples from dogs, cats and horses that had been previously been tested for coronaviruses from 2018-2019 were obtained from Cornell Animal Health Diagnostic Center. Negative samples were further screened with a pan-coronavirus nested PCR targeting the RNA-dependent RNA polymerase (RdRp) gene, which is highly con- served across coronavirus species. Amplicons of the appropriate length were further analyzed with Sanger sequencing and confirmed to be a coronavirus by BLAST search. Positive equine (fecal) and feline (ascites) samples were used to evaluate novel tiled amplicon primer schemes for WGS. The genetic data from all sequencing was analyzed to determine relatedness to other known coronaviruses. A long term goal of the work is to study the evolutionary history and intrahost variation of these species in order to assess their potential zoonotic risk. These results will also enhance our understanding of the prevalence and diversity of veterinary coronaviruses for the benefit of animal health.

Research Grant: This study was funded (FOA PAR-18-604) from the U.S. Food and Drug Administration's Veterinary Laboratory Investigation and Response Network (FDA Vet-LIRN) under grant 1U18FD006993-01 Student Support: NIH T35 AI007227

Establishing the role of the lateral habenula in central itch processing

Suna L. Cranfill, Emma Janke, Minghong Ma, Wenqin Luo

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

Itch is a complex sensory experience that also encompasses affective and behavioral components, as evidenced by the common presentation of comorbid depression, anxiety, and other psychological disorders in chronic itch patients. How- ever, the cellular and circuit mechanisms linking pathological itch to affective disorders are poorly understood. Here, we identified a novel role for the lateral habenula (LHb), a brain region important for negative valence, aversive behaviors, and depression, in mediating acute itch behavior. Using c-Fos immunostaining in mice, we found that the LHb is highly activated by both itch and pain stimuli. Using optogenetic and chemogenetic approaches to reactivate itch-activated LHb neurons, we found that this population suppresses active behavioral responses to itch stimulation, such as scratching, and promotes passive immobility. Moreover, chemogenetic activation or inhibition of LHb neurons did not affect general loco- motion, suggesting that the itch-evoked immobility behavioral response is context-specific.Activation also did not affect pain-related behaviors, raising the possibility that discrete LHb ensembles process different aversive sensory modalities. Preliminary evidence using chemogenetic inhibition of LHb neurons suggests that this structure is required for the pro- motion of itch-evoked passive immobility, but does not appear to function as a brake on itch-evoked active scratching. Collectively, these results establish the LHb’s role in promoting a passive behavioral state during acute itch challenge, and support a model in which the LHb mediates the development of depressive symptoms in association with chronic itch disorders.

Research Grant: NIH R01NS083702, NIH R01NS094224 Student Support: NIH F31DE-29361

2021 National Veterinary Scholars Symposium • 103 Relationship between parity and reproductive disease in managed African Painted Dogs (Lycaon pictus)

Megan Crawford, Anneke Moresco, Dalen Agnew

Michigan State University College of Veterinary Medicine, East Lansing, MI (Crawford, Agnew); Reproductive Health Surveillance Program (Moresco, Agnew)

The managed female African Painted Dog (APD) population has been found to have high prevalence of reproductive disease, including cystic endometrial hyperplasia, adenomyosis and pyometra1. These diseases can result in infertility and even death2,3. It is important to have females that can reproduce in order to maintain genetic diversity and sustain the managed APD population. The purpose of this study was to identify risk factors (e.g., parity, age, contraceptive use), associated with different reproductive diseases in order to provide zoos with management recommendations for female APDs. After a female APD died or was ovariohysterectomized, tissues were fixed in 10% formalin and shipped to the Re- productive Health Surveillance Program (RHSP). Tissues were photographed, dissected, embedded, sectioned, and stained with hematoxylin & eosin. Microscopic evaluation identified reproductive lesions. Pearson correlation coefficients were produced using GraphPad Prism . Parity was not significantly correlated with cystic endometrial hyperplasia or adeno- myosis. Parity was negatively correlated with pyometra (r2 = -0.3; P = 0.035) and moderately positively correlated with uterine neoplasia (r2 = 0.54, P ≤ 0.0001). Additionally, the results showed there was a positive correlation between age and presence of cystic endometrial hyperplasia (r2 = 0.52, P ≤ 0.0001), and a moderate positive correlation between age and presence of adenomyosis (r2 = 0.52, P ≤ 0.0001). Preliminary data suggests that cystic endometrial hyperplasia and ade- nomyosis are degenerative changes associated with age and not associated with parity. Breeding female APD at a younger age may allow them to reproduce before cystic endometrial hyperplasia and adenomyosis develop.

Research Grant: Association of Zoos and Aquariums (AZA) Conservation Grant Fund and the Disney Conservation Fund Student Support: Boehringer Ingelheim and the Graduate School at Michigan State University

CircRNA expression in parasitic nematodes

Niurka Cubero Martinez, Sarah Minkler, Michael Kimber

Doctor of Veterinary Medicine Candidate 2024 (Cubero Martinez), Professor and Chair Department of Biomedical Sciences (Kimber), College of Veterinary Medicine, Iowa State University, Ames, Iowa

Circular RNAs (circRNA) are a recently discovered RNA type that are transcribed from protein-coding genes and formed into covalently-closed structures by backsplicing. Known to be expressed in a wide range of eukaryotes, circRNAs are believed to function primarily as miRNA sponges to regulate gene expression. They are also reported to have roles in protein binding and recruitment and may even be translated under some circumstances. We have previously used an RNA- seq approach to characterize circRNA expression in the gastrointestinal parasite, Ascaris suum. The purpose of this current study is to validate this RNA-seq analysis by confirming tissue expression of circRNAs found inA . suum body wall mus- cle, ovaries and extracellular vesicles (EVs). A. suum adult females were collected from an abattoir in Marshalltown, IA and maintained in the lab. To quantify tissue-specific expression of select circRNAs, RNA was isolated and extracted from ovary, body wall and secreted EVs and then treated with RNAse R and validated with qPCR. We also examined the effect of ivermectin on circRNA expression and secretion as this antiparasitic drug is believed to work, in part, by inhibiting host-modulatory secretions from parasitic nematodes. Worms were treated with 0.1 mM and 1 mM ivermectin, the results remain in progress. This study will confirm the expression of circRNA in nematodes and reveal the complexity of gene regulation in these important parasites.

Research Grant: NIH T35 Training Grant Student Support: None

104 • 2021 National Veterinary Scholars Symposium The role of bystander CD8+ T cells in Chlamydia-induced upper genital tract pathology

Emily Daigneault, Srikanth Manam, and Ashlesh K. Murthy

College of Veterinary Medicine, Midwestern University, Glendale, Az

Currently, the leading cause of sexually transmitted bacterial disease is Chlamydia trachomatis. When left untreated, a subset of women may experience an ascending upper genital tract infection, that leading to severe immunopathology in the uterus and fallopian tubes, such as pelvic inflammatory disease, and complications including ectopic pregnancies and infertility. Chlamydia muridarum, the mouse pathogen equivalent to C. trachomatis, causes both genital infection and reproductive pathology. Our lab has previously shown that Chlamydia-specific CD8+ T cells cause upper genital tract pa- thology. The objective of this experiment is to determine the role of bystander CD8+ T cells in upper genital tract pathol- ogy during Chlamydia infections. To accomplish this, we will be using OT1 mice where CD8+ T cells mount an immune response only against SIINFEKL peptide in ovalbumin, but not against chlamydial antigens. On days 0, 14, and 28, mice will be immunized intranasally with 100mg of ovalbumin. On day 60, mice will be infected intravaginally with 5X104 IFU of C. muridarum, then monitored for bacterial shedding every 3 to 4 days until infection is cleared. On day 74, some of the mice will be euthanized. Spleens will be collected and stimulated in vitro; total splenic and CD8+ specific immune responses will be measured. On day 150, genital tracts are collected, and pathology will be quantified.

Research Grant: 2R15AI101920-03 Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study Program

Minimally invasive repair of acetabular fractures in dogs: an ex-vivo feasibility study

Cambrie L. Dalton, Stanley E. Kim

Department of Small Animal Clinical Sciences (Dalton, Kim), College of Veterinary Medicine, University of Florida, Gainesville, Florida

Objective: To develop and describe minimally invasive plate osteosynthesis (MIPO) for acetabular fracture repair in dogs using plates contoured to 3D printed hemipelvic models and to assess the accuracy and feasibility of the technique in cadaver testing. Study Design: Ex-vivo feasibility study. Animals: Adult canine cadavers (n = 5). Methods: Bone plates were contoured to 3D printed hemipelvic models derived from computed tomographic scans of each dog. Acetabular, ischial, and pubic osteotomies were performed. A small craniolateral approach to the ilial body and caudal approach to the ischium were made and connected through epiperiosteal tunnels. Under fluoroscopic guidance, fractures were reduced, and pre-contoured bone plates were applied with cortical and/or locking screws. Postoperative computed tomographic images and resultant 3D renderings were used to assess fracture gap, step defect, and sagittal and coronal angulation. Cadavers were dissected for subjective assessment of sciatic nerve injury. Results: Small (< 2 mm) fracture gaps, minute step defects (< 1 mm), low (< 5°) pelvic angulation, and minimal (none [n = 4] and mild [n = 1]) sciatic nerve injuries were observed. Conclusion: The developed acetabular MIPO technique shows promise as a feasible procedure for future acetabular fracture repair. Clinical Significance: Further studies must be performed before this technique for acetabular MIPO can be recommended for clinical practice.

Research Grant: Edward Debartolo Gift, College of Veterinary Medicine, University of Florida Student Support: Edward Debartolo Gift, College of Veterinary Medicine, University of Florida

2021 National Veterinary Scholars Symposium • 105 Investigation of intrinsic nervous system dysfunction of the gallbladder in dogs with mucocele formation

Kaitlin V. Daly, Chloe Mariant, Stephen H. Stauffer, Laurianne Van Landeghem, Jody L. Gookin

Department of Clinical Sciences (Daly, Stauffer, Gookin), Department of Molecular Biomedical Sciences (Mariant, Van Landeghem), College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Gallbladder mucocele formation is one of the most common and deadly biliary diseases of dogs. The pathogenesis is poorly understood and treatment options are limited. Hallmark features are excessive secretion of abnormal mucus by the epithelium and poor gallbladder contractility. Secretion and contractility are both influenced by the intrinsic nervous system of the gallbladder. Our preliminary data identify fewer neuronal extensions in the mucocele gallbladder compared to healthy gallbladders. To comprehensively understand changes occurring in the intrinsic nervous system of gallbladder mucoceles, our study objective was to perform transcription profiling to identify, quantify, and compare the neurotrans- mitter content of ganglia from healthy and mucocele gallbladders. Healthy canine gallbladders were dissected to separate the epithelium, submucosa and muscle layers. To localize the ganglia, each layer was stained as a whole mount using immunofluorescence with antibodies specific for neuronal markers (Protein Gene Product 9.5,Tubulin b3). To confirm tissue viability, the submucosa and muscle layers were digested and cultured for glia, whose isolation was confirmed by immunofluorescence using antibodies specific for glia (glial fibrillary acidic protein, Sox10, S100-b). Moving forward with this project, we will dissect control and mucocele gallbladders and digest each tissue layer to liberate and collect viable ganglia. These ganglia will undergo mRNA extraction and sequencing to identify differences in type or expression of neurotransmitter synthesizing proteins. Our hypothesis is that specific neurotransmitter deficiencies in intrinsic nervous system function can be targeted to help restore normal gallbladder function.

Research Grant: Morris Animal Foundation Grant D17CA-068 Student Support: George H. Hitchings New Investigator Award, CGS/IDEXX Veterinary Student Summer Scholar Award

Opening a can of (lung)worms: molecular characterization of Dictyocaulus of North American bison

Hannah A. Danks, Caroline Sobotyk, Meriam N. Saleh, Matthew Kulpa, Joe Luksovsky, Lee C. Jones, Guilherme G. Verocai.

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX (Danks, Sobotyk, Saleh, Kulpa, Luksovsky, Verocai); US Fish and Wildlife Service, Wildlife Health, Natural Resource Program Center Bozeman, MT (Jones)

Dictyocaulus is a globally distributed genus of lungworms of domestic and wild ungulates. Dictyocaulus adults inhabit the bronchi, potentially causing subclinical and clinical disease, and impacting animal health and production. North Ameri- can bison and cattle share various parasitic nematode species, particularly in areas where co-grazing occurs. The current assumption is that the North American bison (Bison bison) share the lungworm D. viviparus with cattle, but this has yet to be confirmed on a molecular basis. The aim of this study was to molecularly characterizeDictyocaulus lungworm isolates from bison across the United States. Fecal samples were collected from 5 wild bison herds from Iowa, North Dakota, Oklahoma, Colorado, and Montana in 2019 and 2020, and privately-owned bison from Oklahoma and Texas. First-stage larvae (L1) were isolated via Baermann technique. Genomic DNA was extracted from L1 of up to 3 animals per herd, and followed by PCR and sequencing targeting the partial cox1 and ITS2 genes. Phylogenetic analyses were performed in MegaX. Sequences of North American bison Dictyocaulus belong to a single, uncharacterized species, clustering in well-supported clades (100% and 99% bootstrap support for cox1 and ITS2, respectively), differing fromD. viviparus of cattle in North America and Europe, and European bison (Bison bonasus). Our results contradict previous assumptions regarding parasite identity, highlighting the need for characterization of this species through morphological and molecular methods, elucidating its biology and host range, and potential impact on host health. Further investigations into the biodi- versity of Dictyocaulus species infecting bovids and cervids in North America is warranted.

Research Grant: None Student Support: Boehringer Ingelheim VSP, Texas A&M College of Vet Med and Biomed Sci

106 • 2021 National Veterinary Scholars Symposium Comparison of agents to maintain hydration of Yucatan minipig skin

Camryn Davis, Julie Brown, Lee Matthews, Amanda Darbyshire

College of Veterinary Medicine (Davis); Department of the Executive Vice President for Research and Partnerships, Laboratory Animal Program (Brown, Matthews, Darbyshire); Department of Comparative Pathobiology, College of Veterinary Medicine (Darbyshire)

College of Veterinary Medicine (Davis); Department of the Executive Vice President for Research and Partnerships, Laboratory Animal Program (Brown, Matthews, Darbyshire); Department of Comparative Pathobiology, College of Veterinary Medicine (Darbyshire), Purdue University, West Lafayette, Indiana

Yucatan minipigs are commonly used in biomedical research. These animals are prone to dry skin (xerosis) accompanied by pruritus. In previous work, minipigs housed by the Purdue Laboratory Animal Program were oiled twice weekly with lanolin to mitigate this condition, however, this resulted in excessive debris along the dorsum sometimes accompanied by exfoliative lesions. In this study, alternatives (glycerol and coconut oil) were used to compare their abilities to hydrate skin appropriately. The agents were applied twice a week over a four week period in a crossover design so that each pig was treated with each agent for one week with a 4 day washout period. Skin was assessed using a corneometer and both visual and tactile scoring at various timepoints. Results from visual and tactile scoring 24 and 48 hours after each application showed that the ideal skin condition was achieved in the following order: glycerol (83.3% visual,75% tactile), coconut oil (79.2%,64.6%), control (33.3%,39.6%), followed by lanolin (29.2%,12.5%). No significant difference was found from the results of the corneometer; it was concluded that the device was inaccurate for this purpose. Lanolin pooled on the skin and left the pigs greasy days after the agent was applied while glycerol had the highest frequency of ideal scores. In conclusion glycerol was found to be the best option for hydrating Yucatan minipig skin in the research setting.

Research Grant: None Student Support: Boehringer Ingelheim and Purdue College of Veterinary Medicine

Immunomodulatory effects of high-frequency irreversible electroporation on canine primary lung cancer

Elaina Davis, Alayna Hay, Kenneth Aycock, Kailee David, Kelsey Murphy, Rafael Davalos, Joanne Tuohy

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN (Davis), Department of Small Animal Clinical Sciences, VA-MD College of Veterinary Medicine, Blacksburg, VA (Davis, Hay, Murphy, Tuohy), Department of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA (Aycock, David, Davalos)

Canine primary lung cancer accounts for approximately 1% of canine neoplasia and is reported to metastasize in more than 80% of cases. Patients that undergo treatment have a mean survival of approximately 1 year. A non-thermal, minimal- ly invasive tumor ablation modality termed high-frequency irreversible electroporation (H-FIRE) has been investigated for its ablative and immunomodulatory abilities. The aim of this study is to evaluate the immune response after H-FIRE treatment on canine primary lung tumor cells in vivo and in vitro. For the in vivo experiment, paired H-FIRE treated and untreated lung tumor samples from three previously treated canine patients were analyzed for gene expression changes via the cancer inflammation and immunity crosstalk Qiagen RT2 PCR array. For the in vitro experiment, canine lung adeno- carcinoma (CLAC) cells were treated with H-FIRE at 1500 V/cm and 3000 V/cm using bipolar square waves with 2 ms width and 5 ms delay. At two hours post 1500 V/cm treatment, gene expression changes were evaluated with the previ- ously mentioned arrays and cytokine production was evaluated by a MILLIPLEX magnetic bead array. For the in vivo experiment, there was an increase in expression for 38 of the genes associated with inflammation and immunity in treat- ment samples compared to untreated samples. For the in vitro experiment, there was an increase in expression for 7 genes and decrease in expression of 23 genes associated with inflammation and immunity when compared to both the control and sham treatment groups. The concentration of TNF-a was increased in the treatment group compared to the sham and untreated groups. The results of this study suggest that H-FIRE changes the tumor microenvironment.

Research Grant: Veterinary Memorial Fund Student Support: Boehringer Ingelheim

2021 National Veterinary Scholars Symposium • 107 Evaluation of inactive vaccines against Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss)

Tryssa de Ruyter, Eric Littman, Mark Adkinson, Alvin Camus, Susan Yun, Zeinab Yazdi, Timothy J. Welch, Bill Keleher, Esteban Soto

University of California-Davis, CA (de Ruyter, Littman, Yun, Yazdi, Soto); University of Pennsylvania, PA (de Ruyter); California Department of Fish and Wildlife, CA (Adkinson); University of Georgia, GA (Camus); National Center for Cool and Coldwater Aquaculture, WV (Welch), Kennebec River Bioscience, ME (Keleher)

Lactococcus garvieae is an important emergent bacterial pathogen of farmed fish in the USA. A lack of efficacious -vac cines, increasing antimicrobial resistance, and limited treatment options have led to mortalities approaching 90% in some epizootics. Direct economic losses are compounded by the costs of delayed production and growth, chemotherapeutants, and added labor. Furthermore, when antibiotic therapy is attempted, treatment failure is common. These factors illustrate an urgent need for the development of effective prophylactic measures. The purpose of this study was to evaluate protec- tion conferred to rainbow trout (Oncorhynchus mykiss) against L. garvieae by a formalin-killed vaccine augmented by immersion and injectable forms of the adjuvant MontanideTM as well as enhanced protection afforded by booster vaccina- tion. Initial immersion vaccination was followed by booster vaccination via immersion or intracoelomic injection routes along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. garvieae by cohabitation with diseased fish 30d post-booster administration.Additionally, we investigated the immune response of rainbow trout to immunization supplemented with the injectable and immersion adjuvants using transcriptome analysis. Further research is needed to better understand the epizootiology of L. garvieae in rainbow trout aquaculture and the pathogenesis of lactococcosis to develop safe and effective prophylactic and therapeutic protocols against this serious pathogen.

Research Grant: California Department of Fish and Wildlife Statewide Fish Disease Research Program Student Support: None

The Role of Piezo1 in Pre-Adipocyte Differentiation

Andrew P. Decker, Javier Rendon Mora, Andres Contreras

Michigan State University College of Veterinary Medicine Department of Large Animal Clinical Sciences

Cardiovascular disease (CVD) is currently the leading cause of death in the United States every year. One such disease with high prevalence is hypertension, which also has poorly understood mechanisms making it difficult to control. One factor contributing to high blood pressure is the elasticity and compliance of the aortic perivascular adipose tissue (aP- VAT), tissue immediately surrounding the aorta. The aorta periodically expands and contracts in response to changes in blood flow, which is compromised by a loss of aPVAT compliance. Mesenchymal stem cells from the aPVAT can dif- ferentiate into different cell types, fibroblasts, with low compliance, and adipocytes, with higher compliance.Adipocyte accumulation in the aPVAT is favorable as this can increase compliance of the tissue. Piezo1, a mechanically gated cation channel, has the potential to impair adipogenesis, which would reduce compliance. Pre-adipocytes were induced for adipogenesis under three variables: induction/maintenance media with the Yoda1 agonist, induction/maintenance media without the Yoda1 agonist, and with preadipocyte media without Yoda1 as a control. For each variable, two conditions existed of induction without mechanical strain (MS-) or, applying mechanical strain (MS+) via the FlexCell machine. Our data indicates that mechanical stimulation of Piezo1 reduces adipogenesis of pre-adipocytes. These results reveal the impact of mechanical stimulation on the differentiation of preadipocytes that could aggravate hypertension. Understanding the relationship that mechanical forces have on adipogenesis through Piezo1 signaling can lead to treatments being offered with the potential to combat the epidemic of heart disease in this country.

Research Grant: NIH 2R25HL103156-11 Student Support: NIH 2R25HL103156-11

108 • 2021 National Veterinary Scholars Symposium Stimulator of Interferon Genes (STING) is essential for TLR-mediated upregulation of Type I IFN genes

Abigail E. DeJohn, Cynthia A. Leifer

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY

Type I Interferons (IFNs), comprised of IFNa and IFNb, are critical for host defense against viral infections but can also contribute to autoimmune diseases. It is well known that innate immune Toll-like receptors (TLRs) induce IFN by a mech- anism dependent on the transcription factor family interferon response factor (IRF), but it is thought to be independent of the cytosolic protein Stimulator of Interferon Genes (STING). STING is present in both non-immune and immune cells, including the human monocytic cell lines THP-1 and U937, and upon activation, STING upregulates IFNa and IFNb via IRF. Recent data from the Leifer lab showed that cells deficient in STING lack TLR-induced IRF activation. Thus, STING may play a previously undescribed role in TLR signaling. We hypothesized that TLR signaling would induce IFNa and IFNb with different kinetics than a STING agonist but would depend on STING expression and activity. To address this, we used a STING inhibitor and STING deficient cells to measure TLR-induced IFN mRNA expression. I treated U937 cells with either STING inhibitor H151 or a vehicle control, then with TLR4 or TLR8 ligands, STING agonist cGAMP, or IFNb. Similarly, wild-type and STING deficient THP-1 cells were stimulated with TLR agonists, STING agonist or IFNb. I collected mRNA after 0, 4, 8, or 24 hours. Using positive control stimuli, I first validated primer sets for IFNb, IFNa4, and a degenerate IFNa primer set that detects multiple IFNa genes. I then used reverse transcriptase to make cDNA and ran a qPCR to measure the levels of control 18S as well as the type I IFNs. Results from these studies will advance our understanding of IFN gene regulation and elucidate the role of STING in TLR signaling.

Research Grant: none Student Support: NIH T35 AI007227

Protective efficacy of a yeast byproduct diet against APEC challenge in poultry

Anne C. Devorak, Breck N. Peterson, Yu-Yang Tsai, Meaghan M. Young, Hilary Hsieh, Charlize S. Nakatsu, Nicolle L. Barbieri, Catherine M. Logue

Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA

Avian pathogenic Escherichia coli (APEC) causes systemic extraintestinal infections in poultry, clinically known as coli- bacillosis, resulting in considerable economic losses for the poultry industry worldwide. APEC-associated disease results in significant morbidity, mortality and carcass condemnation at slaughter. Live vaccines and probiotics are commonly used by producers to control bacterial infections; however, vaccines and probiotics may be ineffective against the diversity of disease-causing pathogens, including APEC. In this study, we tested whether a proprietary feed containing a specif- ic yeast byproduct could be used to enhance gut health and offer protection against challenge with a well-characterized highly pathogenic APEC O78 strain. In each of two identical trials, 120 chickens were split into eight groups: four groups were fed a control diet while the other four groups were fed the test diet containing a yeast byproduct for 21 days. On day 14 of the study, birds were challenged with either Phosphate Buffered Saline (PBS) (control) or APEC O78 (intratracheal- ly or orally) at a concentration of 1X108cfu . On day 21, the birds were euthanized and necropsied. Swabs of heart blood and air sac, and tissues of the liver, spleen, lung and ceca were collected for bacterial counts. It was found that APEC was detected in birds fed the control diet at a similar rate to the supplemented feed suggesting that the yeast byproduct had little protective effect in reducing the rate of contamination detected. However, the route of challenge resulted in greater systemic disease for intratracheally challenged birds. This study opens the door for future studies to investigate the protec- tive efficacy of other feed additives.

Research Grant: Private company – to C.M. Logue Student Support: Boehringer Ingelheim, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 109 Likelihood of Infection in Veterinary Patients Treated for Sepsis in the Emergency Room

Vidharshi N. Dharmasena, Ian Destefano, Katrina Manson, Annie Wayne

Cummings School of Veterinary Medicine at Tufts University (Dharmasena), Department of Emergency and Critical Care at The Foster Hospital for Small Animals (Destefano, Manson, Wayne)

In this retrospective study, we aimed to evaluate the likelihood of sepsis and septic shock in dogs treated with a sepsis protocol in the emergency room at the Foster Hospital for Small Animals (FHSA) at the Cummings School of Veterinary Medicine at Tufts University. Within this study, we characterized dogs treated with a septic kit from 2009 - 2017 as con- firmed sepsis, possible sepsis, or no sepsis. Sepsis was defined in this study as dogs that meet the Systemic Inflammatory Response Syndrome (SIRS) and had a confirmed infection. Infection was confirmed through a variety of methods includ- ing imaging, positive cultures, cytology or surgery. Septic shock is defined as sepsis with evidence of hypotension (less than 70mmHg) or a lactate greater than 2.5mmol. Descriptive statistics will be used to analyze the percentage of cases that received septic kits that did not have an infection requiring antibiotics. Our main objective is to bring awareness to inappropriate antimicrobial use in veterinary medicine as well as highlight the importance of antimicrobial stewardship. Antimicrobial resistance is one of the biggest threats facing the world - both in veterinary medicine and human medicine today. Being cognizant of the damage of overuse and inappropriate use of antimicrobials is imperative to being successful against the looming threat of antimicrobial resistance.

Research Grant: None Student Support: Cummings School of Veterinary Medicine at Tufts University

Alpha-enolase as a biomarker for early diagnosis of acute kidney injury and chronic kidney disease in cats

Amanda Diaz, Jennifer Hawley, Michael Lappin

Center for Companion Animal Studies, Department Clinical Sciences, College of Veterinary Medicine and Biological Sciences, and Colorado State University, Fort Collins, CO

Kidney disease is one of the most common health issues seen in domestic cats. Whether it be an acute kidney injury that is fatal, an acute kidney injury that leads to chronic kidney disease (CKD), or CKD itself. For years, research has focused on diagnosing kidney disease earlier than with currently available tests like serum creatinine concentration. As the creatinine concentration rises, the glomerular filtration rate goes down. The primary problem with this test is that by the time abnor- mal results are detected, there is already a 75% decrease in kidney function. Tests that detect kidney abnormalities earlier would allow for earlier supportive care to attempt to slow down this progressive and irreversible disease. Alpha-enolase is a glycolytic enzyme with a wide distribution in tissues, including the kidneys. Studies in our laboratory have shown that alpha-enolase may be a viable biomarker of kidney damage. For example, immunohistochemistry was used to demonstrate the distribution of alpha-enolase in feline kidneys in different life stages. Cats over 10 years of age and cats in different IRIS stages of CKD have a different distribution of alpha-enolase within the kidney tissues than young, healthy cats.The goal of this study is to evaluate alpha-enolase as an early biomarker for diagnosing kidney disease in cats. The hypothesis of this study is that as the kidneys are damaged, the degeneration of the renal tubules will lead to increased concentrations of alpha-enolase in the urine and serum. The hypothesis will be tested using results from an alpha-enolase antigen assay performed on banked serum and urine samples from normal cats, cats with acute kidney injury, and cats in different IRIS stages of CKD.

Research Grant: The Kenneth W. Smith Professorship in the Center for Companion Animal Studies Student Support: Center for Companion Animal Studies, Department of Clinical Sciences, CVMBS, CSU

110 • 2021 National Veterinary Scholars Symposium A comparison of Amazon parrot blood gas values on the i-STAT and EPOC

Jena M. Dicharry, Shannon D. Dehghanpir, Mark A. Mitchell, Thomas N. Tully Jr.

Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Point-of-care handheld units that provide blood-gas analysis of animal blood samples are a valuable diagnostic technique in canine and feline medicine. This technology allows clinicians to determine the acid-base and respiratory and metabolic state of patients. However, the use of these devices, Element Point-of-Care (EPOC) and the i-STAT, relative to compan- ion avian species has yet to be determined. The aim of this study was to collect venous blood-gas data from 26 healthy Hispanolian Amazon parrots (Amazona ventralis) on two point-of-care analyzers (EPOC and i-STAT) and to determine if this is a viable diagnostic testing method for these animals. The results allowed for the establishment of reference ranges and agreement and correlation analysis between the two units. All data was evaluated to determine if significant differenc- es existed between sexes, and no bias was determined. Data collected from both devices for the venous partial pressure - of carbon dioxide (pCO2), pH, and bicarbonate (HCO3 ) demonstrated Gaussian distribution. Thus, reference ranges were calculated. Only the i-STAT analyzer generated values that exhibited a normal distribution for venous partial pressure of oxygen (pO2). Due to the large range of the data for pO2 on both devices, reference ranges were not calculated. Results th th for pO2 are reported as the range, median, and percentiles (25 -75 ). When compared, the results from each device were highly correlated (r > 0.9), but the data for pO2, pCO2, and pH were in poor agreement. While the two devices should not be used interchangeably, the results of this study indicate they are clinically useful in assessing the acid-base status in Hispanolian Amazon parrots and other avian species.

Research Grant: None Student Support: Kenneth F. Burns Veterinary Student Award

Transcriptional response of Aedes aegypti Malpighian tubules to silencing of the Toll pathway inhibitor Cactus

Cameron E. DiGate, and Michael Povelones

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Heartworm disease in canids can be fatal if left untreated, placing high value on prevention. However, owner non-com- pliance, evidence of drug resistance to preventatives, and the inability to reach wild canid populations highlights the need for new interventions. Mosquito borne pathogens, such as Dirofilaria immitis, the causative agent of heartworm disease, must complete a lifecycle in the insect before becoming capable of infecting another vertebrate host. This requirement makes targeting pathogens in mosquitoes an attractive possibility for blocking pathogen transmission. Recent studies have shown that activation of the Toll immune signaling pathway of the mosquito Aedes aegypti potently blocks development of transmission stage D. immitis larvae. Arrested larvae are retained at their site of development, the Malpighian tubules, the mosquito’s renal organ. Immune activation was achieved by RNAi-mediated gene silencing by injecting mosquitoes with dsRNA targeting the Toll pathway inhibitor Cactus (vertebrate IkB). Despite the strong block of D. immitis develop- ment, it is not known if the injected dsRNA mediates its effect directly in the Malpighian tubules or indirectly by affecting another tissue that in turn affects the tubules. To address this question we are performing mRNA sequencing on dissected Malpighian tubules following injection of dsCactus RNA. We will assay differentially expressed Toll pathway targets us- ing mosquitoes treated with a non-specific dsRNA as a control. Determining whether theD. immitis block in Malpighian tubules is caused by the Toll or another pathway will inform future experiments to generate mosquitoes that are refractory to infection by D. immitis.

Research Grant: Boehringer Ingelheim, and the Penn Institute for Immunology Student Support: Boehringer Ingelheim, and the Penn Institute for Immunology

2021 National Veterinary Scholars Symposium • 111 Retrospective evaluation of the indication for and outcome following sildenafil administration in cats

Samantha R. Dolan, John E. Rush, Emily T. Karlin, Elizabeth A. Rozanski

Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts

Sildenafil, a phosphodiesterase type 5 inhibitor, is a vasodilator that acts preferentially on the pulmonary vasculature to decrease pulmonary arterial pressure. It is approved for the treatment of pulmonary hypertension in people. In dogs with pulmonary hypertension, sildenafil has been associated with an improvement of clinical signs and increased survival times. Sildenafil has also been shown to potentially mitigate the effects of certain causes of congestive heart failure in peo- ple, and research on its use for this indication is ongoing. In dogs with myxomatous mitral valve disease and pulmonary hypertension secondary to left-sided CHF, sildenafil administration resulted in improved clinical status and a drop in esti- mated pulmonary artery pressure. As there are few reports of sildenafil use in cats, the aim of this retrospective study is to examine the indications for and use of sildenafil in cats with heart disease, and to evaluate medical records for side effects that might be attributed to sildenafil. Searching electronic medical records and pharmacy prescription records at the Foster Hospital for Small Animals at Tufts University, data was collected to evaluate the effects of sildenafil in cats. In total there have been 101 cats identified with mention of sildenafil in their medical records. Of these, at least 22 have received one or more doses of sildenafil. Data collection on echocardiographic findings, evidence of side effects possibly associated with sildenafil, and long-term outcome is ongoing.

Research Grant: Barkley Fund Student Support: Cummings School of Veterinary Medicine at Tufts University Summer Research Training Program

Characterizing the maternal contribution to the gut microbiome of one-week-old mice

Erin Donovan, Nikki Seilhamer, Amber Russell, Kevin Gustafson, Aaron Ericsson

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

The transfer of maternal microbes to offspring plays an important role in long term health and immune function, and the gut microbiome in particular has become the focus of many studies in recent years. The murine gut microbiome evolves rapidly in the first few weeks of life, but the contribution of various maternal microbiomes to the neonatal gut microbiome composition at one week of age has not been comprehensively examined. While the neonatal gut microbiome does not resemble the maternal gut microbiome at this early stage, it is still of biological relevance. The goal of the present study is therefore to characterize the contribution of the maternal vaginal, oral, and fecal microbiomes on the neonatal gut microbi- ome composition at one week of age. Mice will be obtained from two of the primary vendors used in research, as previous studies have characterized the difference in gut microbiomes in mice from each of these vendors.The neonatal mice will be euthanized at approximately one week of age, and the dams will be euthanized with their respective litters. Neonatal and maternal fecal samples will be taken, as well as oral and vaginal swabs from the dams. DNA will be extracted, and used to generate 16S rRNA libraries, used to define the microbial populations. We hypothesize that the neonatal gut mi- crobiome will most closely resemble the maternal vaginal microbiome, as compared to the maternal oral and fecal micro- biomes. Such results would emphasize the role of vaginal delivery in the maintenance of the gut microbiome in neonates as old as one week of age.

Research Grant: MU Metagenomics Center Student Support: Stipend for Erin Donovan is supported by an endowment established by IDEXX-BioAnalytics

112 • 2021 National Veterinary Scholars Symposium Variation in tick species abundance between two collection methods at three central Illinois study sites

Alexandra K. Doran, Heather Kopsco, Nohra Mateus-Pinilla, and Rebecca L. Smith

Department of Pathobiology, College of Veterinary Medicine (Doran, Kopsco, Mateus-Pinilla), Illinois Natural History Survey-Prairie Research Institute (Mateus-Pinilla), University of Illinois at Urbana-Champaign, Urbana, IL

As global climate change continues to facilitate the spread of ticks and tick-borne illnesses, it is more important than ever to understand the habitat preferences of these tiny yet dangerous . There are currently at least 15 species of ticks known to inhabit Illinois. These species spread many serious illnesses, including Lyme disease, to humans and domestic animals. Habitat preferences of these tick species are well-documented, but previously collected data suggest a surprising lack of ticks at three central Illinois study sites with habitats that appear conducive to ticks. The purpose of this study is to determine if collecting ticks via small mammal trapping will yield different relative tick species abundances compared to using a drag cloth in these sites. The study will also provide the opportunity to examine the prevalence of Babesia microti, Borrelia burgdorferi, Anaplasma phagocytophylum, and Ehrlichia spp. in small mammals to determine if the number and species of ticks found on a particular host can be used to predict disease status. We will trap field mice using live Sherman traps baited with bread and peanut butter. Traps will be placed at sites with prior approval from site managers. Animals will be restrained within a clear plastic tube that limits movement during sample collection, then released. Ear punches and blood samples will be collected for parasite identification. Ectoparasites will be removed and placed in 70% ethanol for subsequent identification. Understanding why seemingly ideal locations do not have the expected abundance of ticks may yield clues about how the spread of ticks may be mitigated.

Research Grant: CDC Cooperative Agreement U01 CK000505; DOD TB180052 Student Support: Office of the Director, NIH, T35 OD00011145

Transferability of IncN and IncI plasmids among field strains of Salmonella enterica and Escherichia coli

Mackenzie E. Dorr, Ananta Arukha, Arye Silver, Dylan Smurlick, Subhashinie Kariyawasam, Thomas N. Denagamage

Department of Comparative, Diagnostic, and Population Medicine (Arukha, Silver, Smurlick, Kariyawasam) and Department of Large Animal Clinical Sciences (Dorr, Denagamage), College of Veterinary Medicine, University of Florida, Gainesville, Florida

Resistance to extended spectrum cephalosporins (ESC) in is a major public health concern. We report- ed CTX-M-1 type ESC-resistant nontyphoidal Salmonella and Escherichia coli in poultry. This study aimed to sequence the IncN and IncI plasmids carrying the blaCTX-M-1 in these isolates and assess their transmissibility rates between homolo- gous and heterologous bacteria. Plasmids were transferred from three donor strains (E. coli/IncN, S. Heidelberg/IncN, and

S. Heidelberg/IncI) into nine recipient strains. Lastly, blaCTX-M-1 gene PCR, plasmid isolation, and antimicrobial suscepti- bility testing were performed on the transconjugants to confirm the successful transfer of ESBL plasmids. Sequencing of plasmids confirmed IncN plasmids are 40 kb in size and the IncI plasmid is 100 kb in size. Successful plasmid transfer was observed between donor E. coli (IncN) and all recipient strains except for E. coli 50. Transfer efficiency betweenE. coli donor and Salmonella recipients was higher than E. coli donor and E. coli recipients. Successful plasmid transfer was also seen between S. Heidelberg (IncN) and all recipient strains, with efficiency higher betweenSalmonella donors and recipients than between Salmonella donors and E. coli recipients. Successful gene transfer was lastly observed between S. Heidelberg (IncI) and E. coli 50 as well as the lab strain of E. coli DH5a, though not observed with any Salmonella recipients. Based on our findings, we conclude that transfer of thebla CTX-M-1 gene via conjugation is possible yet occurs at different frequencies depending on the donor and recipient strain of bacteria.This study emphasized the possible zoonotic transfer of ESC resistance via the food chain.

Research Grant: University of Florida College of Veterinary Medicine Faculty Start-up Funds, Dr. Thomas Denagamage Student Support: Florida Veterinary Scholars Program Boehringer Ingelheim Veterinary Scholar Program

2021 National Veterinary Scholars Symposium • 113 Defining the PD1+ and PDL1+ populations in response to targeted therapy treatment in a murine melanoma model

Matthew Drescher, Andrew White

Cornell University College of Veterinary Medicine, Ithaca, NY

Nearly half of all patients with advanced melanoma suffer from a constitutive mutation to the Braf-Mek pathway. Initial treatment has been shown to be effective, but resistance to inhibition treatment is considered inevitable, thus making the pursuit of secondary treatments urgently needed. By using a transgenic murine model to induce melanoma growth, we can model the growth and response of tumors to blockage of the Programmed Death (PD-1) protein pathway that exists as a mechanism for tumor cells to inhibit cytotoxic T-cells. The overall aim of this study was to assess PD-1 and PDL-1 levels as a foundational groundwork for further exploration into this promising therapy. By using indirect antibodies and immunofluorescent techniques, we were able to map out the frequency of PD-1 and PDL-1 at different stages in tumor development, including post-resistance to treatment with conventional therapeutics. To do this, we have studied the co-localization of PD-1 and PDL-1 with all T-cells (CD3), cytotoxic T-cells (CD8), T-regulatory lymphocytes (FoxP3), and macrophages (F4/80). Qualitative and quantitative results show that PD-1 exists in cytotoxic T cells at lower levels than expected, compared to those of other lymphocytes, suggesting that further exploration and experimentation is needed before administering treatment. A second study was also conducted to evaluate levels of immune cells in early tumorigen- esis also using transgenic murine models. Both studies offer valuable insight to the treatment of melanoma for both human and animal health.

Research Grant: Cornell University Start-up Funds Student Support: Boehringer Ingelheim

The contributions of different functions of herpes simplex virus ICP22 to viral replication

Rachel Dufour, Claire Birkenheuer, Joel Baines

Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

HSV-1 transcription is mediated by host RNA polymerase II (Pol II) that is recruited to initiate a temporal cascade of viral gene expression. The immediate early (a) genes are transcribed first, and their protein products are required to continue viral replication. ICP22, encoded by the a22 gene, is proposed to have two main roles: modulation of Pol II phosphoryla- tion via the ICP22 C-terminal domain, and formation of intranuclear virus-induced chaperone-enhanced (VICE) domains via its N-terminal region. The relative contributions of VICE domain formation and Pol II phosphorylation to viral repli- cation is unclear. We have created an ICP22 truncation mutant virus using BAC technology bearing 3 stop codons inserted into the middle of the PVUII site at codon 180 of 401 of a22. This virus should retain the ability to form VICE domains but lack the ability to alter Pol II phosphorylation. VICE domain formation and viral replication will be compared between the virus with truncated ICP22, a virus lacking ICP22, and the wild type virus using titration with crystal violet staining, fluorescence microscopy, and bright field microscopy. If VICE domain formation is sufficient to support viral replication, we would expect robust replication of the truncation mutant. If Pol II phosphorylation is important, we would expect that replication of the truncation mutant should be significantly impaired relative to wild type virus. Future experiments will use deep sequencing techniques to elucidate potential roles in Pol II initiation and/or elongation.

Research Grant: NIH R21AI148926 and NIH R01 AI141968 Student Support: National Institute of Health

114 • 2021 National Veterinary Scholars Symposium Prevalence of upper respiratory pathogens in Colorado Front Range rescue horses

Conner Dugan, Elsbeth O’Fallon, Carson Zweck-Bronner, Gabriele Landolt

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

The Colorado Front Range unwanted horse population is an important group of equids that present a risk to national horse populations. The stressful and transitional environments with commingling of mixed origin equids play host to various contagious pathogens that may or may not produce clinical signs. Infections of Equine Influenza, strangles Streptococ( - cus equi ssp. equi), Streptococcus equi ssp. zooepidemicus, and equine herpesviruses 1 and 4 (EHV-1 and 4) may present with systemic signs of illness. However, subclinical infections are possible and are often undiagnosed. This includes the gammaherpesviruses, equine herpesviruses 2 and 5 (EHV-2 and 5). We hypothesize that equids rescued from feedlots and auctions with clinical and subclinical infections of respiratory pathogens present a contagious risk to the general equine population as these horses are rehomed. Existing data from polymerase chain reaction (PCR) analysis of nasal swabs of clinical and subclinical horses from various rescue organizations across the front range will be analyzed to characterize the prevalence of upper respiratory infections in this target population. The data represents a diverse population of equids by including ages ranging from newborn foals to 28 years, and various breeds including ponies, donkeys, Quarter Horses, and Draft breeds. Quantifying the prevalence of infections in this population will provide necessary and beneficial infor- mation about the risk level these horses present to other horse populations upon dispersal. This information will guide veterinarians, rescue operations, and horse owners in risk assessment and implementation of biosecurity and quarantine considerations.

Research Grant: None Student Support: USDA Animal Health & Disease Scholar

Face mask debris - potential risk to face mask wearers

Shiori Echizenya, Amanda Burns, Jenessa Gjeltema, Kent Pinkerton

School of Veterinary Medicine, University of California, Davis, Davis, CA

The COVID-19 pandemic along with wildfires have sparked an urgent need for improved guidelines regarding face mask use. The United States Centers for Disease Prevention and Control recommends reuse of filtering face-piece respirators, such as home-made masks, to conserve supplies for essential workers. Although previous studies have demonstrated the protective nature of wearing masks to limit the dispersal of exhaled respiratory droplets from the wearer to other individu- als, there remains the potential to face mask-wearers of inhaling particles generated from the mask itself. This risk has not been investigated. The goal of this project is to examine whether inhalable face mask debris is present for new or mechan- ically aged masks using scanning electron microscopy (SEM) and Raman Spectroscopy. We identified the presence of inhalable particles from new masks as well as aged masks. To determine their biological effects, particles collected from these masks were instilled intranasally in mice. Pulmonary function testing was performed, as well as bronchoalveolar lavage to determine if face mask debris particles alter the physiologic function of the lungs and/or produce inflammatory changes in the lungs of mice. To date, our studies demonstrated by SEM images that fabric masks release the greatest number of particles, followed by surgical masks, with the least number of particles released by N-95 masks.

Research Grant: None Student Support: NIH T35 Training Grant

2021 National Veterinary Scholars Symposium • 115 Use of a yeast fermentation-based probiotic to reduce Salmonella prevalence in lymph nodes of dairy cattle

Stephen E. Edache, Natalia Cernicchiaro, Diana M. Dewsbury, Leigh A. George, Xiaorong Shi and T.G. Nagaraja

Department of Veterinary Medicine, Federal University of Agriculture, Makurdi, PMB 2373, Benue State, Nigeria (Edache); Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States (Cernicchiaro, Dewsbury, George, Shi, Nagaraja)

Salmonella carriage in lymph nodes has been identified as a potential source of ground beef contamination, and thus of human illness. This study evaluated whether the whole-farm use of an in-feed yeast fermentation-based probiotic (Saccha- romyces cerevisiae) is associated with the reduction in prevalence of Salmonella in the subiliac lymph nodes (LN) of dairy cattle during summer months. With cooperation from two commercial processing plants in the Southwest and Northeast US regions, lots of harvested cattle from dairy farms feeding the fermentation product (cases) were identified. Control farms (not feeding the fermentation product) were matched to the case farms based on the lot’s average body weight, type of cattle and week of processing. Up to 20 bovine LNs were collected from each farm. Samples were analyzed for the presence of Salmonella by culture-based and quantitative PCR methods. A total of 227 LNs were collected between May 28 and July 13, 2021. Overall Salmonella LN prevalence (across both plants and weeks) was 5.7% (13/227); among case farms, Salmonella LN prevalence was 3.1% (3/114), whereas among control farms prevalence was 9.4% (10/113). Higher prevalence of Salmonella was observed in samples from the Southwest (12.2%, 9/74) compared to the Northeast (2.6%, 4/153) region. Our preliminary results indicate a numerical reduction in the prevalence of Salmonella carriage in the LN of culled dairy cattle fed the yeast fermentation-based probiotic compared to those not fed the probiotic during summer months. Identifying effective pre-harvest strategies that reduce the burden of Salmonella are essential to ensuring food safety and protecting public health.

Research Grant: National Cattlemen's Beef Association Student Support: Boehringer Ingelheim Veterinary Scholars Program and the KSU CVM Office of Research

Development of fecal IgA assay for detection of segmented filamentous bacteria infection in laboratory mice

Kerri Edwards, Marcia Hart, David Birk, Greg Purdy, Craig Franklin

Department of Veterinary Pathobiology & Mutant Mouse Resource and Research Center, University of Missouri College of Veterinary Medicine, (Edwards, Franklin) and IDEXX-BioAnalytics (Hart, Birk, Purdy), Columbia, MO

The microbiota of laboratory mouse models is very complex, and we are only beginning to understand its effects on the research models. Research suggests that differences in microbiota have potential effects on outcomes of murine studies. One bacterium known to modulate models is nonpathogenic segmented filamentous bacterium (SFB) which colonizes the ileum. Its presence promotes proliferation of Th17 cells and increased IgA levels that correlate with changes in phenotype of several models. With age, SFB levels wane to the point of becoming undetectable by fecal PCR and/or postmortem ileal PCR and histologic examination suggesting that the bacteria is ultimately cleared. This can be problematic for researchers who recognize a potential impact of SFB on long term studies after the PCR-based window of detection has occurred. The objective of the study is to develop a fecal IgA assay and assess its utility for the detection of current or prior SFB infec- tion in laboratory mice. SFB was isolated and inoculated by oral gavage into 8 males, and 8 female weanling C57BL/6 mice. Fecal samples are collected weekly from these mice as well as 4 negative controls of each sex for two months. SFB status will be determined by PCR and the novel fecal IgA assay. Terminal necropsy will be performed, and SFB status will be determined by fecal and ileal PCR, fecal and ileal IgA, serum IgA and IgG and ileal histology. We hypothesize IgA will persist in mice that have cleared infection and be detectable at ages where testing by PCR and histology is negative. This will provide for a more accurate test in determining SFB status, including situations where SFB has been cleared.

Research Grant: This work is supported by NIH grant U42 OD010918, Mutant Mouse Resource and Research Center. Student Support: IDEXX-BioAnalytics

116 • 2021 National Veterinary Scholars Symposium Prevalence of the RAPGEF5 c.2624C>A variant associated with EFIH in the United States Thoroughbred population

Megan E. Elcombe, Rebecca R. Bellone, K. Gary Magdesian, and Carrie J. Finno

Department of Population Health and Reproduction (Elcombe, Bellone, Finno) Veterinary Genetics Laboratory (Elcombe, Bellone), and Department of Medicine and Epidemiology (Magdesian), School of Veterinary Medicine, University of California-Davis, Davis, CA

Equine familial isolated hypoparathyroidism (EFIH) is a fatal condition in Thoroughbred foals characterized by muscle contractions and seizures resulting from severe hypocalcemia. In 2019, a nonsense variant in RAPGEF5 (c.2624C>A p.Ser875*) was identified as the genetic basis, and an allele frequency of 1.8% was identified in a non-random set of 82 Thoroughbreds. This disorder has only been reported in the Thoroughbred breed and mode of inheritance is autosomal re- cessive. Approximately 20,000 Thoroughbred foals are registered annually, with $77,000 as the average yearling sale price in 2019. Thus, loss of one foal from EFIH can have a significant economic impact. Objectives of this study were to;(1) determine an accurate estimate of allele frequency of the RAPGEF5 c.2624C>A variant in a large random cohort of horses between 1989-1995 (n = 378) and 2005-2019 (n = 810) and (2) identify if this is a recent variant in the Thoroughbred pop- ulation and is undergoing positive selection. DNA was isolated from hair follicles and serum samples banked at the UC Davis Veterinary Genetics Laboratory and genotyped for the RAPGEF5 c.2624C>A variant using the commercially avail- able assay. Allele frequencies of 0.4% in the older population (95% CI, 0.08% to 1.23%) and 0.67% in the recent popula- tion (95% CI, 0.36% to 1.23%) were not significantly differentP ( = 0.56). The oldest sample with the variant detected was born in 1992. No EFIH homozygotes were detected. This study demonstrated that the RAPGEF5 c.2624C>A variant is present at very low frequency in the United States Thoroughbred population but is not a recent mutation. Genetic testing is recommended to further monitor the trend of variant frequency within the Thoroughbred population.

Research Grant: UC Davis Center for Equine Health Student Support: NIH T35 Training Grant 5T35OD010956-22

Investigating beaver dam analogues as a novel method to reduce Cryptosporidium and Giardia load in wet meadows

Stephanie A. Elliott, Ariel I. Loredo, Andrea E. Packham, Woutrina A. Smith

One Health Institute, School of Veterinary Medicine, University of California, Davis, CA (Elliott, Loredo, Packham, Smith)

Beaver dam analogues (BDAs) are a human-made ecological intervention, assembled using native flora, which are placed in degraded wet meadows to help restore the environment. Ecological restoration in the wetland setting is important, as healthy wetlands are more efficient at removing protozoal pathogens than degraded wetlands due to their high vegetation density and slow water flow rate. The aim of this pilot study is to determine if BDAs, through their vegetative construc- tion, remove protozoal pathogens while simultaneously helping to repair wetlands. It is hypothesized that BDAs will reduce the downstream load of inactivated Cryptosporidium and Giardia (oo)cysts by 70% when compared to the initial amount released. At our study site in the high Sierra, a protozoal transport study was performed where 4.8x106 of inac- tivated Cryptosporidium and Giardia (oo)cysts were released upstream of the BDA. Surface water samples were taken downstream of the BDA at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 18 hours, and 24 hours post-release. An upstream sample was taken 15 minutes and 24 hours post-release. These water samples were filtered in- field, then (oo)cysts were separated using immunomagnetic separation in the laboratory. They were stained using fluo- rescent antibody staining and enumerated via fluorescent microscopy. Data are being analyzed and preliminary findings support the hypothesis that at least 70% of parasites were captured by the beaver dam analogues, thereby contributing to cleaner surface waters downstream along with their function to slow water that increases availability to wet meadow vege- tation in the riparian areas around the streams.

Research Grant: Phil and Karen Drayer Wildlife Health Center Fellowship Award Student Support: UC Davis School of Veterinary Medicine Endowment fund

2021 National Veterinary Scholars Symposium • 117 Defining the inflammatory cytokine profile in acute tendon injury to enhance mesenchymal stem cell therapy

Ilene M. Ellis, Jessica M. Gilbertie, Kristen M. Messenger, Alix K. Berglund, Drew W. Koch, Lauren V. Schnabel

College of Veterinary Medicine (Ellis, Gilbertie, Messenger, Berglund, Koch, Schnabel), Comparative Medicine Institute (Gilbertie, Messenger, Berglund, Koch, Schnabel), North Carolina State University, Raleigh, North Carolina

Tendons, specifically digital flexors, are common sites of trauma for performance animals and athletes. Translational research seeks to improve the efficacy of tendon healing via the manipulation of innate cellular components and their cytokinetic cross-talk with infiltrating immune modulatory cells. However, progress has been hindered by a lack of in vivo examination of the timing and presence of cytokines relevant to the tendon healing process. The aim of this study was to assess in vivo temporal expression of inflammatory cytokines during acute tendon injury using an equine model with the goal of using the obtained data to enhance tendon targeted mesenchymal stem cell (MSC) therapy. For the study, novel BASi 100 kDa ultrafiltration probes were inserted into surgically created superficial digital flexor tendon (SDFT) core lesions in six horses. Tendon ultrafiltrate was collected over 21 days; levels of IL-1b, IL-6, IL-8, IL-10, TNF-a, PGE2 and TGF-b isoforms were measured via multiplex immunoassay and mapped according to concentration and timing of detec- tion. Statistical analysis was performed using SAS 9.4 and R software with statistical significance declared at P < 0.05. Median concentrations of 2097 pg/mL of IL-1b, 1971 pg/mL of IL-6, and 110 pg/mL of IL-8 expression were recorded within the first 48 hours of collection. Going forward with this data, MSCs will be stimulated with IL-1b and IL-6 in vitro; gene expression will be analyzed via RNA sequencing to measure changes in their secretome compared to unstimulated controls. This data will then be used to determine if further benefit can be conferred with inflammatory priming of MSCs for treatment of tendon injuries in an effort to revise current clinical practice.

Research Grant: The Grayson-Jockey Club Research Foundation, The Fund for Orthopedic Research in honor of Gus and Equine Athletes, and The Barton & Marie-Claude White Equine Orthopedic Research Endowment Student Support: NIH: T35OD011070 (IE) IBRTP, North Carolina State University

Transfection of bovine preputial keratinocytes for expression of antibody against Tritrichomonas foetus

Lauren Ellison, Ella Swales, Merrilee Thoresen, E. Heath King, Darcie Sidelinger, Daryll Vannover, Hannah Peck, Philip J. Santangelo, Amelia R. Woolums

Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University (Ellison, Swales, Thoresen, King, Sidelinger, Woolums); Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University (Vannover, Peck, Santangelo) mRNA therapy, which induces cells to produce proteins, has recently come into focus as a possible treatment for many diseases. The method is potentially powerful because synthetic mRNA can theoretically code for any protein a cell can produce. Tritrichomonas foetus, despite having a large economic impact on cattle production, does not have an approved effective treatment. Synthetic mRNA therapy to induce protective antibodies on bovine preputial epithelium could offer a solution. mRNA was obtained for the TF1.15 and TF 1.17 antibodies that have been shown to bind to 2 different epitopes of a T. foetus attachment protein. An in vitro transfection methodology with bovine primary preputial keratinocytes (PPK) was used to determine the synthetic RNA concentration (1mg, 2mg, or 4mg), time from transfection to T. foetus exposure (24 or 48 hours), and mRNA construct (TF 1.15 or TF1.17) that produced a sufficient amount of antibody to bind T. foetus. Antibody production was quantified using immunofluorescence (IFA) to count the proportion of PPK producing antibody. IFA was also used to show that antibodies produced by transfected PPK bound to T. foetus. Antibody production in PPK cytoplasm was greatest at 24 hours, while more antibody was present in PPK culture supernatants at 48 hours. IFA demon- strated binding of expressed antibody to T. foetus. The optimized transfection conditions were 1mg of RNA, introduction of T. foetus after 24 hours of transfection, and the TF1.15 antibody construct. Further work to determine if binding of antibody impairs T. foetus attachment and pathogenicity is underway.

Research Grant: CRIS Grant Funding (MSU-CVM, USDA Section 1433 Formula Funds, 2019-2020) Student Support: National Institutes of Health 5T35OD010432

118 • 2021 National Veterinary Scholars Symposium Client Perceptions of an Educational Partnership to Provide Low Cost Veterinary Care to an Underserved Area

Stella Elwood, Emily McCobb, Erin King

Department of Clinical Sciences and the Center for Animals and Public Policy, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA.

Multiple studies cite cost of service as the most prevalent barrier blocking pet owners from access to veterinary care. Tufts at Tech (T@T) has made great progress in lessening this widespread hardship. The organization accomplishes this goal by offering care at 25% the national average cost to individuals who rely upon governmental assistance, like food stamps. At the same time, the clinic allows fourth year veterinary students to complete a primary care rotation, which strengthens their communication skills and enhances their cultural humility. Furthermore, the program trains high school students, many of whom qualify for federal subsidies for breakfast and lunch, to become certified veterinary assistants. In this way, since its establishment in 2012, T@T has benefited the community in many different ways.However, despite these achievements, those involved in the operations of T@T acknowledge the need for continued improvement in order to serve the clinic’s patients, as well as their families, in the best way possible. As a result, this project aims to foster cli- ent-centered/driven progress by creating and employing an accessible client feedback system.Through questions that ask clients about their experiences at T@T, we can identify areas in which further growth is necessary, which in turn, deter- mine where future funding and research endeavors should be allocated. Consequently, client opinion will drive practice changes, as the clients themselves know and understand their own needs, and the ways in which we may best assist them and their pets, better than anyone else.

Research Grant: PetSmart Charities grant Student Support: None

Inhibition of canine glioma cells by MAP4K4 knockdown and treatment with the metastasis inhibitor metarrestin

Abby English, Claudia Baumann, Simon Platt, Rabindranath De La Fuente

College of Veterinary Medicine (English), Department of Physiology and Pharmacology (Baumann, De La Fuente), and Small Animal Medicine and Surgery (Platt), University of Georgia College of Veterinary Medicine, Athens, GA

Gliomas are the second most common primary brain tumor in dogs and show many similarities to pediatric gliomas. For high grade gliomas, survival time is relatively short due to the aggressive nature of the disease and the lack of effective treatments. This study aims to test the effects of two potential anticancer compounds that have shown promising results in human cancer cells on canine glioma cells. Expression of MAP4K4, a serine/threonine kinase upregulated in many types of cancer cells and indicative of poor prognosis for survival, was targeted using specific siRNAs (200nM). Incubation for 48h resulted in high rates of siRNA transfection and led to significantly decreased MAP4K4 protein expression (P < 0.05) as determined using a specific MAP4K4 antibody. Notably, live cell imaging revealed that downregulation of MAP4K4 results in decreased cell migration as indicated by a significant delay in wound healing of m5 m/15mins (P < 0.05) com- pared to untreated control cultures at 6d post transfection. In addition, cells were treated with a novel, first in class inhibi- tor metarrestin, which disrupts the perinucleolar compartment, a subnuclear body that is selectively formed in highly met- astatic cancer cells but not in normal cells. Cultures treated with 5mM metarrestin for 24 hours showed significantly less growth (P < 0.01) compared to control glioma cells, suggesting an effect of this novel compound on glioma cells of canine origin. Our results suggest the potential applicability of both strategies for targeting canine glioma cells. Further analyses are underway to establish whether a two-pronged approach, using siMAP4K4 and metarrestin in combination, can further inhibit glioma cell proliferation and migration.

Research Grant: National Institutes of Health (HD093383), National Science Foundation Center for Cell Manufacturing (CMaT; 164805), U.S.D.A-NIH (2020-67015-30882), Georgia Partners in Regenerative Medicine Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

2021 National Veterinary Scholars Symposium • 119 Immune response elicited by implantation of a novel polyanhydride-based single dose vaccine

Micah Esala, Brett Sponseller, Sam Humphrey, Sean M. Kelly, Paola Boggiatto, Balaji Narasimhan, David Verhoeven, Douglas E. Jones

Dept. Vet. Pathology (Esala, Jones), Vet. Microbiology & Preventative Medicine (Sponseller, Verhoeven), College of Vet. Med., ISU, Ames, IA; USDA- ARS (Humphrey, Boggiatto), USDA, Ames, IA; Dept. Chemical & Biological Engineering (Kelly, Narasimhan), Nanovaccine Institute (Boggiatto, Narasimhan, Verhoeven, Jones), ISU, Ames, IA.

Historically, soluble vaccines with multiple booster vaccinations have been used to generate an effective immunity against infectious diseases. Concomitant immunity is the phenomenon that after a single infection, a host can be protected from subsequent disease from the same pathogen if the host remains chronically infected with the pathogen. In an effort to recapitulate concomitant immunity, implantable single-dose vaccines were surgically placed within C57BL/6 mice. Two experiments were done to determine the effectiveness of these implants.The first of which involved comparing different ratios of antigen and adjuvant with 2 polyanhydride chemistries with different degradation rates to observe which were most effective in eliciting an immune response. The second involved tracking antigen-specific CD4+ T cell populations after different methods of vaccination: a) bolus of soluble vaccine, b) an implant with a pressed vaccine rod that degrades from one surface, or c) a naked vaccine rod that degrades at all surfaces. We hypothesize that the soluble vaccine will induce a strong primary immune T cell response against the antigen in question but will decrease more rapidly compared to the implant which will release antigen and adjuvant at a steadier rate, imitating concomitant infection. In this way, we will discover how the altered CD4+ T cell and B cell response and the antigen release kinetics of these implants affect the overall immune response.

Research Grant: Research Grant: None Student Support: Student Support: Boehringer-Ingelheim Vetmedica, Inc. (BIVI)

What is our impact? Examining accessible veterinary services at Western University of Health Sciences’ CVM

Simon E. Ettenger, Rhea Hanselmann, Gillian G. Angliss

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA

Low-income and unhoused individuals face significant barriers in accessing veterinary care for their companion animals. Inadequate access to veterinary care has multiple negative consequences that include adverse public health outcomes, stress to the human-animal bond, and the prevention of unhoused individuals with pets from accessing shelter. The city of Pomona, California, home to Western University of Health Sciences’ College of Veterinary Medicine (WesternU CVM), has a large proportion of economically disadvantaged residents and evidence that many in need of veterinary services are unable to obtain them. The purposes of this project were to create an inventory of low-cost veterinary services currently provided by WesternU CVM, characterize the populations that have accessed veterinary care through these services, and illustrate the types of veterinary services provided. Medical records from WesternU sites offering low-cost veterinary services were reviewed for pertinent information, including geographic location of clients and specific preventive care ser- vices provided. This data will be used to assess the degree to which WesternU CVM’s programs benefit Pomona residents and guide the development of new or adjusted accessible veterinary healthcare offerings for companion animals in Pomona.

Research Grant: None Student Support: WesternU Student Summer Research Grant, WesternU CVM 2021 Veterinary Summer Research Program

120 • 2021 National Veterinary Scholars Symposium Histologic Classification ofTursiops truncatus Regarding Age and Freshwater Lesions

Ian J Evans, Christa Barrett, Debra Moore, Timothy W Morgan

Department of Pathobiology and Population Medicine (Barrett, Moore, Morgan), Institute for Marine Mammal Studies (Barrett, Moore), College of Veterinary Medicine (Evans), Mississippi State University, Starkville, MS

In 2019, the Bonnet Carre spillway and diversions for the Mississippi River released trillions of gallons of contaminated freshwater, from 31 states and 2 Canadian provinces that drain numerous agricultural and industrial sites, into the Mis- sissippi Sound. This caused a marked drop in the salinity of the Mississippi Sound as well as an influx of novel bacteria and fungi that contributed to increased Bottlenose Dolphin mortality and led to the declaration of an Unusual Mortality Event (UME) by the National Oceanic and Atmospheric Administration (NOAA). To better understand the pathogene- sis of dolphin mortality associated with fresh water incursion, it is important to understand how certain lesions correlate with known causes of mortality such as sepsis. Equally important is to determine how age correlates with morbidity and mortality in fresh water incursions. With this in mind, we studied decalcification methods (EDTA and Kristenson’s) for Tursiops truncatus teeth to determine if these methods result in increased conservation of histologic structure and clarity in the dental specimens to help with aging by counting Growth Layer Groups present in the dentin. Additionally, we clas- sified freshwater lesions histologically from stranded dolphins from the Mississippi Sound during the UME in 2019 and tested for any association between the presence of certain freshwater lesions and other indicators of sepsis, such as hepatic extra-medullary hematopoiesis (EMH) or pneumonia.

Research Grant: Mississippi Marine Mammal and Turtle Conservation, Recovery, and Monitoring Program, National Fish and Wildlife Foundation under Mississippi Department of Environmental Quality Agreement No. 18-00081 Student Support: Mississippi Department of Environmental Quality Agreement No. 18-00081

Dog with MRSP pyoderma converted to MSSP after Staphylococcus pseudintermedius vaccine administration

Emma F. Faddoul, Stephen A. Kania and Linda A. Frank

Department of Biomedical and Diagnostic Sciences (Kania), Department of Small Animal Clinical Sciences (Frank), College of Veterinary Medicine (Faddoul), University of Tennessee, Knoxville, TN

Staphylococcus pseudintermedius is a gram positive, coagulase positive, opportunistic pathogen that is the leading cause of canine pyoderma; it is also a common pathogen of otitis externa and normal flora on the skin and mucous membranes. Methicillin-resistant S. pseudintermedius (MRSP) is an emerging and challenging problem in the veterinary communi- ty, with some MRSP patients being left with no antibiotics to treat their infection. Our study documents that a dog with pyoderma infected with MRSP and vaccinated with a four-protein S. pseudintermedius vaccine over a 4-week period converted to having methicillin-susceptible S. pseudintermedius (MSSP). The dog had a strong antibody response to each component of the vaccine with the strongest response from the last sample taken following three injections. Western blot analysis confirmed the presence of the vaccine components in the susceptible bacteria and multilocus typing suggested that the resistant bacteria were replaced with susceptible bacteria rather than a change in resistance within the MRSP.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 121 A comparative anatomical study of the muscles of facial expression between Lycaon pictus and other canids

Mia A. Felix, Felicia A. Rocco, and Heather F. Smith

Department of Anatomy, College of Veterinary Medicine, Midwestern University, Glendale, AZ

African wild dogs, Lycaon pictus, are canids that are recognized for their high sociality and hypercarnivory. Detailed dis- sections were completed to assess the anatomical adaptations of facial musculature in three different canid species,Canis latrans (coyote), Canis familiaris (domestic dog), and L. pictus (African wild dog). A facial region of particular interest are the muscles surrounding the orbit as these muscles play a crucial role in facial expression. The three main muscles, mm. retractor anguli oculi lateralis (RAOL), orbicularis oculi, and levator anguli oculi medialis (LAOM) were identified. However, in the L. pictus specimen, the oculi were previously removed and portions of orbicularis oculi and RAOL were damaged, but still partially visible. Contrary to previous hypotheses, the wild canids L. latrans and L. pictus do possess m. LAOM just deep to the skin. The function of LAOM, to intensely raise the inner eyebrow, is closely associated with facial expression of domestic dogs as a social cue to their human counterparts. However, these anatomical findings may suggest an adaptation in wild canids to facilitate intraspecies communication. In L. latrans, m. zygomaticus terminated at the level of the commissure of the lip and met m. platysma without diving deep to it, in contrast to a deep condition in L. pictus and C. familiaris. This variation may be due to the young developmental age or compromised state of the specimen. In addi- tion, m. frontalis was present in all canids and sat just rostral to m. interscutularis overlying the frontal and parietal bones. The location of m. frontalis is debated in the literature however, we identified m. frontoscutularis as a separate muscle in each canid.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

Mass Antimicrobial Administration and Multi-Drug Resistance in Bovine Bacterial Respiratory Pathogens

Lindsey Fenster, Brent Credille

Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA

Antimicrobial resistance (AMR) in M. haemolytica and P. multocida, bacteria commonly associated with bovine respira- tory disease (BRD), is increasing in prevalence. Recent work would suggest that metaphylaxis might be one of the drivers of AMR in bacterial respiratory pathogens. The hypotheses of this study were that metaphylaxis is a primary driver of AMR in bacterial respiratory pathogens and AMR will negatively impact the outcome of antimicrobial therapy in cattle diagnosed with BRD. Therefore, the objectives of this project were to evaluate the impact of metaphylaxis on the prev- alence of multi-drug antimicrobial resistance (MDR) in M. haemolytica, P. multocida, and H. somni isolated from beef stocker calves at high risk of developing BRD and to determine how MDR impacts the outcome of antimicrobial therapy. For this randomized, double-blinded, placebo-controlled study, high-risk, sale barn origin beef calves were transported to a research stocker facility in Athens, GA. Calves were randomly assigned to receive either saline or tulathromycin at arrival processing and monitored for the development of signs consistent with BRD for 42 days. Serum and body weights were obtained from each calf at arrival processing and on days 14, 28, and 42. A deep nasopharyngeal swab (DNP) was collected from each calf at arrival processing and 14 days after arrival and submitted for bacterial culture and susceptibil- ity testing. Bronchoalveolar lavage (BAL) was performed on five randomly selected calves from each treatment group at arrival processing and 14 days after arrival to determine the prevalence of common viral respiratory pathogens. Addition- ally, serum, DNP and BAL were collected from calves diagnosed and treated for BRD.

Research Grant: Georgia Commodity Commission for Beef Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

122 • 2021 National Veterinary Scholars Symposium Hematology reference intervals for zoo-housed neonatal giraffe calves Giraffa( camelopardalis)

Hunter Ferchaw, Jennifer Thomas, Ronan Eustace, and Kimberly A. Thompson

College of Veterinary Medicine (Ferchaw, Eustace, Thompson) and Department of Pathobiology and Diagnostic Investigation (Thomas), Michigan State University, East Lansing, MI; Potter Park Zoo, Lansing, MI (Eustace); and Binder Park Zoo, Battle Creek, MI (Thompson).

Species-specific reference intervals are necessary for the proper interpretation of hematological values in zoo-housed species. However, references intervals may vary between age groups within a species, such as neonates vs. adults. The aim of this study is to provide age-specific reference intervals for neonatal zoo-housed giraffeGiraffa ( camelopardalis). Complete blood count (CBC) results were obtained by performing a retrospective survey of giraffe calves born at vari- ous zoological institutions throughout the United States between January 2016 and May 2021. Inclusion criteria for the study included healthy giraffe calves with: whole blood samples (EDTA or lithium heparin), collected via jugular vein phlebotomy while under manual restraint, and when less than 72 hours old. Reference intervals were calculated using the Reference Value Advisor freeware according to American Society for Veterinary Clinical Pathology’s (ASVCP) consensus guidelines for determination of de novo reference intervals. The results of this study will enhance the ability of clinicians to interpret hematology values for neonatal giraffes.

Research Grant: None Student Support: Boehringer Ingelheim and the Graduate School at Michigan State University

Development of Evidence Based Antibiotic Stewardship Recommendations For Empiric Canine Skin Infections

Kyle Ferguson, Keun Seok Seo, Margaret Khaitsa, Cory Langston, Cooper Brookshire

Mississippi State University College of Veterinary Medicine

Antibiograms are usually compiled from bacterial culture and antibiotic susceptibility (BCAS) test results from infec- tions that clinicians submit for testing. These data may be biased towards antibiotic resistant, refractory, or otherwise severe infections. There is a need to perform antibiotic susceptibility testing surveillance for infections that are typically treated empirically. This study aimed to create an antibiogram representative of canine skin infections typically treated empirically that included chlorhexidine susceptibility testing. Chlorhexidine topical therapy is the most commonly rec- ommended treatment for canine skin infections, but routine BCAS tests never assess chlorhexidine susceptibility. Twen- ty-four staphylococcal isolates were collected from canine skin infections from patients without a history of antimicrobial resistant infections and no history of antibiotic administration in the previous 6 months. All 24 isolates were susceptible to commonly recommended first-line antibiotics. Chlorhexidine susceptibility was determined using a hybrid MIC/Kirby Bauer drop-test; 6 mL of chlorhexidine solution at various concentrations around the epidemiologic cutoff value of 8m g/ ml were placed on Mueller-Hinton agar plates that were inoculated with each isolate. All 24 isolates were inhibited by 8 mg/ml chlorhexidine. These data suggest that chlorhexidine resistance is uncommon, and currently recommended first line antibiotics are appropriate for empiric treatment when there is a low clinical suspicion of resistance. Owing to reduced selection pressure for extended spectrum betalactamase resistance in bystander bacteria, clindamycin should be considered the preferred first-line therapy.

Research Grant: Research Grant: MSU-CVM ORGS Internal Faculty Grant Program Student Support: Student Support: MSU-CVM Summer Research Experience NIH T35; Boehringer Ingelheim

2021 National Veterinary Scholars Symposium • 123 Retrospective study of fish cancer prevalence and therapies

Emma Ferraro, Tara Harrison, Leigh Duke

Exotic Species Cancer Research Alliance, North Carolina State University College of Veterinary Medicine

This study evaluated fish cancer through medical records from zoos, aquariums and exotic animal veterinarians.The parameters evaluated included geographic location, habitat type, signalment, location of cancer, type of cancer, survival time, and treatments provided for each patient. This data was entered into the Exotic Species Cancer Research Alliance (ESCRA) database, then the resulting statistics were compiled and analyzed. Out of 511 cases, sex was known from 28.4% of patients, and most were female (14.5%). A majority of the submitted animals were from a zoological park or aquarium (57.3%), followed by private ownership (9.8%). The most common species reported was koi (spp. Cyprinus rubrofuscus) (25.0%). The most commonly specified location of primary tumors was the neck (7.0%), with soft tissue being the most commonly affected body system (22.1%). Spindle cell sarcoma was the most common primary histopathol- ogy tumor diagnosis (12.7%). Most of the reported cancers were malignant (65.2%). Only five cases reported any form of treatment for the tumors, with surgery being the only therapeutic method used. Four of these treated patients had their cancer completely excised and one animal had an incomplete excision of their cancer. None of these animals were report- ed to have any complications or had died as a result of their surgical procedures. This data suggests that there is a deficit in documented clinical therapy of cancer in fish species. However, despite this limited data, surgical excision of cancer in fish may be beneficial for clinicians to strongly consider or further investigate when feasible for the patient and client.

Research Grant: None Student Support: NC State Veterinary Scholars Program and the Boehringer Ingleheim Veterinary Scholars Program

Efficacy of tiletamine/zolazepam with butorphanol, medetomidine and azaperone for the immobilization of bison

Angelica Fieglar, Dave McRuer, Stephanie Hamilton, Nigel Caulkett

Department of Pathology and Microbiology (Fieglar, McRuer), Department of Companion Animals (Hamilton), Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI; Department of Veterinary Clinical and Diag- nositc Sciences (Caulkett), University of Calgary faculty of Veterinary Medicine, Calgary, Alberta.

Access to potent opioids for the purpose of wildlife immobilization has become increasingly difficult due to the human opioid crisis, stimulating a need for an alternative drug combination for bison immobilization. Combinations including tiletamine/zolazepam has shown success in this area but has also lead to some traumatic recoveries. In this study we evaluated two doses of tiletamine/zolazepam in a combination of tiletamine/zolazepam, medetomidine, butorphanol, and azaperone (MTZB+A) for reversible field immobilization. Eight healthy, male plains bison (Bison bison bison) from a captive herd were split into two treatment groups: a high dose (1.0mg/kg) or low dose (0.6mg/kg) of tiletamine/zolaze- pam. Induction times between the two groups differed by a few minutes but all immobilizations achieved a sufficient plane of anesthesia to permit surgical castration. All animals were reversed with naltrexone and atipamezole 60 minutes after induction and recoveries were uneventful. The mean time to recovery and the quality of recovery was the same in both groups. All animals became hypoxic while immobilized but were successfully treated with supplemental oxygen. The low- er dose of tiletamine/zolazepam for MTZB+A was sufficient for captive bison chemical immobilization but further study with a larger sample size is required to determine if the high and low doses are statistically significant.

Research Grant: Parks Canada Agency Student Support: Youth Employment and Skills Strategy (YESS)

124 • 2021 National Veterinary Scholars Symposium Can activity monitors improve our ability to assess outcomes and improve animal welfare in research settings?

Brent R. Fink, Jeremiah T. Easley, Lisa C. Hersh, Kevin M. McGhaw

College of Veterinary Medicine and Biological Sciences (Fink) and Preclinical Surgical Research Laboratory, Department of Clinical Sciences (Easley, Hersh) Colorado State University, Fort Collins, CO. College of Agriculture, Environmental and Nutritional Sciences (McGhaw), Tuskegee University, Tuskegee, AL

Behavioral metrics used to evaluate the recovery of sheep undergoing preclinical device testing are generally limited and often reflect a single point in time. Continuous activity monitoring can help to quantify the performance of medical therapies, postoperative recovery, and health status of research animals. Activity monitors designed for use in dogs have become widely available and provide an interesting opportunity for low-cost continuous monitoring of research animals when attached to wearable collars. We evaluated two commercially available accelerometer-based canine activity mon- itors (PitPat and Whistle) in sheep to establish baseline activity levels indoors and out on pasture. Sheep were outfitted with activity monitors and allowed to resume normal behavior. Average distance and time at rest were calculated for each animal and compared across the following groups: 1) PitPat vs. Whistle 2) Indoor vs. Outdoor and 3) Pre-surgery vs. Post-surgery. Both activity monitors were successfully worn on sheep in indoor and outdoor settings. Average recorded distances varied significantly between monitors (PitPat mean = 14.6 miles/day, Whistle mean = 2.7 miles/day), however there was high correlation between the two devices (r2 = 0.86). Understanding baseline activity for individual sheep and monitoring for deviations from these baselines may allow for early identification of subclinically diseased sheep, improv- ing our ability to treat illnesses/injuries earlier and more effectively. In a preclinical setting where novel medical devices are being tested, this activity data may act as a behavioral metric when determining relative advantages or disadvantages of a treatment or procedure over existing methods.

Research Grant: Funded internally through the Preclinical Surgical Research Laboratory, Colorado State University Student Support: USDA Animal Health & Disease Scholar

Determining the effect of neuron-derived extracellular vesicles on axonal regeneration of dorsal root ganglia

Amanda Flanagan, David Lin

Department of Biomedical Sciences (Flanagan, Lin), College of Veterinary Medicine, Cornell University, Ithaca, New York

Peripheral nerve diseases such as traumatic neuropathies and sensory neuropathies have been found in dogs and cats. A problem in this field is finding treatments for nerves that can slow the progress of these diseases. Nerve growth factor has been shown to promote the initial sprouting of axons, but other factors that help guide axons to their appropriate targets are still poorly characterized. Studies with Schwann- and olfactory ensheathing cell-derived extracellular vesicles (EVs) have been shown to promote axonal regeneration after nerve damage. This study tested whether other neuron-derived EVs also promote the growth of dorsal root ganglia (DRG) axons. This was accomplished by isolating individual DRG neurons from mice and culturing them with or without neuron-derived EVs. Additionally, whole DRGs were plated and treated with or without EVs. The DRG neurons and whole DRGs were stained with beta-tubulin antibody to detect neurite growth, and then analyzed by comparing axonal length between EV treated and control conditions. The application of neuron-derived EVs may provide a starting point for developing a treatment that can delay the progression of peripheral nerve diseases.

Research Grant: None Student Support: NIH T35OD010941 & Cornell University College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 125 A review of botanical anti-Inflammatories for the treatment of osteoarthritis

Tasman Flora, Michelle Kutzler

College of Veterinary Medicine (Flora), and Department of Animal and Rangeland Sciences (Kutzler), Oregon State University, Corvallis, Oregon

Botanical treatments for disease have been used for centuries and there has been increased interest in their use for vet- erinary medicine. However, little is known about how and if these compounds work or if they are safe to recommend. The purpose of this study was to evaluate botanical anti-inflammatories for pain management in cases of osteoarthritis. It was hypothesized that the efficacy of botanical treatments for management of osteoarthritis would not differ from the efficacy of using conventional, synthetic treatments (e.g., NSAIDs).The goals of the study were to review published in vivo research and report on the mechanism of action, efficacy, side effects and pharmacokinetics of each botanical. Two hundred and fifty-nine botanical compounds were identified within vivo anti-arthritic, anti-inflammatory, anti-oxidative, or anti-nociceptive properties that improved the symptoms of osteoarthritis. Nearly all of these (257/259) used laboratory animals (rats, mice, rabbits). About a fifth of these studies (46/259) used human patients. There were only four relevant studies in dogs. To serve as a guide for practitioners, the botanicals were grouped according to their mechanism of action and properties displayed. The current project has identified where gaps existed relating to the use of botanicals for osteoar- thritis in veterinary medicine.

Research Grant: None Student Support: Boehringer Ingelheim Graduate Summer Program

Unearthing the elusive Johne’s Disease: a widespread granulomatous and disseminated bacterial disease in goats

Brayden Foley, Brittanie Peake, Timothy Snider, and Jennifer Rudd

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

Johne’s Disease (JD), a widespread, chronic malabsorptive enteritis primarily affecting ruminants, results in malnutrition, chronic diarrhea, muscular wasting, and devastating production losses. The etiologic agent, Mycobacterium avium sub- sp. paratuberculosis (MAP), is a hardy bacterium shed in feces and milk that can survive on pasture or in water for over a year. While MAP infection occurs early in life, detection is difficult before the development of clinical disease, which occurs years later. Recent studies have provided additional insight into the pathogenesis of disease in cattle, but small ru- minant infections remain poorly described and do not directly mimic bovine disease. This preliminary study describes the extent of disease beyond the gastrointestinal tract and associated lymph nodes in the goat. Various tissues (GI, LN, liver, gall bladder, spleen, heart, lung, muscle, brain) were collected from ELISA+ Fecal PCR+ goats (n = 13). Histopathologic changes and severity were scored for each tissue by a board-certified veterinary pathologist. Marked granulomatous dis- ease was not only identified in small intestinal and associated lymph node sections, but also in liver samples from infected animals. In addition, lymphocytic infiltration was present in liver, gall bladder, and heart of several goats. RT-PCR de- tected MAP DNA in several tissues outside the enteric system at varying quantities, suggesting an important bacteremic phase. Results provide valuable data to aid future studies aimed at closely examining the virulence of MAP specifically in small ruminants as well as identifying potential immunologic responses and biomarkers that would aid in earlier detection of this disease.

Research Grant: None. Student Support: Oklahoma State University CVM, Associate Dean for Research & Graduate Education

126 • 2021 National Veterinary Scholars Symposium Effect of feeding sugar beet pulp on microbial butyrate production in healthy adult horses

Tamara D. Ford, Beverly J. Meena, Lynn M. Martin, Kile S. Townsend, Aaron C. Ericsson, Philip J. Johnson

Department of Veterinary Medicine and Surgery (Ford, Meena, Martin, Townsend, Johnson) and Department of Veterinary Pathobiology (Ericsson), College of Veterinary Medicine, University of Missouri, Columbia, MO

Colic is the leading cause of untimely death of horses, as well as the most common reason for emergency veterinary treat- ment among horses. Common conditions associated with hindgut microbial dysbiosis such as colic and colitis are often complicated by gut leakiness and inflammation. Butyrate, a short chain fatty acid produced by mutualistic bacteria in the gut, provides energy to colonocytes and mitigates intestinal hyperpermeability, ultimately promoting the overall health of the large intestine and the integrity of its mucosal barrier. Certain bacterial genera present in the equine gut microbiota uti- lize dietary fiber to produce butyrate. Unmolassed sugar beet pulp is a high fiber feed that is believed to promote microbial butyrate production and thus promote health. The purpose of this study is to determine the effect of the partial replacement of dietary hay with unmolassed sugar beet pulp on butyrate production capacity by bacteria in the gut of healthy adult horses. Fecal samples will be collected from 6 adult horses before and after the introduction of sugar beet pulp into their diet. DNA will be extracted from those samples and undergo 16S rRNA sequencing to determine the number of butyr- ate-producing bacteria present pre- and post-treatment. It is expected that the number of butyrate-producing bacteria, and therefore potential for, butyrate production, will increase as a result of introducing sugar beet pulp. The outcomes of this study could be utilized to improve management practices, reduce the incidence of large intestinal dysbiosis, and decrease emergency health issues in horses through the promotion of general gut wellness.

Research Grant: University of Missouri Metagenomics Center Animal Health Foundation of St. Louis Student Support: USDA National Institute of Food and Agriculture, Animal Project 1025109

Boosting host immunity against bacterial otitis media though disruption of pathogen mediated immunomodulation

Grace K. Forker, Kalyan K. Dewan, Eric T. Harvill

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Otitis media (OM) remains a leading cause of morbidity in children globally, with up to 50% of children receiving at least one diagnosis of OM by two years of age. There are no established vaccines available to prevent OM. Existing animal models of bacterial OM are limited in modeling natural progression of disease: injecting high-doses of human oto-patho- gens directly into middle ear bullae to establish infection. Recent work in the lab has shown that when inoculated with numbers as low as 500 CFU via the nasal cavity, Bordetella bronchiseptica, a well-known natural respiratory pathogen of mice, also infect the middle ears with numbers reaching over 105 CFU by day 7. Adoptively transferred antibodies against B. bronchiseptica failed to protect middle ears from infection, suggesting poor efficacy of conventional vaccine approach- es for prevention. Mice infected with B. bronchiseptica (BbΔbtrS), a mutant strain disrupted of its ability to suppress host immunity, generated a robust and protective immune response which conferred sterilizing and lasting immunity in all respiratory organs. Here, we examined whether immunity conferred by BbΔbtrS would also protect against middle ear in- fection of a natural mouse oto-pathogen. We observed that BbΔbtrS provided complete protection in all respiratory organs and the middle ears in convalescent mice. This highlights a potential for the development of vaccines based upon disrupt- ing pathogen mediated immunomodulation to prevent middle ear infection.

Research Grant: NIH R21 DC018496, Bacterial factors and host response in chronic otitis media Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

2021 National Veterinary Scholars Symposium • 127 Behavioural and physiological indicators of stress in horses participating in equine-assisted psychotherapy

Zoe Fournier, Chuyan Wei, Laurie McDuffee, William Montelpare

Departments of Health Management (Fournier, Wei, McDuffee) and Applied Human Sciences (Montelpare), University of Prince Edward Island, Charlottetown, PE

Equine-facilitated psychotherapy (EFP) is a branch of animal assisted therapy that focuses on treating mental illnesses by using horses as a therapy tool. EFP has been shown to be beneficial to humans; however, research on the participat- ing animals’ welfare is limited. The purpose of this study was to determine if participation in EFP causes stress in hors- es. Eight adult horses of varying breeds were used for the study. Each horse was paired with a veteran diagnosed with post-traumatic stress disorder for eight weekly sessions, 1.5 hours in duration. Therapy sessions consisted of grooming and leading the horses. Horses were instrumented with heart rate monitors (Polar ), which recorded heart rate variability (HRV) throughout the session. Saliva was also collected before and after each session to obtain cortisol measures. Heart rate variability data was analyzed using the Kubios HRV software. Cortisol concentrations were obtained using an ELISA technique. Behaviour scores were obtained through video analysis of the sessions, where frequency of stress behaviours was recorded based on a previously established stress ethogram. Descriptive statistics, comparison of measures before and after EFP sessions, and Bland-Altman plots were used to analyze the data. Results showed a significant decline in salivary cortisol levels in horses after the session (t-value 5.34, P < 0.001), and there were no significant changes in HRV measures indicative of stress. In addition, HRV and cortisol measures of stress were in agreement. Overall, the findings indicated that horses are not stressed during participation in EFP.

Research Grant: Horses and Humans Research Foundation Student Support: AVC Veterinary Summer Research Award

Melanomacrophage centers as a measure of lead toxicity in Lepomis megalotis from southeast Missouri

Jacob J Franzak, Katrina K Knott

College of Veterinary Medicine, University of Missouri, Columbia, MO (Franzak); Ecotoxicology and Conservation Physiology Program, Ecological Health Unit, Science Branch, Missouri Department of Conservation, Columbia, MO (Knott)

Lead (Pb) mining has a long history in Missouri, beginning in 1740 and once leading the globe in Pb production. While the footprint of active mining sites for Pb has decreased, historic contamination has raised concerns regarding the poten- tial negative ecological responses to the remaining mining waste. The Big River Watershed, located in southeast MO, was designated a Superfund site by the EPA in 1992 due to elevated Pb concentrations in sediment and fish tissue. Pb levels change dramatically at different points throughout the Big River, as distance from upstream chat piles increases and new rivers merge with the Big River downstream. The gradient of Pb levels in the sediment has caused a similar phenomenon to be observed in collected fillet and blood Pb levels of the longear sunfish (LSF, Lepomis megalotis). This study will measure melanomacrophage centers (MMCs) in the liver, an aggregation of phagocytic cells documented to be a histolog- ic indicator of toxicity and stress. The number, size, and density of MMCs in livers of LSF from the Big River Watershed will be compared to known concentrations of Pb in fillet, blood, and sediment. Fish (n = 15/site) were collected at three Pb-contaminated sites (2 sites on the Big River and 1 site on the Meramec River downstream of the Big River confluence) and 1 reference site (Caster River). We hypothesize that the fish with elevated concentrations of Pb in filet and blood sam- ples will exhibit a higher density of MMCs, indicating a dose-response relationship between Pb exposure and histologic toxic changes. This data will inform ongoing remediation efforts to improve the ecological health of the Big River, and support recreational and subsistence opportunities for all Missourians.

Research Grant: MO Department of Conservation, MO Department of Health and Senior Services (Center of Disease Control's Environmental Public Health Tracking & Agency for Toxic Substances and Disease Registry) Student Support: Stipend for Jacob Franzak is supported by an endowment established by IDEXX-BioAnalytics

128 • 2021 National Veterinary Scholars Symposium Efficacy of Meloxicam as an Analgesic in Mice Surgical Procedures

Layne Freeman, Brinley Cannon, Sarah Tan, Jeffrey Kim, Lon Kendall

Colorado State University (Freeman, Cannon, Tan, Kim, Kendall), Louisiana State University School of Veterinary Medicine (Freeman)

Mice are one of the most commonly used species in biomedical research and are often used as animal models in surgical procedures. Meloxicam is a non-steroidal anti-inflammatory drug (NSAID) that is frequently used for pain management in post-operative patients, but the effective dosages are not well understood or documented for mice surgical procedures. In this study, we measured the analgesic properties of meloxicam in female CD1 mice after undergoing an ovariectomy with a ventral laparotomy. The mice were treated with an initial dose of either saline solution (n = 8), meloxicam at 2.5mg/ kg (n = 8), or meloxicam at 10mg/kg (n = 8) prior to surgery with a maintenance dose every 12 hours up to 36 hours. Post-operatively, their pain was assessed by observing the following behaviors at 3, 6, 12, 24, and 48 hour time points: nesting, orbital tightness, rearing, wound licking, grooming, general activity, and arched posture. These findings should provide an effective protocol for pain management in mice surgical procedures.

Research Grant: None Student Support: Kenneth F. Burns Clerkship Program In Laboratory Animal Medicine and Pathology

Differentiation of PK resistant prion protein allelotypes in heterozygous sheep with classical scrapie

Rylie D. Frese, Justin J. Greenlee, M. Heather West Greenlee, Quazetta L. Brown, Eric D. Cassmann

Biomedical Sciences, Iowa State University College of Veterinary Medicine, Iowa State University Ames, IA (Frese, H Greenlee); Virus and Prion Research Unit, United States Department of Agriculture, ARS, NADC (J Greenlee, Brown, Cassmann)

The prion protein gene (PRNP) has a significant impact on the susceptibility of sheep to the scrapie agent. Sheep homo- zygous for lysine (K) at codon 171 are relatively resistant to scrapie in experimental and naturally infected flocks of sheep while sheep homozygous for glutamine (Q) at codon 171 are more susceptible. We previously reported that sheep with KK171 genotype did not develop clinical signs or detectable PrPSc after oronasal inoculation of the scrapie agent, and QK171 genotype sheep had prolonged incubation periods. We hypothesized that PrPSc in the brain of heterozygous QK171 sheep is mostly composed of PrPSc from the Q allele rather than the K allele. Our preliminary data demonstrates the ability to discriminate between QQ171 and KK171 PrPSc based on differential antibody binding; this was demonstrated by a western blot technique using two monoclonal antibodies, SAF84 and SHA31. SAF84 does not detect PrPSc from the K171 allele, whereas SHA31 binds to both the Q and K allelotypes. Therefore, this differential binding will allow us to quantify the relative amounts of PrPSc allelotype in heterozygous QK171 sheep. These findings will be important for expanding our knowledge on the scrapie resistance properties of the K171 polymorphism.

Research Grant: Congressionally appropriated funds to the USDA, ARS. The funders of the work did not influence study design, data collection and analysis. Student Support: Boehringer Ingelheim Summer Scholars Program

2021 National Veterinary Scholars Symposium • 129 Pharmacokinetics of acetylsalicylic acid and salicylic acid in dairy cattle

Bailey R. Fritz, Michael D. Kleinhenz, Shawnee R. Montgomery, Geraldine Magnin, and Johann F. Coetzee

Department of Clinical Sciences (Fritz, Kleinhenz) and Department of Anatomy and Physiology (Montgomery, Magnin, Coetzee), College of Veterinary Medicine, Kansas State University, Manhattan, KS

Administration of non-steroidal anti-inflammatory drugs (NSAID) may improve health and milk production in postpartum dairy cattle. Aspirin, or acetylsalicylic acid (ASA), is an NSAID that is available for use without prescription in the dairy industry. However, pharmacokinetic (PK) data for ASA in plasma are outdated, and there are currently no published milk residue or pharmacodynamic data available for ASA in dairy cows. The objectives of this study were to (1) describe the pharmacokinetics of ASA and salicylic acid (SA) in the milk and plasma of postpartum dairy cattle following oral admin- istration of aspirin; (2) to establish a recommended milk withdrawal period for dairy cattle treated with ASA; and (3) to determine the impact of ASA administration on plasma prostaglandin E2 metabolite (PGEM) concentrations compared to baseline values. Primiparous (n = 3) and multiparous (n = 7) postpartum Holstein dairy cattle were enrolled in the study. Animals received 2 oral treatments with ASA at 200 mg/kg, 24-h apart. Acetylsalicylic acid and SA concentrations in plasma and milk samples from 0-h through 120-h after ASA administration were analyzed using ultra performance liquid chromatography triple quadrupole mass spectrometry. Plasma PGEM concentrations in samples from 0-h to 24-h after ASA administration were determined using ELISA. The average change in PGEM concentrations compared to baseline for 2-h, 6-h, 10-h, and 24-h after administration of ASA was -64.7% 6 13.4, -48.3% 6 12.9, -31.0% 6 17.0, and -39.6% 6 17.6, respectively. These findings suggest that ASA administration may mitigate postpartum inflammation through reduction in prostaglandin production.

Research Grant: Kansas State University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program; KSU CVM Office of Research

The effects of passive vs. active enrichment on the stress levels of ball pythons

Courtney M. Fukushima, Manel Lopez-Bejar, Curtis Eng

College of Veterinary Medicine, Western University of Health Sciences, Pomona, California

Reptiles have become an increasingly popular pet, with over one million American households owning pet snakes (War- wick, 2018). Among all the various species of reptiles, we have chosen for our study the ball python (Python regius), one of the most popular household pet snake species owned in the United States. Given their increase in popularity along with a higher demand for veterinary care, it becomes even more vital to thoroughly educate future and present pet owners on proper care and husbandry. Proper education is so impactful that the life of the animal may even be extended, as inappro- priate husbandry has been demonstrated to be one of the leading causes of stress, disease, and impaired welfare in captive reptiles (Waeyenberge, 2018). With currently limited research published on this topic, this study will aim to demonstrate if the welfare and chronic stress levels in ball pythons (Python regius) will improve with provision of enrichment in enclo- sures and/or an increase in the frequency of handling. Ten ball pythons will be used in this study, in which five will receive a different enrichment item in their cage every other day (passive enrichment) while the other five will receive handling for a total of five minutes (active enrichment) also every other day. A baseline blood draw will be taken on day 0 and after a 2 week acclimation period, as well as a post-enrichment blood draw after our 47-day study to measure corticosterone. Corticosterone has been shown to be a useful biomarker indicative of stress levels in snakes (Waeyenberge, 2018). Our hope of this study is to provide objective evidence that enrichment, whether passive or active, lowers stress levels and therefore improves the welfare in captive snakes.

Research Grant: None Student Support: None

130 • 2021 National Veterinary Scholars Symposium Distribution of Coccidioides spp. lesions in necropsy cases at a veterinary teaching hospital

Brenley D. Fuller, Heather Reid, Alice Wang, Sylvia Ferguson, and Kathryn L. Wycislo

Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Coccidioidomycosis is a fungal infection endemic to the Southwestern United States. While initial infection with Coccid- ioides spp. typically occurs via inhalation of arthroconidia, the yeast form can disseminate widely throughout the body. Our study aimed to characterize the tissue tropism of Coccidioides spp. identified in necropsy cases submitted to the Mid- western University Diagnostic Pathology Center. Records were reviewed to identify necropsy cases that had confirmed Coccidioides spp. infection, based on the presence of intact spherules on histopathology. Over a five-year period (July 2016-2021), a total of 96 cases met the inclusion criteria. Necropsy case data was organized according to species, loca- tion of fungal spherules, and cause of death. Canine cases constituted the majority (60/96, 62.5%), with lesser numbers of equine (12/96, 12.5%), ruminant (12/96, 12.5%), feline (7/96, 7.3%), non-human primate (3/96, 3.1%) and exotic (2/96, 2.1%). Coccidioidomycosis was determined to be the cause of death in 67/96 (69.8%) of cases; however, in horses, Coc- cidioides spp. infection was often an incidental finding. As expected, the respiratory tract was the most commonly affected organ system amongst species; frequency of infection within other organ systems was also quantified. The information gained from this study will help contribute to the body of knowledge regarding Coccidioidomycosis in numerous veteri- nary species.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

Mathematical modelling to explore the role of environmental factors in avian cholera outbreaks in snow geese

Heather Fumia, Jess Dennehy, Harrison Clark, Wendy Beauvais

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana (Fumia, Clark, Beauvais); London School of Hygiene and Tropical Medicine, University of London, London, United (Dennehy)

Avian cholera, caused by the bacterium Pasteurella multocida, is a significant infectious disease affecting birds world- wide, including commercial poultry. Carrier birds act as reservoirs for P. multocida, but the role of the environment as a reservoir has not been fully elucidated. A recent study suggested that P. multocida may exist within Acanthamoebae, which are abundant in water sources and soil, but further proof of this interaction is needed. To explore this hypothesis, we utilized a mathematical model to determine if this mechanism could play a role in avian cholera outbreaks in snow geese (Anser caerulescens). We studied two snow geese migration sites in Indiana and Nebraska, which both have had more than one outbreak in the last 10 years. Publicly available data on snow geese sightings, occurrence of avian chol- era outbreaks, and hourly soil temperatures were collected from 2011-2021 and plotted to explore year-to-year trends and compare outbreak and non-outbreak years. An existing compartmental model was adapted to simulate the dynamics of Acanthamoebae and P. multocida at different temperatures. Based on the results from the model, which assumes that Acanthamoebae exist in the active trophozoite form between 15-368C, these amoebae in the soil are unlikely to play a role in avian cholera outbreaks in our study sites because the temperatures are not warm enough for the amoebae to be infected by P. multocida. The hypothesis may be supported in warmer climates and it is possible that there are other contributing environmental factors, like biofilms, other free-living amoebae, or vectors, but more research must be conducted.

Research Grant: None Student Support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

2021 National Veterinary Scholars Symposium • 131 Surgical approaches to the canine liver hilus: a comparison of five techniques and evaluating diaphragmotomy

Cristina A. Gabaldon and Judit Bertran

Department of Surgical Oncology (Bertran), College of Veterinary Medicine, University of Florida, Gainesville, FL

Canine hepatocellular carcinoma comprises 35-60% of all canine primary hepatic tumors. Over 60% of these tumors pres- ent as a single, massive tumor that can arise from different areas of the liver. Treatment of choice includes surgical resec- tion with prognosis dependent on the size and location of the tumor and surgical outcomes. Massive liver tumors arising from the hilus of the liver present a particular challenge in surgical resection, as the common surgical practice of midline approach does not allow for optimal access to the liver hilus located deep to the rib cage. We described and compared five different surgical approaches to the liver that use median laparotomy, median laparotomy with caudal sternotomy, right paracostal, left paracostal and mercedes incisions. We calculated the visual field offered, as well as, scored import- ant anatomical structures based on their visibility using each approach. Data thus far demonstrates that amongst the five approaches, the conventional median laparotomy offers the smallest visual field and the most limited access to the liver hilus. Furthermore, performing a diaphragmotomy has revealed the dynamic effects between the liver and thoracic cavity. By inducing pneumothorax, further access to the liver hilus has been gained through additional caudal manipulation of the liver. Additional testing of the five surgical approaches and diaphragmotomy to effectively permit a complete view of the liver hilus and perihepatic structures are underway.

Research Grant: Boehringer Ingelheim Veterinary Scholars Programs and UF’s College of Veterinary Medicine Department of Surgical Oncology Student Support: BI Veterinary Scholars Programs and UFCVM’s Department of Surgical Oncology

The effect of BIO-PLY, a novel PRP lysate, on synoviocyte/chondrocyte co-culture supernatant viscosity

Rachel Gagliardi, Jessica M. Gilbertie, Alicia G. Schubert, Julie M. Long, Lauren V. Schnabel, Heidi L. Reesink

Department of Clinical Sciences North Carolina State University College of Veterinary Medicine (Gagliardi, Gilbertie, Schubert, Long, Schnabel) and Department of Clinical Sciences Cornell University College of Veterinary Medicine (Gagliardi, Reesink)

Osteoarthritis is a debilitating disease that affects both humans and animals. It is a progressive disease that often results in significant pain, limited mobility, and reduces overall quality of life. Currently, there are no therapeutics in human or veterinary medicine that markedly alter the progression of OA and none that are able to reverse the negative effects of this disease. The purpose of this study was to investigate the effect of BIO-PLY, a novel platelet-rich plasma lysate, on sy- noviocyte/chondrocyte co-culture supernatant viscosity. Previously, BIO-PLY has been shown to have significant anti-in- flammatory properties and increase synoviocyte hyaluronic acid (HA) production.We hypothesize that supernatant from synoviocyte/chondrocyte co-cultures treated with BIO-PLY will have a greater viscosity than those untreated or those treated with conventional treatments (exogenous HA or steroid). Further, we hypothesize that the supernatant viscosity will increase over time (across our 24h, 48h, and 72h timepoints). In order to test these hypotheses, a co-culture system of donor matched equine synoviocytes and chondrocytes was utilized. LPS stimulation was used to induce an OA-like phenotype. Treatments were implemented 24h post-LPS stimulation: no treatment control; exogenous HA; steroid; and BIO-PLY. Samples were collected at 24h, 48h, and 72h. Supernatants were analyzed on a viscometer-rheometer on a chip (m-VROC, RheoSense). Apparent viscosity measurements were averaged to give an estimated apparent viscosity of each sample. Preliminary results demonstrate that BIO-PLY may increase the supernatant viscosity as compared to the other treatment groups previously mentioned. Further investigation is underway.

Research Grant: Fund for Orthopedic Research in honor of Gus and Equine athletes (F.O.R.G.E.) Student Support: NIH T35 AI007227

132 • 2021 National Veterinary Scholars Symposium Radiation lowers humeral cortical bone density but also osteoarthritis incidence in non-human primates

Nicole A. Gagnon, Nequesha S. Mohamed, J. Mark Cline, John Olson, Jeffrey S. Willey

College of Veterinary Medicine (Gagnon), North Carolina State University, Raleigh, NC; Department of Orthopedic Surgery (Mohamed), Pathology (Cline, Olson), Radiation Oncology (Willey), Wake Forest School of Medicine, Winston Salem, NC

Total body irradiation (TBI) has long-term health effects; childhood cancer survivors treated with TBI are more likely to develop diabetes mellitus (DM). DM induces a pro-inflammatory environment conducive to developing osteoarthritis (OA). Radiation is associated with arthropathy and lowered bone mineral density (BMD). This study utilized a non-human primate (NHP) model to test the hypothesis that osteoporotic changes and OA measured by computerized tomography (CT) occur with increased incidence in DM TBI NHPs when compared to TBI-only, DM-only, and control NHPs. Rhesus macaques (n = 134) were previously exposed to 1.1-8.5 Gy; other NHPs (n = 32) were unexposed. At the time of imaging, 9 irradiated NHPs had DM, while zero non-irradiated NHPs were diabetic; however, two developed DM fifteen months later. CT analysis of the proximal humerus revealed a lower cortical volume (P < 0.001), lower cortical BMD (P < 0.001), and shorter humeral length (P = 0.003) in TBI-only primates vs other groups. Trabecular BMD was higher (P = 0.006) in TBI-only NHPs vs control. Greater cortical volume, marginally higher cortical BMD, and similar trabecular BMD for DM TBI vs control indicated a differential response related to DM status.After sub-cohort analyses for OA (n = 86), NHPs with shoulder OA had higher cortical and lower trabecular BMD values (P < 0.05 for all). Shoulder OA severity was high- er in DM-only and control, while reduced in TBI-only NHPs (P = 0.009). TBI in non-DM NHPs resulted in expected bone deficits. However, irradiated NHPs with DM did not exhibit similar bone changes but rather opposite responses. As OA was not associated with TBI, a correlative link between TBI and DM status with skeletal deficits was not supported.

Research Grant: NIH U01AI150578 Student Support: T35 OD010946

Endoparasite survey of foxes and coyotes on Prince Edward Island, Canada

Olivia Galeuzzi, Will Robbins, Gary Conboy, Spencer Greenwood, Melanie Buote, Megan Jones

Canadian Wildlife Health Cooperative, Atlantic Region (Galeuzzi, Jones), Pathology and Microbiology (Galeuzzi, Conboy, Buote, Jones), Biomedical Sciences (Robbins, Greenwood), Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada

The Red Fox (Vulpes vulpes) and the Eastern Coyote (Canis latrans), which inhabit Prince Edward Island (PEI), Canada, are hosts to various parasite species. The diversity and distribution of helminths within these wild canids on PEI remains largely unknown. For instance, the French Heartworm (Angiostrongylus vasorum), which causes potentially fatal disease in dogs, has recently been identified in coyotes in Nova Scotia, a precedent for its likely spread to PEI.Additionally, in the last several years, sarcoptic mange has become an important emerging ectoparasitic disease and management challenge for both foxes and coyotes in PEI, and the potential relationship between helminth infection intensity and diversity, and risk of sarcoptic mange, has never been investigated. The aims of this study were to describe the diversity and distribution of helminths in wild canids on PEI; perform targeted surveillance for Angiostrongylus vasorum; and investigate a possible relationship between helminth burden and incidence of sarcoptic mange. In order to identify and quantify helminth diver- sity in wild canids, lung and GI parasite recovery procedures were conducted on trapper-sourced coyotes (n = 30) and fox- es (n = 60) from PEI collected during the 2020-2021 trapping season. Results from a parallel sarcoptic mange study will be used to assess the relationship between sarcoptic mange and endoparasite load. Final results will follow once parasite recovery procedures on all specimens are complete. Preliminary findings include the discovery of the zoonotic tapeworm, Echinococcus sp., the first report of this important parasite in Prince Edward Island.

Research Grant: Canadian Wildlife Health Cooperative, Atlantic Region Student Support: Canadian Wildlife Health Cooperative, Atlantic Region; AVC VetSRA Program

2021 National Veterinary Scholars Symposium • 133 The effects of Losartan use in dogs on stifle joint pain and progressive osteoarthritis following TPLO

Alondra M. Gallego, Dr. Loic Dejardin, Dr. WillIam Chung, Char Panek, Dr. Karen Perry, Dr. Aimee Colbath

DVM Student (Gallego), Head of Orthopedic Surgery, Small Animal Clinical Sciences (Dejardin), Small Animal Clinical Sciences (Chung, Perry), Large Animal Clinical Sciences (Panek, Colbath), College of Veterinary Medicine, Michigan State University, East Lansing, MI

The aim of this study is to evaluate postoperative progression of osteoarthritis (OA) and joint pain in dogs that have undergone a tibial plateau leveling osteotomy (TPLO) procedure and received Losartan. Fifty dogs will be randomly assigned and evenly divided to receive either Losartan or a placebo every 12 hours by mouth, along with a Non Steroi- dal Anti-Inflammatory Drug (NSAID) typically given following a TPLO. At the initial appointment and at recheck ap- pointments held at 4, 8 and 16 weeks after surgery, an orthopedic physical exam, subjects will be assigned a veterinarian assignment numerical score based on lameness, weight-bearing and pain response to joint flexion and extension. Pain will also be assessed by the veterinarian and owner using a visual analogue scale (VAS). Force-platform gait analysis will be used to objectively assess weight-bearing. Radiographic images of the stifle will be taken to assess severity of OA and monitor progression over time. It is hypothesized that dogs receiving Losartan following a TPLO will have lower (closer to normal) veterinarian assignment numerical lameness, weight-bearing, and pain scores, lower visual analogue scores (closer to no pain), higher peak vertical ground reaction forces and vertical impulses and less osteophytes and effusion radiographically. Since this is an ongoing clinical trial in which the recruitment phase has just begun, there is currently no data available to interpret. If our hypothesis is correct, Losartan can revolutionize the treatment of OA and joint pain in dogs following a TPLO, as there are currently no disease-modifying drugs available for the treatment of OA and long-term use of NSAIDs can cause harmful effects to multiple organ systems.

Research Grant: Endowed Research Funds Project Student Support: NIH T35

Impact of age and weaning stress on histamine receptor subtypes localization and expression in GI tract

Carmen-Maria Garcia, Kyan Thelen, Mahsa Fardisi, Adam J. Moeser

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Early weaning (EW) is a common practice in pig production but causes high levels of stress during a critical period of gas- trointestinal (GI) immune development can lead to long-term deleterious changes in GI barrier and immune development and increased disease risk. The mechanisms by which EW stress alters long-term disease risk remain poorly understood and interventions are lacking. Recently our lab showed EW piglets exhibit a significant elevation in plasma histamine levels, heightened intestinal mast cell (MC) activation and upregulation of histamine receptor subtypes 2 (H2R) and 4 (H4R) gene expression, but not H1R expression. To gain a better understanding of how H2R is mediating EW-associated intestinal inflammation, the objective of this study is to determine the expression pattern and localization of HR subtypes within the intestinal mucosa. We hypothesize that the EW piglets will exhibit increased intestinal numbers of HR2- and HR4-positive cells following weaning stress and HR subtypes will exhibit different localization patterns. Jejunum was harvested from 15d old (EW) and 28d old (LW) piglets at 0hr and 24hr post-weaning, and then stained with via immuno- histochemistry with specific antibodies to identify HR1, HR2 and HR4 receptors after which the numbers of HR-positive cells and their localization within the intestinal mucosa will be quantified and categorized. Learning how age and weaning influences the HR expression and their localization could provide insight into the mechanistic role of histamine and HRs in early and long-term gut development.

Research Grant: Unknown Student Support: Foundation for Food and Agriculture Research, and BRUSH

134 • 2021 National Veterinary Scholars Symposium Exposure-response of amlodipine on the renin-angiotensin-aldosterone system and blood pressure in healthy cats

Tatiana M. Garcia Marrero, Jessica L. Ward, Melissa A. Tropf, Agnes Bourgois-Mochel, Emilie Guillot, Lingnan Yuan, and Jonathan P. Mochel

Department of Veterinary Clinical Sciences (Garcia, Ward, Tropf, Bourgois-Mochel) and Biomedical Sciences (Yuan, Mochel), College of Veterinary Medicine, Iowa State University, Ames, IA; Ceva Sante Animale (Guillot), Libourne, France

Systemic hypertension is a common cardiovascular pathology in older cats that is treated primarily with the calcium channel blocker amlodipine besylate (AML). In dogs, AML is known to activate the renin-angiotensin-aldosterone sys- tem (RAAS), which can lead to negative long-term cardiovascular consequences, such as excessive arterial stiffening, and myocardial fibrosis. Currently, little is known about the effect of AML on biomarkers of the feline RAAS, nor about chronobiological variations of the RAAS in healthy cats. The objectives of the present study were to evaluate pharma- cokinetics of AML administration in healthy cats, to investigate the pharmacodynamic response of AML on blood pres- sure and RAAS biomarkers, and to characterize patterns of diurnal variation in the feline RAAS. This study enrolled 20 healthy cats older than 5 years randomized by sex to two treatment groups using a cross-over design. Cats received each treatment (AML 0.625mg PO once daily or identical placebo) for a period of two weeks with a washout period of 4 weeks in between treatments. Data collected at baseline and after each treatment period included non-invasive blood pressure, hematology and biochemistry panels, and echocardiography. At the conclusion of each treatment period, multiple blood samples were collected over a 24-hour period for serial quantification of plasmaAML and serum RAAS biomarker levels. Blood pressure and RAAS biomarker data will be compared between treatment groups and correlated with plasma AML levels. We expect that AML-treated cats will have lower blood pressure and higher levels of all RAAS biomarkers com- pared to placebo-treated cats, suggesting that AML activates both the classical and alternative RAAS pathways.

Research Grant: Ceva Sante Animale Student Support: National Institutes of Health T35 Grant

Alterations in the Gut Microbiota and Immune Responses Associated with NADF Induced by PFOS

Jasmine S. Gardner, Jacob Siracusa, Tai L. Guo

1)Department of Veterinary Medicine, Tuskegee University College of Veterinary Medicine DVM Candidate class of 2024, Tuskegee, AL. 2)Department of Veterinary Biosciences, College of Veterinary Medicine, University of Georgia, Athens, GA

An increase of fat ($5%) within hepatocytes, which is not a direct result of alcohol consumption, is used to clinically define Non-Alcoholic Fatty Liver Disease (NAFLD), the most common cause of liver disease globally. This study aims to observe how perflourooctane sulfonic acid (PFOS), affects the progression of NAFLD via changes in the gut microbiota and associated changes in the innate and adaptive immune systems leading to NAFLD progression. In this study, NODEF male mice were randomly divided into 2 groups based on blood glucose levels (BGLs) at baseline: non-diabetic (ND), dosed with vehicle control (VC), 1 mg/kg PFOS (PFOS-L), or diabetic (D) at baseline and dosed with VC, PFOS-L, or 10 mg/kg PFOS (PFOS-H). Mice were given access to a Western Diet (WD) and orally treated by gavage daily for 6 months. Weekly measurements of BGLs and body weights (BW) would be taken. Monthly, we conducted glucose tolerance tests (GTT) and insulin tolerance tests (ITT) and measured food and water consumption. Also, we conducted a series of behav- ior tests. After 1 month of exposure there were no significant changes in the BGLs, BWs, or food and water consumption rates between treatment groups and respective controls. GTT revealed significant increases in BGLs at 15-min time point after glucose administration in both the D-and ND-PFOS-L mice after 1 month. Additionally, BGLs in the D-PFOS-H mice were significantly increased at 0-min time point compared to controls, but this effect was mitigated after glucose test. In contrast, ITT revealed no significant changes in BGLs obtained at any of the time points analyzed when compared to their controls. Overall, PFOS is shown to dysregulate glucose metabolism suggesting metabolic alterations.

Research Grant: None Student Support: NIH Grant

2021 National Veterinary Scholars Symposium • 135 Evaluating the causes and consequences of canine food motivation within the Dog Aging Project Pack cohort

Kathleen Gartner, Kellyn McNulty, Annette L. Fitzpatrick, Audrey Ruple, Zihan Zheng, Kate E. Creevy

Small Animal Clinical Sci (Gartner, McNulty, Creevy), College of Vet Med & Biomed Sci, Texas A&M, College Station, TX; Epidem, Family Med (Fitzpatrick), Biostats (Zheng), Schools of Med and Public Health, University of Washington, Seattle, WA; Public Health (Ruple), College of Health and Human Sci, Purdue, West Lafayette, IN

Obesity in dogs is a growing health crisis and a complex multifactorial disease. While recent data suggest that eating behavior in dogs influences the development of obesity, little is known about the particular factors that may affect a dog’s eating behavior. This study utilized various surveys including the validated Dog Obesity Risk Assessment (DORA) ques- tionnaire to evaluate the influence of several intrinsic and extrinsic factors on a dog’s level of food motivation. Addition- ally, the same surveys were used to assess the impact of dog food motivation and owner management factors on canine obesity. Dogs were obtained from a nationwide, observational, prospective study, the Dog Aging Project (DAP). All dogs in the study (n = 410) completed the same set of relevant questionnaires and had a current obesity status assigned by their owner. Additionally, a subset of dogs (n = 120) also had current (within the calendar years 2020-2021) body condition scores (BCS) as determined from their veterinary medical records. We expect to see associations between food motivation and one or more dog demographic (intrinsic) and/or environmental (extrinsic) factors. We also anticipate that it will be more likely for dogs with a higher food motivation score to be overweight and that in comparison to veterinarians, owners will underreport their dogs as being overweight. By improving our understanding of canine eating behavior, veterinari- ans will be better equipped to recognize dogs at innate risk of developing obesity and to identify interventional targets to minimize that risk.

Research Grant: This study is supported by the U19AG057377 grant from the National Institute on Aging, a part of the National Institutes of Health, and by private donations. Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Evaluation of CD8+ T cell exhaustion markers following Chlamydia genital infection

Je’Pierah Gasper, Weidang Li, and Ashlesh K. Murthy

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Chlamydia trachomatis is an obligate intracellular bacterial agent that is considered one of the most widespread sexually transmitted infections. C. trachomatis can lead to serious sequelae among women, including pelvic inflammatory disease, tubal factor infertility, ectopic pregnancy, and chronic pelvic pain; however, in some populations, C. trachomatis can be asymptomatic and left undetected. Using the Chlamydia muridarum mouse model of infection, we have previously shown that CD8+ T cells are important for protective immunity against intracellular pathogens. During chronic infection, CD8+ T cells are exposed to persistent antigenic and inflammatory signals that can gradually lead to the deterioration of T cell function. CD8+ T cells express multiple inhibitory receptors, such as programmed cell death protein-1 (PD-1), lympho- cyte-activation gene-3 (LAG3), and T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3). The aim of this study is to determine how specific inhibitory receptor signals integrate to promote and maintainT cell exhaustion, and further understand how chlamydial infections persist and cause pathology. Twenty mice will be injected with 15mg of medroxyprogesterone (Depo-Provera), an inhibiting agent that suppresses ovulation and pregnancy. The injection will occur 5 days prior to intravaginal challenge with C. muridarum (5x104 CFU/mouse). An additional twenty mice will be unchallenged and act as the control. Four groups of infected mice (~5 mice/group) will be euthanized at weeks 2, 4, and 5 (ten infected and five uninfected at week 5) to extract CD8+ T cells from the spleen and genital tract for analysis of T cell exhaustion surface markers.

Research Grant: NIH NIAID 2R15AI101920-03 Student Support: Boehringer Ingelheim Veterinary Scholars Program

136 • 2021 National Veterinary Scholars Symposium Effect of water extract from Euglena gracilis on granulocytic differentiation and activation of promyeloblasts

Damilola Gbore, Susumu Ishiguro, Deepa Upreti, Masaaki Tamura

Department of Anatomy and Physiology (Gbore, Ishiguro, Upreti, Tamura), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas; Faculty of Veterinary Medicine (Gbore), University of Ibadan, Ibadan, Oyo State, Nigeria

Euglena gracilis is a green alga which has been shown to have potential medicinal properties. This team’s recent study in- dicated that treatment with a water extract from Euglena gracilis (EWE) may link the inhibition of murine lung carcinoma growth and the attenuation of granulocyte function. The current study aims to further investigate the immunomodulatory properties of EWE by assessing its effect on granulocytic differentiation and activation using a human promyeloblast cell line (HL-60). Differentiation of HL-60 cells was induced with Dimethylsulfoxide (DMSO) for 4 days and cells were then stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours in the presence of EWE at 1, 10 and 100 mg/ml. Both differentiation and activation were evaluated by analyzing changes in cell morphology and surface marker expression us- ing optical microscopy and flow cytometry respectively. CD11b, CD16 and CD116 were selected as markers for granulo- cytes, monocyte/macrophage and neutrophils respectively. Actin polymerization was used as a functional assay. Although some minor morphological changes were observed after treatment, these changes were unable to be quantified. Flow cyto- metric analysis revealed that treatment with EWE significantly decreased populations of both CD11b+ and CD116+ cells regardless of the PMA treatment. Spontaneous actin polymerization was also decreased by treatments of all three EWE concentrations. However, PMA-induced actin polymerization in CD11b+ cells was significantly inhibited by only the high concentration of EWE. No significant changes were observed in CD16+ and CD116+ populations. These results suggest that EWE is a potent inhibitor of both granulocytic differentiation and functional activation.

Research Grant: Kansas State University, Johnson Cancer Research Center Innovative Research Award and research grant from euglena Co. Ltd Student Support: IDEXX Laboratories, Inc

Detection of pathogenic Leptospira spp. in fish

Allison Geer, Conner Duke, Scott Brovarney, Emily Willems, Joey Morgan, Aggy Vanderpool, LaRoy Brandt, and Ashutosh Verma

College of Veterinary Medicine (Geer, Duke, Brovarney, Willems, Morgan, Verma), School of Mathematics and Science (Vanderpool, Brandt), Cumberland Mountain Research Center (Vanderpool Brandt), Center for Infectious, Zoonotic and Vector-borne diseases (Verma), Lincoln Memorial University, Harrogate, TN

Leptospirosis is an important zoonotic disease that accounts for significant morbidity and mortality in animals and hu- mans, and is maintained in a population due to chronic kidney infection of reservoir mammals. Earlier work from our lab has shown that rodents, voles, shrews, chipmunks, cottontails and several species of amphibians and reptiles are reservoirs of Leptospira spp. in the Cumberland Gap Region of KY, TN and VA. The aim of this study is to determine if fish con- tribute to the maintenance of leptospirosis in the aquatic environment. We will be collecting 300 fish belonging to various species from the Powell River in Harrogate, TN, and their kidneys will be screened with a highly sensitive and specific Taq-Man quantitative PCR (qPCR). So far, we have collected 110 fish belonging to the following species: bass Amblop( - lites (17), MIcropterus (7)), stone rollers (Campostoma (16)), darters (Percina (5), Etheostoma (2)), chubs (Erimystax (2), Semotilus (5), Nacomis (5)), sunfish (Lepomis (22)), catfish Pylodictus( (1)), hogsuckers (Hypentelium) (3)), shiners (Luxilus (9), Notemigonus (6), Cyprinella (1)), longnose gar (Lepisoteus (3)), minnow (Pimephales (1)), Redhorse (Mox- ostoma (5)). Genomic DNA from kidneys of 81 fish were extracted and screened by qPCR. Only one kidney was positive for leptospiral DNA and contained 1 x 104 GE/g (genomic equivalents per gram) of kidney tissue. Multi locus sequence typing (MLST) will be used to genotype leptospires present in positive kidney. Multiple gene loci will be PCR amplified, sequenced and compared with available homologous gene sequences in the databases. This study will provide information on the role of fish in the epidemiology of leptospirosis in the region.

Research Grant: LMU-CVM intramural grant to AV Student Support: BIVSP scholar, LMU CVM summer research program

2021 National Veterinary Scholars Symposium • 137 Biological and hematological profiles of bighorn sheep (Ovis canadensis)

Morgan F. Gentzkow, Brianna Beechler, Robert Spaan

Department of Biomedical Sciences, Carlson College of Veterinary Medicine (Gentzkow, Beechler), Department of Fisheries, Wildlife, and Conservation Science (Spaan), Oregon State University, Corvallis, Oregon

Many wildlife species are lacking reference ranges for biochemical parameters. Having an established reference range for each species can aid in predicting the health and survival of the animal. The objectives of this study were to (1) establish reference ranges for biochemical parameters on wild California and Desert bighorn sheep subspecies (Ovis canadensis), (2) determine whether both subspecies have similar reference ranges, (3) determine whether biochemical parameters can be used to predict the sheep’s survival eight months post capture, and (4) determine how the biochemical parameters of an adult ewe may predict whether she can raise her lamb to a weanling. Reference intervals for California bighorn sheep (n = 135) were calculated using a non-parametric method from blood samples taken during 2016 and 2017 capture events. The sheep ranged from age 0-11, with a mix of males and females. The calculated reference ranges tended to vary from recorded ovine reference ranges from OSU-VDL and the reference ranges that Borjesson et. al (2000) calculated for free ranging desert bighorn sheep. Reference ranges for the Desert bighorn sheep blood samples taken during 2020 still need to be processed and calculated. Once, that is completed, we will go through and compare the calculated reference ranges of the two subspecies and survival of sheep and their young if applicable. Reference ranges from this study should be able to be used on other California and Desert bighorn sheep.

Research Grant: Oregon Department of Fish and Wildlife, Nevada Department of Fish and Wildlife, and California Department of Fish and Wildlife Student Support: Boehringer-Ingelheim Veterinary Scholars Program (BIVSP)

PFAS bioaccumulation and associated wild fish and human health impacts in the Detroit River, Michigan USA

Serena E. George, Bridget B. Baker, Alex Haimbaugh, Tracie R. Baker

School of Veterinary Medicine, University of Wisconsin-Madison (George), Madison, WI; Institute of Environmental Health Sciences (B. Baker, T. Baker), Department of Pharmacology School of Medicine (Haimbaugh, T.Baker), Wayne State University, Detroit, MI

Per- and polyfluoroalkyl substances (PFAS) are artificial endocrine disruptors that are particularly stable and pervasive due to strong carbon-fluorine bonds. They are known to bioaccumulate in protein-rich tissues of fish and do not degrade with cooking. Despite controlled animal experiments linking PFAS to adverse health outcomes, there are no national regulations of PFAS as a hazardous material at this time. To assess bioaccumulation of PFAS in wild fish and the potential human health implications, we analyzed the concentration of 40 PFAS from muscle and serum samples of fish represent- ing different trophic levels along the Lake Huron - Lake Erie Corridor. In Summer 2021, walleye (Sander vitreus), gizzard shad (Dorosoma cepedianum), yellow perch (Perca flavescens) and white sucker (Catostomus commersonii) were collect- ed for analysis from the Detroit River (contaminated site) and St. Clair River (reference site). Additionally, invasive round gobies (Neogobius melanostomus) were humanely euthanized to obtain gonad and liver histology samples as well as data to assess morphological (hepatosomatic index, gonadosomatic index, Fulton’s condition factor) PFAS exposure effects. Correlations between PFAS concentrations in wild fish tissue and biomarkers identified through controlled zebrafish trials will be analyzed. We hypothesize that PFAS tissue concentration will be associated with gene alterations involving immune system function, endocrine system function, and reproduction in wild fish. Understanding genetic and morpho- logical alterations as well as evaluating PFAS levels in wild fish collected for human consumption will allow for a better understanding of potential ecological and human health impacts of PFAS exposure.

Research Grant: Great Lakes Water Authority Student Support: UW-Madison SVM Summer Scholars Research Program, WSU WATER Lab Summer Internship Fellowship

138 • 2021 National Veterinary Scholars Symposium Evaluation of contamination risk associated with forced-air warming units in canine surgery

Jordan P. Gerras, Kyle G. Mathews, Megan E. Jacob, James B. Robertson

Department of Clinical Sciences (Mathews), Department of Population Health and Pathobiology (Jacob), and Office of Research (Robertson), College of Veterinary Medicine, North Carolina State University, Raleigh, NC

This pilot study utilized 43 medium-large (median 26.0 kg, range 17.9-31.7 kg) recently euthanized canine cadavers over a 10 week period. The abdominal region of cadavers was aseptically prepared and a forced-air warming unit was turned on under the surgical drape. Cultures were obtained after placing agar plates on the sterile field for 2 hours. Agar plates were incubated for 48 hours and bacterial colonies were spectated. Those that were unable to be spectated were character- ized by microscopy and gram staining. Surgical drape attachment method, forced-air warming unit fan speed and operat- ing room (OR) ventilation type were variables. We hypothesized there would be increased contamination with the towel clamp drape attachment method compared to skin staples, higher warm air unit fan speed, and absence of positive pressure within the OR. We were unable to identify a difference in contamination risk between controls and any combination of variables. A post hoc power analysis was done after the first 34 treated cadavers, 26 plate and 9 cadaveric controls which showed that a minimum of 120 cadavers would be needed to detect a fourfold difference between cadaveric controls and any treatment group at 70% power. A minimum of 270 cadavers would be needed to detect a fourfold difference between controls and any treatment group at 90% power. Given that there were no significant differences in results between any of the groups in this pilot study, and given the results of the power analysis, continuing the study was not feasible. However, because the two treatment dogs with large numbers of CFUs detected had long hair, further analysis of potential warm air unit related contamination in long-haired breeds may be warranted.

Research Grant: None Student Support: NC State Veterinary Scholars Program

Differential gene expression analysis of newborn mouse kidneys lacking Prkaca (PKAca)

Laurielle Gibson, Rawah Faraj, Garfield Grimmett, and Pawan Puri

Department of Biomedical Sciences, Tuskegee College of Veterinary Medicine (Gibson), Tuskegee University, Tuskegee, AL

Understanding of signaling networks regulating renal development can provide insights into the mechanisms of renal disease. Protein Kinase A (PKA) is one of the main effectors of the second messenger cAMP and regulates various cellular functions. Previous ex vivo studies using embryonic kidney explants showed that PKA is a key regulator of tubulogen- esis and ureteric branching. However, the role of PKA in renal development in vivo remains undefined. We showed that in wildtype (WT) kidney, p-Creb, a well-known read out of cAMP-PKA signaling, is present in nephron progenitors and their early epithelial derivatives, however, disappears in proximal tubules (PT) suggesting a role of PKA in early nephro- genesis. Furthermore, we showed that PTs of WT embryonic explants undergo cystic dysplasia and dedifferentiation fol- lowing stimulation by a cAMP analogue, 8-Br-cAMP and become p-Creb+ve. Goal for this project was to understand how the lack of PKAca affects gene expression in the developing kidney. mRNA-sequencing was performed on the kidneys isolated from newborn PKAca knockout (KO) mice and wild-type littermates followed by functional enrichment by GO and KEGG analyses. 5046 were differentially expressed between the two groups. For 2797 upregulated genes, some of the top categories identified by GO and KEGG analyses were epithelial tube morphogenesis, urogenital development, cova- lent chromatin modification and cell matrix adhesion. For 2249 downregulated genes, some of the top categories identified were mitochondrial inner membrane protein complex, oxidative phosphorylation, ribonucleotide metabolic processes and ribosome. These results reveal PKAca-regulated cellular processes and associated genes in the developing kidney.

Research Grant: National Institutes of Health project # 2T35OD010432, National Institute of General Medical Sciences project # 1SC2GM130475 and RCMI core facility grant # U54MD007585 Student Support: National Institutes of Health project # 2T35OD010432

2021 National Veterinary Scholars Symposium • 139 Dolphins to horses (but not seahorses) - prevalence, type, and susceptibility of Staphylococcus delphini

Christopher Given Jr, Shannara Welch, Logan Ardrey, Jing Wu, Sara D. Lawhon

Department of Clinical Microbiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Staphylococcus delphini colonizes the skin and nostrils of horses and other species; it also causes opportunistic infections of the integumentary and respiratory systems. Data on methicillin resistance and mupirocin resistance in S. delphini are lacking. The aim of this study was to determine the prevalence of S. delphini type A and B. We also determined prevalence of mupirocin and methicillin resistance in each type of S. delphini. Bacterial isolates from horses presented to the Texas A&M University Veterinary Medical Teaching Hospital between 1/1/2010 and 12/31/2020 were identified using MAL- DI-TOF and then differentiated into type A or B using PCR. Staphylococcus delphini isolates were tested for mupirocin resistance, and minimum inhibitory concentration (MIC) data were collected and summarized to determine susceptibility. Of 28,988 horses presented to the hospital, 2,524 had cultures submitted. A total of 26 horses had infections in which S. delphini was isolated. Ten (38.5%) of the isolates were type A and 16 (61.5%) were type B. None of the isolates were found to have mupirocin resistance or methicillin resistance. Beta-lactamase production was noted in six of the type A and four of the type B isolates. Otherwise the isolates were susceptible to the antimicrobials tested with the exception of one type B that was resistant to macrolides. The isolates were further classified by the site or tissue that was cultured. The majority of isolates were collected from skin (n = 9) and the upper respiratory tract (n = 9). Other sites included joints (n = 3), the eye (n = 3), and single isolates from the blood and bone. There was no significant difference in theS. delphini type at each of these locations.

Research Grant: FDA grant U18FD006171, the Texas A&M Clinical Science and Translational Research (CSTR) Institute Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Defining Tissue Expression and Isotype of Ultralong CDR3 “Cattlebodies” by Real Time qPCR

Nicole Glenn, Cody Horton, Jeannine A. Ott, Michael F. Criscitiello

College of Veterinary Medicine and Biomedical Sciences, Department of Veterinary Pathobiology, Texas A&M University, College Station, TX

Alongside typical antibodies (Typ), cattle express unique ultralong antibodies (UL) that develop in gut-associated lym- phoid tissue (GALT) rather than just bone marrow. These UL contain a CDR H3 comprised of a b-ribbon “stalk” and disulfide-linked “knob” that reaches over 70 amino acids in length. UL exhibit extensive reach to recessed pathogen epitopes and provide broadly neutralizing immune responses to elusive antigens. To date, no broadly-neutralizing anti- bodies have been manufactured that reliably combat antigens of pathogens such as HIV or SARS-CoV-2, which poses the question of how UL may play a role in developing prophylactic and therapeutic treatment options, which may be informed by understanding their physiology in the cow. This study quantifies the relative amounts of UL, compared to Typ, in 6 tissue types (bone marrow, spleen, Peyer’s patch, mesenteric lymph nodes, circulating PMBCs) extracted from a single steer, and quantifies the relative expression of 5 antibody isotypes (IgG, IgA, IgM, IgE, IgD) within each tissue. We syn- thesized cDNA from preserved RNA samples and quantified relative expression of each antibody isotype using RT-qPCR. We hypothesized that UL are found in higher concentrations in GALT compared to non-lymphoid tissues, while isotype prevalence fluctuates based on the antigen-specific immune response.With a more complete understanding of tissue-spe- cific expression patterns and isotype-specific immune responses of these special antibodies, we can better understand their function in the cattle immune response. Further, these data open the door for strategic application of these antibodies in immunotherapy in other species, including humans.

Research Grant: NSF IOS-16568702. Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

140 • 2021 National Veterinary Scholars Symposium Central and peripheral contribution to heat stress intolerance in wild type and MH susceptible mice

Ryan Gluck, Shane Antrobus, Gennady Cherednichenko, Wayne Wei Feng, Isaac Pessah, Monica Aleman, Chelsea Crowe

Department of Molecular Biosciences (Gluck, Antrobus, Cherednichenko, Feng, Pessah) and Department of Medicine and Epidemiology, The William R. Pritchard Veterinary Medical Teaching Hospital (Aleman, Crowe), School of Veterinary Medicine, University of California, Davis, CA

Adverse outcomes to heat stress are emerging as a major concern in both humans and animals due to rising global tem- peratures, especially for those with genetic risk factors promoting heat stress intolerance (HSI). Ryanodine receptor type 1 (RYR1) mutations are a major monogenic risk factor for HSI and are responsible for the pharmacogenetic disorder malig- nant hyperthermia (MH). Dietary caffeine (CAF) intake as a risk factor for HSI in individuals expressing MH susceptible RYR1 mutations has not been investigated. We use a knock-in mouse model with mutation RYR1-R163C (HET) to study differential responses to a moderate heat stress protocol with and without dietary levels of CAF compared toWT. Contin- uous recording of brain EEG, heart ECG and core temperature are used to assess physiological responses in HET and WT mice. Blood, muscle, and brain tissue samples are collected to elucidate central and peripheral mechanisms contributing to HSI. WT mice exhibit a monotonic rise in core temperature (0.124 °C/min) and survived 60 minutes of heat stress. HET mice show a biphasic rise in core temperature with a steep second phase (0.366 °C/min) not observed in WT mice and invariably trigger with a fulminant MH crisis. Dietary CAF failed to significantly influence outcomes in either sex (male & female, median survival = 34.5 & 37.2 min, respectively [P = 0.3]). Electrophysiological recordings from WT and HET mice undergoing moderate heat stress are ongoing and pending analysis, as are histological and clinical chemistry analyses. HET mice expressing a human MH susceptibility mutation are exquisitely sensitive to HSI and likely reveal an underappreciated genetic risk factor, although dietary CAF intake does not appear to influence HSI.

Research Grant: National Science Foundation (NSF) Grant 1840842 and National Institutes of Health (NIH) Grant 1R01 ES030318 Student Support: NIH T35 Training Grant 5T35OD010956-22

Macrophages and prostate cancer racial disparities

Taylor N. Godwin, Angelo M. De Marzo, Karen S. Sfanos, and Janielle P. Maynard

Department of Pathology, Johns Hopkins University, Baltimore, MD (Godwin, De Marzo, Sfanos, Maynard), Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD (De Marzo, Sfanos, Maynard), Department of Urology, James Buchanan Brady Urological Institute, Johns Hopkins University, Baltimore, MD (De Marzo, Sfanos).

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. African American (AA) men have 76% higher incidence and 2.3 times higher mortality than that of European American (EA) men. Genetic alterations, African ancestry, increased inflammation-related gene expression, and increased innate immune cells in the tumor microenviron- ment are proposed contributors to PCa aggressiveness in AA men. Studies suggest that cancer cells secrete factors that change innate immune macrophages from a tumor-inhibiting (M1) to a tumor-promoting (M2) phenotype. Our previous work identified differential expression of CD80 (M1 marker) and CD163 (M2 marker) in tissue microarrays (TMA) constructed from radical prostatectomy tissues from AA and EA men. In the current study, we evaluated CD163 expres- sion in whole tissues sections from radical prostatectomy PCa specimens (n = 19 AA; n = 19 EA) and organ donor normal prostates (n = 3 AA; n = 6 EA) using RNA in situ Hybridization (RISH). The HALO image analysis platform was used to estimate the area of positive signal per unit tissue area. Results indicate a significant increase in CD163+ macrophages in normal prostate tissues compared to PCa tissues (P < 0.01), particularly among AA men (P < 0.04). Interestingly, the mean CD163 expression of normal prostates was higher in AA compared to EA men, while there was decreased CD163+ macrophages in PCa tissues from AA men compared to EA men. However, these were not statistically different. Our find- ings suggest different macrophage patterns in the prostate microenvironment ofAA versus EA men with and without PCa. Future studies will investigate whether these differences contribute to disparities in PCa outcomes.

Research Grant: Department of Defense PCRP awards W81XWH-14-1-0364, W81XWH-17-1-0286, W81X- WH-17-1-0292, W81XWH-18-2-0013 and W81XWH-18-2-0015. Prostate Cancer Foundation award 19CHAS03 Student Support: 5T35OD024982-03- Training Veterinary Students for Careers in Biomedical Research

2021 National Veterinary Scholars Symposium • 141 20-year trend in type of references used for small mammal drug dosages in an exotic formulary

Olivia Golden, Nicola Di Girolamo, João Brandão, Stephanie Kline, Layna Tarpalechee

Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma

Formularies are publications including a list of appropriate medications and therapies along with their dosages and other medical and biological information. Veterinary formularies are intended to aid clinicians in rational drug prescription, but referencing inferior quality sources hinders diagnosis and treatment. The purpose of this study is (1) to assess the charac- teristics of sources used to support drug dosages and reference intervals for small mammals in such formulary and (2) to evaluate how the sources changed over five editions of this formulary. A cross-sectional evaluation of the references cited in the chapters related to small mammals for all five editions of the formulary was performed. Each reference was evaluat- ed and classified by the type of source cited then journal articles and conference proceedings were further classified based on study design. References were grouped according to their location in the formulary tables to exclude those not utilized in drug and reference interval tables. Unadjusted analyses showed fourth (OR: 1.67; 95% CI: 1.02-2.74; P = 0.04) and fifth (OR: 3.40; 95% CI: 2.07-5.57; P < 0.001) editions had a significant association with increased use of primary drug sources as compared to the first edition. Upon multivariable adjustment, only the association between the fifth edition and primary drug sources persisted. Unadjusted analyses showed odds of reference interval references citing secondary sourc- es were similar for all editions as compared to the first. Most references cited in small mammal drug and reference interval tables of the formulary were secondary sources. Exotic practitioners should heed limitations of the formulary to evaluate the information within it more critically.

Research Grant: None Student Support: Oklahoma State University, College of Veterinary Medicine, Veterinary Clinical Sciences

Increased immune responses by intradermal and intranasal immunization with a novel adjuvant in mice

Malaycia Goldsmith, Juan F. Hernandez-Franco, Harm HogenEsch

Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, IN

Vaccines are commonly formulated with adjuvants, which stimulate immunologic pathways to improve protective immu- nity against infectious diseases. The most widely used adjuvants in human and veterinary vaccines are aluminum and oil- in-water emulsion adjuvants. However, these adjuvants can cause adverse reactions and are only approved for intramus- cular administration of vaccines. Intradermal (ID) and Intranasal (IN) vaccination can stimulate different types of immune responses and can be done without hypodermic needles. However, there is a need for new adjuvants that can be used in ID and IN vaccines. In this study, we investigated the immune response in mice following ID and IN vaccination with a combination adjuvant composed of Nano-11 and ADU-S100. Nano-11 is a phytoglycogen-based nanoparticle that is safe for ID and IN vaccination. It can be formulated to deliver diverse antigens and cyclic dinucleotides, such as ADU-S100. This cyclic dinucleotide bind and activates the stimulator of interferon genes (STING) receptor which leads to the produc- tion of type 1 interferons. Mice were immunized twice with ovalbumin (OVA) only or OVA with Nano-11/ADU-S100. The combination adjuvant increased the titers of OVA-specific serum IgG subclasses and serum and intrabronchial IgA, as well as plasma cells in the bone marrow. Mice immunized with the combination adjuvant had increased numbers of OVA-specific Th1 and Th17 cells as well as CD8 T cells in the spleen. Immunization via the IN, but not ID route, in- creased the number of resident memory T cells in the lungs. These findings demonstrate that the combination adjuvant induced robust humoral and cell-mediated immunity and support its use in ID and IN vaccines.

Research Grant: USDA/NIFA Grant 2019-67015-29814; USDA Hatch Project IND020164H Student Support: Boehringer Ingelheim and the Purdue College of Veterinary Medicine

142 • 2021 National Veterinary Scholars Symposium Restoration of spermatogenesis after germ cell transplantation into irradiated recipient piglet testes

Savannah Goldstein, Yanfei Yang, Awang Hazmi Awang-Junaidi, Willow Burnes, Fahar Ibtisham, Monique N. Mayer, and Ali Honaramooz

Department of Veterinary Biomedical Sciences (Goldstein, Yang, Burnes, Ibtisham, Awang-Junaidi, Honaramooz), Department of Small Animal Clinical Sciences (Mayer), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada

In mice, germ cell transplantation (GCT) into testes of infertile recipients results in spermatogenesis. Cancer treatment in preadolescent boys can cause future infertility due to gonadotoxic damage. Pre-treatment testicular biopsies can be cryopreserved and hypothetically used for autologous GCT to restore fertility. Using piglets as a model, we examined 1) the effects of local irradiation on testis development and endogenous germ cell depletion in piglets, and 2) the potential of gonocytes (as the only germ cell type in neonatal testes) to initiate spermatogenesis in pre-irradiated testes. Nine-day old piglets (n = 6/group) underwent localized testis irradiation for 3 consecutive days using doses of 0 (control), 1, 2 or 3 Grays (Gy). Two months post-irradiation (PI), half of the piglets in each group were euthanized and their testes collected. Meanwhile, remaining piglets underwent GCT, receiving gonocytes from unrelated donors (into left testis) and a control saline infusion (right testis). At 4 months PI, the remaining piglets were euthanized. Compared with the control (0 Gy), at 2- and/or 4 months post-irradiation, testis weight indices from the 3 Gy group were smaller, seminiferous tubule den- sity from 2- and 3 Gy groups were lower, germ cell numbers lower, and tubule diameters in all irradiated testes were also lower (P < 0.05). While no spermatogenic development was found in any of the irradiated testes injected with only saline, after gonocyte transplantation, a small number of tubules in the groups of 1- and 2 Gy contained spermatogenesis up to elongating and round spermatids. Hence, in piglets, even 1 Gy of irradiation for 3 days depletes the endogenous germ cell pool but subsequent GCT results in spermatogenesis.

Research Grant: Natural Science and Engineering Research Council (NSERC) of Canada (Honaramooz) Student Support: Boehringer Ingelheim Veterinary Scholars Research Grant (Goldstein)

IL-27 Inhibits the anti-Mycobacterium tuberculosis Activity of Primary Human Macrophages

Hailey R Gollnick, Katarzyna Rzepka, Ankita Garg

Department of immunology, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), is a leading cause of death by an in- fectious pathogen worldwide. Macrophages (Mf) are the main host for Mtb infections. However, despite robust innate immune responses, Mf are often unable to control Mtb replication for reasons that remain elusive. The aim of this study was to investigate the regulation of anti-Mtb activity by the cytokine IL-27. We found infection of primary human Mf’s with Mtb induces the secretion IL-27 as well as anti-Mtb cytokines TNFa and IL-6 (P = 0.01, P = 0.04 and P = 0.04, respectively). We also found that blocking Toll-like-receptor-2 (TLR-2) with antibodies prior to infection inhibited IL-27 secretion (P = 0.01). Additionally, Mtb infection increases the expression of phosphorylated (p) PP2A phosphatase (P = 0.04) in Mf, known to inhibit autophagy mediated control of M bovis. With regard to the effect of IL-27, treatment of Mtb infected Mf with recombinant IL-27 (rIL-27) inhibited the expression of anti-Mtb peptide cathelicidin (hCAP) (P = 0.006), with a concomitant decrease in hCAP quantity in the culture supernatant (P = 0.03). However neutralizing IL-27 did not increase hCAP expression (P = 0.06). Also, neutralizing IL-27 increased immune suppressive cytokine IL-10 (P = 0.05), and neutralizing IL-27 and IL-10 increased the expression of hCAP in Mtb infected Mf (P = 0.75). Our results suggest TLR activation results in induction of the immune suppressive cytokine IL-27. IL-27 inhibits anti-Mtb activity of Mf by suppressing innate immune pathway and increasing phosphatases. These findings provide important information on TLR mediated regulation of anti-Mtb response that is central in defining the outcome of Mtb infection.

Research Grant: The University of Georgia Research Foundation, JFSG2019 Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA college of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 143 Methods of Separation and Characterization of L1CAM-associated Extracellular Vesicles; A Systematic Review

Dimitria E. Gomes, Kenneth W. Witwer

College of Veterinary Medicine, Cornell University, Ithaca, New York, USA (Gomes); Departments of Molecular and Comparative Pathobiology and Neurology (Gomes, Witwer), Johns Hopkins University School of Medicine, Baltimore, MD

When released into biological fluids like blood plasma or serum, brain extracellular vesicles (EVs) and their cargo might serve as useful biomarkers of neurodegenerative and other central nervous system (CNS) diseases such as Parkinson’s and Alzheimer’s. EVs are a heterogeneous group of membrane-delimited particles that are released from cells and are most abundant in the 30-100 nm diameter range. Functioning in cell-cell communication by transporting RNAs, lipids, and proteins, EVs have been reported to cross the blood-brain barrier. Cell specific markers may be used to enrich brain-de- rived EVs from biofluids for biomarker discovery. Most prominently, antibodies that recognize the transmembrane L1 cell adhesion molecule (L1CAM) have been employed to pull down putatively neuronal EVs, leading to publication of numer- ous biomarker signatures of CNS disease. Despite the apparent value of these findings, L1CAM is not 100% specific to neurons, being found on several cell types throughout the body and also upregulated during cancer progression. A recent publication (Norman et al, 2021) has even suggested that L1CAM epitopes are present in biofluids mostly or entirely as cleaved, soluble protein. To understand the landscape of L1CAM EV studies, we conducted a systematic review of the literature as represented in three databases. Our results indicate trends in L1CAM-mediated EV separation, enrichment, and characterization, yielding insights into the rigor of the science published to date and suggesting avenues for further exploration.

Research Grant: KWW is supported in part by the US NIH through DA040385, DA047807, MH118164, AI144997, and CA241694, and by the Michael J. Fox Foundation (00900821) Student Support: Boehringer Ingelheim and NIH T35 OD024982

Visual Acuity in Python regius

Zaira P. Gomez, Curtis Eng, Donald J. Cameron

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA

Snakes are known for their unique abilities including infrared reception and their heavy reliance on heat sensors and vibrations. Infrared reception of snakes has gone under immense investigation; however, there have been very few studies that elaborate on their capacity to see. The goal of this study is to determine visual acuity of Ball Pythons (Python regius) by observing their optokinetic response (OKR). The OKR is a series of rapid saccadic and smooth pursuit movements of the eyes. It has been used for decades to determine the visual acuity in multiple species such as humans, rats, and other non-mammalian species such as zebra fish and box turtles. In this study the snakes were placed in a clear container in a dark room and black and white grating were projected upon the container. The size and velocity of the grating was ma- nipulated which allowed the limitations of their visual acuity to be determined. Past studies have discovered that birds, reptiles, and amphibians achieved gaze stabilization by head and body movements, whereas in mammals and fish, gaze stabilization is conducted by eye movements. Our hypothesis is that the snakes would perform poorly compared to previ- ous studies due to their heavy reliance on other senses. Preliminary results show that P. regius do respond to visual stimuli and show saccadic movement in the direction of their stimuli. Along with observing their visual acuity, a highly sensitive pupillary light reflex (PLR) was observed. There has been no previous documentation on the PLR in snakes. Investigating further into the visual acuity along with the PLR of snakes will provide new information that will allow us to better care for and have a better understanding of this species.

Research Grant: None Student Support: None

144 • 2021 National Veterinary Scholars Symposium Glucocorticoid receptor in 1st trimester human trophoblast cells as tool to study GR signaling in placentation

Gabriella Gonzalez, Seunga Min, Agata M. Parsons Aubone, Gerrit J. Bouma

CSU College of Veterinary Medicine, Department of Clinical Sciences (Gonzalez, Min), Department of Biomedical Sciences (Bouma, Parsons)

Glucocorticoids (GCs) are necessary for fetal organ development and used to promote fetal lung maturation in cases of preterm deliveries. It is known that excess or lack of GC exposure during late gestation is harmful for the fetus. Excessive GC levels correlate with intrauterine growth restriction (IUGR). IUGR develops from adverse maternal, placental, or fetal conditions and can lead to both immediate or long-term consequences. Of concern, IUGR cases have been associated with higher perinatal mortality and morbidity rates. Although glucocorticoid receptor (GR) is known to be present in the placenta, little is known about GR function and signaling in the placenta. Our aim is to further investigate GR signaling in the placenta. First we identified the presence of GR in two first trimester trophoblast cell lines:ACH-3P cells and Swan-71 cells. In order to study the effects of GC in the human placenta, we first must identify an appropriate human trophoblast cell model. To further test GR signaling in placenta, the synthetic glucocorticoid, dexamethasone, will be used as a ligand for GR. We hypothesize that GR signaling stimulates proliferation and cell fusion of human trophoblast cells. GR mRNA and protein will be quantified by qPCR and Western blot respectively. With the outcomes of these studies, we will learn how GR-signaling impacts placental development, which eventually may help provide insight into complicated pregnan- cies associated with IUGR.

Research Grant: CVMBS College Research Council Student Support: USDA Animal Health & Disease Scholar Biomedical Sciences Department

Characterization of injuries associated with fishing gear in California seabirds

Abrielle A. Goodwein, Curtis Eng, and Rebecca S. Duerr

College of Veterinary Medicine (Goodwein, Eng), Western University of Health Sciences, Pomona, California; Department of Research and Veterinary Science (Duerr), International Bird Rescue, San Pedro, California

Recreational fishing gear has negative impacts on the marine environment, with entanglements in fishing gear resulting in significant morbidity and mortality of marine birds. Some of the most common materials involved in entanglements include various fishing lines and hooks. While the prevalence of fishing gear related injuries in seabirds has been inves- tigated regionally along the California coast, details regarding the nature and/or severity of these injuries have not been objectively evaluated or compared. The aim of this study was to objectively characterize fishing gear-associated injuries and describe the nature of these injuries associated with different line and hook types in order to create a standardized characterization chart. Medical records and fishing gear of patients admitted for fishing gear-related injuries at Interna- tional Bird Rescue (IBR) and cooperating rehabilitation facilities between 2018 and 2021 were evaluated. Injury descrip- tions found in these medical records, in conjunction with injury staging methods found in literature, were used to create a characterization scheme specific to fishing gear injuries in seabirds.All gear-related injuries between 2018 and 2021 were then reclassified using this fishing gear injury characterization scheme. Findings will be reviewed in the context of anthro- pogenically induced injuries of seabirds and the morbidity and mortality associated with fishing gear. These results will aid in standardizing the documentation and treatment of fishing gear related injuries in rehabilitating seabirds, as well as contribute to describing the adverse effects of recreational fishing gear.

Research Grant: None Student Support: WesternU Summer Fellowship Grant, WesternU CVM Veterinary Summer Research Program

2021 National Veterinary Scholars Symposium • 145 Comparing Methodologies for the Detection of Leptospira in Environmental Samples

Myranda Gorman, Dhani Prakoso, and Sreekumari Rajeev

Biomedical and Diagnostic Sciences, College of Veterinary Medicine University of Tennessee, Knoxville

Leptospirosis, a life-threatening disease of humans and animals, is one of the most widespread global zoonosis. Pathogen- ic Leptospira are maintained in the kidneys of animals. Contact with urine from infected animals and the contaminated environment are major sources of transmission to animals and humans. Clusters of disease outbreak are common after the rainy season and after recreational water activities. In this study we explored the usefulness of next-generation sequenc- ing on a MinION platform when compared to traditional culture-based methods and PCR to detect Leptospira in water and soil samples obtained from a local creek. Direct PCR on DNA extracted from water and soil samples were positive for Leptospira genus specific 23S and 16S ribosomal DNA targets but was negative for LipL32 gene which is present in pathogenic Leptospira. However, direct PCR performed on water and soil samples that were spiked with a pathogenic Leptospira were positive for the LipL32 gene. We evaluated multiple Leptospira culture media (Korthof, Stuart, Fletcher, and EMJH) and different processing procedures for recovery ofLeptospira from soil and water samples. EMJH media containing antibacterial agents (STAFF or 5-FU) was superior in recovering Leptospira compared to other media used. We have completed the next-generation sequencing of water and soil samples using Nanopore MinION device and currently analyzing the data. Our preliminary data supports the need for enrichment using traditional or molecular methods to detect Leptospira in environmental samples. Our study shows that the soil and the water tested harbors Leptospira spp. Further characterization of Leptospira isolates by PCR and sequencing is also underway.

Research Grant: College of Veterinary Medicine, University of Tennessee Student Support: BI Veterinary Scholars Program

Characterization of host immune responses to Clostridium perfringens affecting broiler chickens

Kaitlin Gorrell, Carissa Gaghan and Ravi Kulkarni

Department of Population Health and Pathobiology, College of Veterinary Medicine, NCSU

Clostridium perfringens type A bacteria cause necrotic enteritis (NE), an economically important disease, in chickens. Although the NE pathogenesis is well-studied, the immune responses against C. perfringens are poorly understood. In the present study, we used an experimental NE model of chickens to characterize immune responses against C. perfringens isolates that varied in their pathogenicity. To this end, broiler chickens were challenged with three isolates, namely, CP5 (avirulent), CP1 (virulent) and JP26 (very virulent) along with controls to measure expression of immune genes (IL-1b, IL-6, IFNg and IL-10) in the cecal tonsils (CT), bursa and harderian gland (HG) tissues as well as measure antibody responses. Additionally, macrophages, in-vitro, were stimulated with C. perfringens bacilli to measure expression of IFNg gene and nitric oxide production. The results indicated that while JP26 induced an upregulation of IFNg and IL-6 genes in CT, the CP1 infection led to an increase in the expression of genes in HG tissues, indicating a spatially regulated inflam- matory response by C. perfringens that vary in their level of virulence. The humoral response evaluation showed that JP26 could induce the most robust IgM antibody responses compared to controls. Furthermore, macrophage stimulation with C. perfringens in-vitro also led to an increased IFNg transcription as well as nitric oxide production in the cell culture super- natants. In summary, the present study shows that C. perfringens can induce an inflammatory response in certain lymphoid tissues associated with an increased antibody response in-vivo as well as macrophage activation in-vitro, and that these responses depend on the virulence nature of this pathogen.

Research Grant: None Student Support: NIH T35 Training Grant

146 • 2021 National Veterinary Scholars Symposium Reasons for relinquishment to animal shelters survey study

Sarah Goss, Megan Gremling, Sara Bennett

Department of Clinical Sciences (Bennett), College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Pet relinquishment to animal shelters has not been comprehensively studied in over 20 years. Relinquishment reasons should be reevaluated in relation to demographic and geographic factors across the United States to appropriately advise community tailored and nationwide intervention strategies. To update this information, an online survey was developed to capture reasons for dog and cat relinquishment to shelters across six geographical regions (Northeast, Southeast, Midwest, Southwest, Rocky Mountain, and Pacific) in the United States.This survey asks shelters to record the reasons owners listed for relinquishing their pets in the years 2019 and 2020. Categories for relinquishment were based on previ- ous studies, and include animal behavior, socioeconomic and personal factors, and owner or animal health. In addition to relinquishment data, questions to determine the demographic and socioeconomic status of the communities served by each shelter, current intervention strategies being practiced by shelters, and changes in operation resulting from the COVID-19 pandemic in the year 2020 were asked. When possible, information provided by shelters will be cross referenced with the most current US census data. After piloting the survey among 10 shelter professionals, Shelter Animals Count (SAC), a voluntary national database for annual shelter intake and outcome data, was utilized to identify shelters likely to have the organized data necessary to complete the survey. Shelters were randomly selected by geographic location from SAC, with 60 shelters per region invited to participate. Nonprofit, private, and municipal shelters were included in each region.The resulting data is expected in Fall of 2021 for statistical analysis.

Research Grant: None Student Support: NIH-T-35 Interdisciplinary Biomedical Research Training Program (IBRTP)

Exploring clinical use of electroencephalography to differentiate anesthesia depth in horses—a pilot study

Kami Graber, Jeff Ko, Carla Murillo

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Electroencephalography (EEG) provides direct monitoring of the brain activity associated with the depth of anesthesia in humans. The use of such an EEG monitor has not been explored in anesthetized horses. The aims of this study were to 1) explore the clinical usage of the EEG monitor on the anesthetized horse and, 2) evaluate if the EEG waveforms and processed EEG indices could indicate the depth of anesthesia changes in horses. We hypothesized that the human EEG monitor would be able to differentiate the depth of anesthesia in the horse. Six horses subjected to orthopedic or soft tissue surgeries were used. The horses were instrumented for standard hemodynamic monitoring for the entire anesthetic proce- dure. Recovery quality was scored (poor, acceptable, excellent) with a video system. A human EEG monitor (Sedline with Root ) with six needle electrodes was positioned on the Fp1, Fp2, F7, and F8 frontal lead. The SedLine monitor calculated the patient state index- PSI, spectral edge frequency 95%- SEF95%, burst suppression ratio- BS, artifact percentage, and electromyographic activity. Results showed that the EEG monitor can be easily applied to the horses. The EEG moni- tor was able to detect the depth of anesthesia changes before the traditional hemodynamic monitor during the surgery and recovery. In addition, it was found that horses that had a frequent and long duration of BS during the surgery had a rougher recovery quality score. In conclusion, this study demonstrated that Sedline EEG monitor could be used routinely for anesthetic depth titration in horses. Further research is warranted to correlate the duration of BS and the quality of the recovery in horses.

Research Grant: None Student Support: Boehringer Ingelheim and Purdue University College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 147 Pressure-induced wound formation and healing with Staphylococcus aureus infection in a diabetic mouse model

Carly A. Grabner, Reagan McDevitt, Keun Seok Seo, Elizabeth Swanson, George E. Howell III

Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, Mississippi

Approximately 25% of type 2 diabetics will develop foot ulcers in their lifetime. Roughly 60% of ulcers will become infected and 15-20% of ulcers will end in limb amputation. Infection of diabetic foot ulcers by Staphylococcus aureus is the leading cause of lower limb amputation. Therefore, the present study was designed to explore the wound healing kinetics of a polygenic model of type 2 diabetes, the TALLYHO mouse, during active infection with methicillin resistant S. aureus (MRSA). Pressure wound size, as well as the local effects of MRSA on the wounds, was compared between the TALLYHO mouse and a lean control, the SWR mouse. Both mouse strains were subjected to a wounding procedure using ceramic magnets consisting of 3-12 hour on/off cycles then the wounds were inoculated with MRSA (USA300 strain) and followed for 10 days. During wound formation both strains developed similar wound sizes. The TALLYHO had signifi- cantly larger wounds compared to the SWR at days 3 and 7 post-inoculation. The TALLYHO in the cohort with more pronounced hyperglycemia had a significantly greater amount ofS. aureus present at day 10 compared to the SWR. At day 10, the TALLYHO had greater expression of the macrophage marker F4/80 compared to SWR. Expression of wound heal- ing markers displayed an overall decrease in TALLYHO compared to SWR, with Mmp13 being significantly decreased. These data suggest that the diabetic mouse model developed more significant wounds compared to the normal mouse model, as well as a diminished healing response due to the bacterial infection. Future plans for the study will explore the effects of exposure to persistent organic pollutants on the wound healing parameters used in the present study.

Research Grant: NIH 5T35OD010432 Student Support: NIH 5T35OD010432

Next generation sequencing revealed heterogeneity between spatially distinct PanINs

Julie Granger, Alicia Braxton, Laura Wood, Ashley Kiemen, Lily Zheng, Ralph Hruban, Rachel Karchin, Denis Wirtz, Pei-Hsun Wu, Laura Wood

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center (Granger, Braxton, Hruban, Wood), De- partment of Oncology (Zheng, Karchin, Wood), The Johns Hopkins University School of Medicine, Baltimore, Maryland; Whiting School of Engineering, The Johns Hopkins University (Kieman, Wu, Wirtz)

Pancreatic ductal adenocarcinoma (PDAC) is common, is usually diagnosed at advanced stages and has a 5-year survival rate less than 7%. PDAC arises from precursors such as pancreatic intraepithelial neoplasia (PanIN) that are curable if detected early. PDAC is caused by somatic mutations in oncogenes and tumor suppressor genes. Few studies have inves- tigated somatic mutations in PanINs. PanINs are microscopic and complex, so are best studied in 3D. Characterizing the mutations in PanINs is an important step in identifying PanINs at high risk for malignant transformation and implement- ing new cancer prevention strategies. To sample microscopic PanINs, grossly normal pancreatic tissue was harvested from a single patient during surgical resection, formalin-fixed and embedded in paraffin, completely sectioned and histologi- cally evaluated on H&E sections to identify PanIN. 3D modeling using a novel machine learning approach to annotate pancreatic tissue at a single-cell level revealed 4 spatially distinct PanINs. Laser-capture microdissection of spatially distinct PanINs was facilitated by 3D modeling. DNA from the PanINs was sequenced on an Illumina MiSeq using a custom targeted panel covering the coding regions of 155 genes. Somatic mutations were called using Strelka followed by visual inspection in Integrated Genome Viewer. Of the 4 PanINs modeled, 3 PanINs shared a KRASG12D mutation. The 4th PanIN had a unique driver gene mutation, KRASG12V, suggesting that it arose independently. While data analysis is ongoing, preliminary findings indicate that genetic heterogeneity exists in independent PanINs within a given patient, suggesting that multiple, genetically independent PanINs occur in close proximity in the human pancreas.

Research Grant: NIH/NCI P50 CA62924, Allegheny Health Network – Johns Hopkins Cancer Research Fund Student Support: 5T35OD024982-03 Training Veterinary Students for Careers in Biomedical Research PI: Mankowski

148 • 2021 National Veterinary Scholars Symposium Improving the clinical efficacy of butorphanol for analgesia in dogs

Mackenzie Gray, Dariyan Springfield, Poyu Lai, Kate KuKanich, Butch KuKanich

Department of Anatomy and Physiology (Gray, Springfield, B. KuKanich) and Department of Clinical Sciences (Lai, K. KuKanich), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Butorphanol is commonly used for short-term sedation and analgesia in dogs. It is widely available and cost-effective. It is a mixed opioid agonist/antagonist, decreasing administrative burden and abuse potential, but is limited by its short dura- tion of effect. This study evaluated methods to enhance the clinical efficacy of butorphanol. Twelve healthy beagles were enrolled; six dogs were randomly allocated to each treatment. Butorphanol was administered as an IV bolus (0.4 mg/kg), IV loading dose (0.2 mg/kg) followed by IV CRI (0.2 mg/kg/hr for 8 hours), SC (0.4 mg/kg) and SC (0.8 mg/kg) mixed with an equal volume sodium bicarbonate producing a suspension due to precipitation (butorphanol-bicarbonate). We hypothesized the CRI would exhibit a longer duration of effect compared to the IV bolus and the butorphanol-bicarbon- ate would exhibit a longer duration compared to the butorphanol SC. Noninvasive centrally-mediated opioid effects were measured during a 24-hour period (rectal temperature and sedation). Rectal temperature was previously correlated with opioid analgesia in dogs. Blood samples were collected for future pharmacokinetic analysis. Rectal temperatures were significantly lower than baseline from 1.5-4 hours (IV), 1-5 hours (CRI) and 2-7 hours (butorphanol-bicarbonate).There was no significant effect on rectal temperature from SC butorphanol. Some sedation was observed in all treatments.These results suggest that IV, CRI and butorphanol-bicarbonate produced opioid effects previously correlated with analgesia, but SC did not. Butorphanol-bicarbonate may be an easy and effective method for extending the duration of butorphanol in dogs. Further studies are required to determine reproducibility of these results.

Research Grant: None Student Support: National Institutes of Health T35OD029981

Anatomy of the first mandibular molar and the mandibular canal described by dog breed size and skull type

Erica Greene, Stephanie Goldschmidt

College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL (Greene); Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN (Goldschmidt)

The mandibular first molar is the largest tooth of the dog mandible and is adjacent to the mandibular canal. The location of the roots of the mandibular first molar relative to the canal can vary among individuals, and the normal relationship between those structures in a diverse population of dogs has not been established. The aim of this study was to identify associations between skull type and breed size, with the location of the mandibular first molar roots relative to the mandib- ular canal. Computed Tomography (CT) scans performed for reasons unrelated to the study were retrospectively analyzed (n = 192). Subjects were sorted into one of 12 groups based on skull type (Brachycephalic, Mesaticephalic, Dolichoce- phalic) and weight (X-Small, Small, Medium, Large). The roots of the mandibular first molar tooth were categorized as lying lingual, buccal, or dorsal to the mandibular canal on the left and right side separately. Statistical analyses will be performed to determine if there is any significant association between mandibular canal location and breed size/skull type. This information can help avoid complications like hemorrhage, infection, and neurovascular damage during extraction of the mandibular first molar by allowing better prediction of the arrangement of these anatomical structures.

Research Grant: None Student Support: Morris Animal Foundation

2021 National Veterinary Scholars Symposium • 149 Reasons for Relinquishment to Animal Shelters: A Survey Study

Megan Gremling & Sarah Goss, Sara Bennett

Department of Clinical Sciences (Bennett), College of Veterinary Medicine (Gremling and Goss), North Carolina State University, Raleigh, NC

Pet relinquishment to animal shelters has not been comprehensively studied in over 20 years. Relinquishment reasons should be reevaluated in relation to demographic and geographic factors across the United States to appropriately advise community tailored and nationwide intervention strategies. To update this information, an online survey was developed to capture reasons for dog and cat relinquishment to shelters across six geographical regions (Northeast, Southeast, Midwest, Southwest, Rocky Mountain, and Pacific) in the United States.This survey asks shelters to record the reasons owners listed for relinquishing their pets in the years 2019 and 2020. Categories for relinquishment were based on previ- ous studies, and include animal behavior, socioeconomic and personal factors, and owner or animal health. In addition to relinquishment data, questions to determine the demographic and socioeconomic status of the communities served by each shelter, current intervention strategies being practiced by shelters, and changes in operation resulting from the COVID-19 pandemic in the year 2020 were asked. When possible, information provided by shelters will be cross referenced with the most current US census data. After piloting the survey among 10 shelter professionals, Shelter Animals Count (SAC), a voluntary national database for annual shelter intake and outcome data, was utilized to identify shelters likely to have the organized data necessary to complete the survey. Shelters were randomly selected by geographic location from SAC, with 60 shelters per region invited to participate. Nonprofit, private, and municipal shelters were included in each region.The resulting data is expected in Fall of 2021 for statistical analysis.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program (VSP)

Toll-Like Receptor (TLR) 9-signaling and its effects on B cell receptor (BCR) signaling in B-1 cells

Sophie R. Gretler, Fauna L. Smith, Nicole Baumgarth

Veterinary Science Training Program (Gretler), Center for Immunology and Infectious Diseases (Smith, Baumgarth), Integrated Pathobiology Graduate Group (Smith, Baumgarth), Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine (Baumgarth), University of California, Davis, CA

B-1 cells are a subset of fetal and neonatal-derived B cells. They produce circulating natural IgM that help remove altered self and foreign antigens, including many pathogens. Antigen stimulation via the BCR fails to induce B-1 cell prolifer- ation due to BCR-signaling inhibitors such as CD5, however, B-1 cells do proliferate in response to TLR-stimulation. We previously showed that TLR stimulation results in the downregulation of CD5 in B-1 cells, which is critical for their differentiation into plasma cells. However, the exact effects of TLR-mediated BCR-reorganization on the function of B-1 cells is unknown. The aim of this study was to evaluate the effects of these stimuli on BCR and TLR expression of B-1 and conventional B-2 cells using flow cytometry and confocal microscopy. Purified murine B-1 and B-2 cells were stimu- lated with CpG, a TLR9 agonist, and/or the BCR agonist anti-IgM (Fab)2, for 72 hours. While CpG +/- anti-IgM increased TLR9 expression 2.8- and 3.5-fold, respectively, anti-IgM alone had no effect on TLR9. In contrast, B-2 cells increased TLR9 expression 3-to-4-fold after both TLR- and BCR- stimulation. TLR- but not BCR-stimulation induced IgM-secre- tion by B-1 cells, as measured by ELISA on cell culture supernatants. Although B-1 cells do not proliferate after anti-IgM stimulation, confocal image analysis showed rapid internalization of surface BCR. Ongoing analysis assesses the degree of TLR9 and BCR co-localization in the LAMP1+ endosome. The results expand our previous findings, indicating signif- icant effects of TLR-stimulation on BCR internalization in B-1 and B-2 cells. Future studies will explore if TLR-mediated BCR internalization facilitates antigen-processing and presentation by B-1 cells.

Research Grant: NIH T-32 OD011147 (FLS); NIH/NIAID R01AI148652 (NB) and R21 AI159115 (NB) Student Support: Students Training in Advanced Research (STAR) Fellowship (SG)

150 • 2021 National Veterinary Scholars Symposium p38 MAPK and NF-kB pro-inflammatory pathways are upregulated during SARS-CoV-2 infection

Kristen Griffin, Curtis Andrew, Lisa Whitworth, Jerry Ritchey, and Craig Miller

Department of Veterinary Pathobiology (Griffin, Andrew, Ritchey, Miller), College of Veterinary Medicine, Microscopy Laboratory (Whitworth), Oklahoma State University; Stillwater, OK

A major hallmark of severe COVID-19 is the development of a pro-inflammatory cytokine storm due to the increased ex- pression of multiple inflammatory pathways. NF-kB and p38 MAP kinase are two important pathways that are commonly activated during viral infection and result in increased production of pro-inflammatory cytokines.The objective of this study is to evaluate the expression of these pathways during SARS-CoV-2 infection in order to determine the potential for these mechanisms to serve as pharmacological targets. In this study, a feline model of SARS-CoV-2 was used to emulate 6 the pathology of acute COVID-19. Twelve (n = 12) domestic cats were intratracheally inoculated with 1.26 X 10 TCID50 SARS-CoV-2 isolate USA-WA1/2020 and humanely euthanized at 4- and 8-days post-inoculation (n = 6 per timepoint). Six (n = 6) uninfected cats served as negative controls. Lung tissue sections were collected on charged slides and incubat- ed with mouse anti-NF-kB and rabbit anti-p38 MAPK IgG antibodies, followed by anti-mouse IgG-AF647 and anti-rabbit IgG-AF488 fluorophore-conjugated secondary antibodies. Confocal microscopy was performed to visualize and assess the expression of these markers in situ. Results showed differential expression of NF-kB and p38 MAPK in inflammato- ry infiltrates in the lungs of infected cats. Quantitative analysis of the expression of these two pathways is pending.The results of this study will provide insight into the sequential expression of pro-inflammatory pathways during the course of SARS-CoV-2 infection.

Research Grant: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103648 Student Support: Oklahoma State University, College of Veterinary Medicine, Pathobiology

Characterizing the microbiome of chickens fed NSPase producing Aspergillus spp

Jimmy Guan, Woo Kyun Kim, Ilya Tolstorukov, Brian Oakley

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA (Guan, Oakley); Poultry Science Department, University of Georgia, Athens, GA (Kim); Keck Graduate Institute, Claremont, CA (Tolstorukov)

Non-starch polysaccharides are compounds such as cellulose and hemicellulose that make up the structural components of plant cell walls in animal feed. Enzymes that break down non-starch polysaccharides are termed NSPases and can be added to animal feeds to decrease feed viscosity, increase usable nutrients, and increase energy for the host via the mi- crobiota. Our study’s main focus is to understand the effects of NSPases on the gastrointestinal microbiome of chickens and their influence on bird performance. A three-week feeding trial was performed in which chickens were split into four experimental groups: supplemented with NSPase-producing Aspergillus, purified NSPases, both Aspergillus and purified NSPases, or a control group receiving no supplementation. Sections of the GI tracts from birds in each group were used for several analyses. First, we performed DNA extractions for 16S rRNA amplicon sequencing to characterize the micro- biome of each bird. Average DNA concentrations ranged from 219 ng/ul for samples from the jejunum to 523 ng/ul for cecal samples with average OD 260/280 ratios of 1.87-1.88. Next, novel primers for PCR and probes for fluorescence in-situ hybridization (FISH) were designed specifically for the NSPase-producingAspergillus spp. strain used in this study using the software packages ARB and ThermoPhyl. Bench protocols were then optimized to perform FISH with these samples. Work in progress is focused on detection and quantification of Aspergillus and relevant Bacterial taxa using PCR and FISH. We expect to see an increase in the relative abundance of commensal microbes that aid in fermentation and presence of Aspergillus associated with experimental treatment.

Research Grant: Unknown Student Support: Foundation for Food and Agriculture Research

2021 National Veterinary Scholars Symposium • 151 Investigating the Association between ELS and Chronic Abnormalities in Ultrasonic Vocalizations in Rats

Katherine Guarino, J. B. Knight, P.B. Bernard

Atlantic Veterinary College Biomedical Sciences

Many individuals who experience an early life seizure are later diagnosed with socialization deficits (i.e. autism).We speculate alterations in auditory communication may be responsible for the aforementioned social abnormalities. Deter- mining the impact of ELS on long term communicative abilities in an animal model is a first step towards clarifying the nature of this relationship. We have classified ultrasonic vocalizations (USVs) of adult rats that had induced early life seizures and compared them with USVs from placebo control rats to determine if our hypothesis that early life seizures cause long term communication differences can be supported. Communication data between two rats is recorded by a microphone that can pick up ultrasonic frequencies and processed by a software to translate into a spectrogram. The spec- trogram is read and categorized by an analyst who records duration, frequency, and call-type. After all rat recordings are analyzed and recorded, the analyst is unblinded and able to compare call types and communications of early life seizure rats vs control rats.

Research Grant: NIH Student Support: VETSRA, NIH

In vitro evaluation of Staphylococcus felis as a potential probiotic therapy for canine superficial pyoderma

Morgan D. Gunn, Mayane Faccin, Aline Rodrigues Hoffmann

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Canine superficial pyoderma (CSP) is a common disease affecting dogs that is typically caused by overgrowth of Staphy- lococcus pseudintermedius on the skin. CSP often requires repeated and long antibiotic courses, which favors antimicro- bial resistance. A strain of Staphylococcus felis has recently been identified to inhibit disease-associated staphylococci, which may be useful as a probiotic therapy to reduce overuse of antibiotics. We hypothesize that S. felis will inhibit S. pseudintermedius on canine skin explants and will ultimately be a safe and useful alternative therapy to treat CSP. Skin explants from healthy dogs were challenged with S. pseudintermedius, then treated with S. felis or its protein extract for 24 hours. The efficacy was analyzed via bacterial count and qPCR. Novel quantitative PCR primers and probes forS. felis were designed with Primer3 and BLAST software and experimentally validated. Preliminary bacterial count results show S. felis exhibits inhibition of S. pseudintermedius when applied to skin explants at 102 and 104 fold dilutions, respectively. We project qPCR analysis will show inhibition of S. pseudintermedius on the skin explants when compared to a non-treat- ment control. If S. felis is established as an effective inhibitor ofS. pseudintermedius on canine skin, a probiotic therapy can be developed and permit this study to move towards safety and efficacy trials in live dogs.

Research Grant: Morris Animal Foundation, grant number D21CA-820 Student Support: Texas Vet. Med. Foundation, Texas A&M College of Vet. Med. and Biomedical Sciences

152 • 2021 National Veterinary Scholars Symposium Expression of LAMA4, PDPN, CAV-1, and ERG as potential biomarkers for canine splenic hemangiosarcoma

Jennifer R. Hagen, Chinatsu Mukai, Kelly L. Sams, Scott A. Coonrod

Department of Biomedical Sciences (Hagen, Mukai, Sams, Coonrod), College of Veterinary Medicine, Cornell University, Ithaca, NY

Hemangiosarcoma (HSA) is a vascular cancer that accounts for 67% of malignant, splenic masses in dogs. Currently, HSA is diagnosed via histopathology of either a biopsy or a splenectomy and the prognosis, even with treatment or surgery, is considered poor. Although histopathology is an accurate diagnostic method it is not quick. This is problematic because another splenic mass, nodular hyperplasia, may present with similar clinical signs but this benign mass can be considered cured with a splenectomy. Previously, lamanin-4 (LAMA4) and podaplanin (PDPN), associated with the extracellular matrix, were used to significantly distinguish HSA from normal splenic tissue. In this study, we aim to find a potential bio- marker that can readily distinguish HSA from nodular hyperplasia in the splenic tissue of dogs. LAMA4 and PDPN were used alongside caveolin-1 (CAV-1) and ETS transcription factor ERG (ERG). CAV-1 and ERG were genes chosen by their significance as determined by ChRO-seq and their involvement in cancer-specific traits such as angiogenesis. Immunohis- tochemistry was used to detect the antigens for the previously mentioned genes in HSA, nodular hyperplasia, and normal splenic tissues. LAMA4 and CAV-1 produced strong, positive stains in both HSA and nodular hyperplasia with weaker, positive staining in normal tissue. PDPN produced weak positive staining in all three tissues. ERG produced strong, pos- itive staining in normal and nodular hyperplastic tissues and weakly in HSA. Based on observational data, ERG had the most noticeable difference in positive staining between HSA and nodular hyperplasia. Therefore, ERG showed the greatest potential as a biomarker for hemangiosarcoma in dogs.

Research Grant: Unknown Student Support: NIH T35 AI007227

Telomere Attrition as a Biomarker for Animal Welfare in Sows: Role of Types of Leukocytes and Social Hierarchy

Carlin Hagmaier, Jennifer Punt, Thomas Parsons

Department of Pathobiology (Hagmaier, Punt), Swine Teaching and Research Unit (Parsons), University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

Public concern for animal welfare has increased and thus farms have focused on enhancing their welfare to appeal to consumers. Investigators have been working to discover biomarkers that can measure chronic stress levels in individu- al animals as an indicator of their welfare. Telomere attrition has been linked in humans to chronic stress, anxiety, and depression, and has been proposed as a potential biomarker for affective state in animals.We are working to determine if telomere attrition can be a measure of chronic stress in sows by comparing telomere length in blood cell populations from sows in well-defined positions in their social hierarchy of our research colony. We predict that the more socially domi- nant sows will have longer telomeres, indicating higher affective state. Our preliminary data has been promising, as we can successfully detect telomere length differences between sow and mouse immune cells using a flow cytometric assay, FLOW FISH. Mouse spleen cell telomeres are twice as long as the average sow telomeres. Also, we are able to couple telomere length measurements with surface staining to distinguish among the heterogeneous immune cell populations isolated from pig blood. We identified a small population of immune cells with lower telomere length compared to the majority of swine blood cells, and this population differs in frequency among individual sows.We will determine using cell surface staining the cell types in each population and if this assay is robust enough to discern differences in telomere length between swine. Literature on telomere length in human cells raises the possibility that it could represent a group of exhausted T cells, which may be an additional biomarker of an animal’s welfare.

Research Grant: Johnsonville Sausage Company Grant for long-term project “Telomere length as a novel indicator of sow welfare” Student Support: NIH T35 OD010919

2021 National Veterinary Scholars Symposium • 153 Osteosarcoma exosomes selectively home to the lung and elicit pro-tumorigenic changes in resident lung cells

Laurel A. Haines, Sophi J. Schofield, Eric P. Palmer, Kathryn E. Cronies, Chris G. Andretsos, Daniel P. Regan

Department of Microbiology, Immunology, and Pathology (Haines, Schofield, Palmer, Cronise, Andretsos, Regan), Colorado State University College of Veterinary Medicine, Fort Collins, CO

Osteosarcoma (OS), the most common primary malignant tumor of bone, often progresses to a highly fatal metastatic disease with limited treatment options. Following resection of the primary tumor, one-third of OS patients relapse with metastases, almost exclusively in the lung. Metastasis is preceded by the formation of a pre-metastatic niche, a process by which distant sites in the body are “primed” for tumor cell seeding by factors secreted by the primary tumor. Of these secreted factors, nano-sized extracellular vesicles, also known as exosomes, have been shown to mediate pre-metastatic changes in resident cells, and display highly specific organotropism in certain metastatic cancers. Little is known about the role of OS exosomes in modulating the pulmonary microenvironment during OS metastasis. We hypothesize that OS exosomes selectively home to the lung and instruct resident lung cells to create a pro-metastatic microenvironment char- acterized by inflammatory and structural changes. To investigate this, we evaluated human OS exosome biodistribution and cellular uptake in mice using intravital imaging, flow cytometry, and immunofluorescence. We also investigated the immunological effects of OS exosomesin vivo in mice and in primary human donor-derived lung fibroblasts and alveolar macrophages. We show that OS exosomes selectively track to the lung and elicit distinct changes in known tumor-promot- ing cytokines in vivo and in vitro. Our findings demonstrate that OS exosomes can alter the lung microenvironment prior to circulating tumor cell arrival. These pro-tumorigenic changes may promote metastasis during OS and could serve as early indicators and potential therapeutic targets for patients with metastatic disease.

Research Grant: The Boettcher Foundation NIH R03OD028265 Student Support: NIH T32GM136628

Developing a facial grimace scale to assess pain in goats

Madeline G. Hall, Michael D. Kleinhenz, Emily J. Reppert, Mikaela Weeder, Maria Lou, Miriam Martin, Andrew Curtis, Bailey Fritz, Shawnee Montgomery, Johann F. Coetzee, and Abbie V. Viscardi

Anatomy and Physiology (Hall, Lou, Martin, Curtis, Montgomery, Coetzee, Viscardi) and Clinical Sciences (Kleinhenz, Reppert, Weeder, Fritz), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Minimizing is a common goal in ethical research and for veterinarians in clinical practice. Facial grimace assessment based on changes to facial features in response to pain has been explored in several species to evaluate pain more objectively, rapidly, and non-invasively. This study aimed to construct a Goat Grimace Scale (GGS) as part of a larger study assessing pain associated with lameness in goats, a common issue seen in the livestock industry. Twenty-four goats of mixed breed and sex were randomly assigned to one of four possible treatments (n = 6 goats/treatment group): sa- line control, high dose/high volume, high dose/low volume, or low dose/low volume intraarticular injection of amphoter- icin B at the left rear digit. At each time point (-24 [Baseline], 4, 6, 12, 24, 48, and 72 h post-induction), the goats’ facial expressions were recorded with a video camera as they walked across a pressure mat. Facial action units (FAUs) of ear position, nostril shape & dilation, orbital tightening, and cheek tightening were determined based on 351 images captured from 168 videos. Two observers blinded to treatment group and time post-lameness induction will score the images based on the newly developed GGS to obtain a quantitative pain score for each goat. If the scores calculated from the proposed grimace scale mirror the results of other pain parameters collected, then a GGS may be beneficial to producers, research- ers, and veterinarians to rapidly detect pain in goats and improve overall animal welfare.

Research Grant: USDA, NIFA, AFRI Grant #2020-67015-31456 Student Support: NIH T35OD029981

154 • 2021 National Veterinary Scholars Symposium A clinical study of ophthalmic parameters of the Quaker parrot (Myiopsitta monachus)

Emily L. Halsmer, J. Jill Heatley, Erin M. Scott

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN (Halsmer); Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX (Halsmer, Heatley, Scott)

A major challenge to veterinarians treating exotic animal populations is the lack of published medical data for normative values to assist with the diagnosis and treatment of various diseases. Ophthalmic studies on the Quaker parrot (Myiopsitta monachus) have not been reported. The aim of this study was to establish normal ophthalmic parameters for select diag- nostic tests in clinically normal Quaker parrots of varying age, size, and sex. Ninety-six captive, adult Quaker parrots were assessed for weight and body condition scores. Complete ophthalmic examinations were performed including assessment of neuro-ophthalmic reflexes, phenol red thread test, rebound tonometry, fluorescein staining, palpebral fissure length measurements, slit lamp biomicroscopy, indirect fundoscopy, and ocular ultrasound biometry. All Quaker parrots had positive menace responses, dazzle reflexes, and direct pupillary light reflexes. Mean6 SD tear production and intraocular pressure in healthy parrots was 13.3 6 4.0 mm/15 sec and 10.6 6 1.4 mmHg, respectively. Mean 6 SD ocular ultrasound measurements include axial globe length (8.1 6 0.2 mm), anterior chamber depth (0.96 6 0.2 mm), lens thickness (2.6 6 0.1 mm), vitreous length (4.5 6 0.2 mm), axial pectin length (3.3 6 0.3 mm), and oblique pectin length (3.6 6 0.4 mm). Measures of association and descriptive statistics were analyzed using Analyse-it v5.81. Age, weight, and sex did not af- fect tear production or intraocular pressures. This work provides references and clinical findings to assist with monitoring the health of wild populations and maintaining the health of captive colonies of Quaker parrots.

Research Grant: None Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Efficacy of appetite stimulants in normorexic and hyporexic chinchillas

Emily Hamilton, Christoph Mans

Department of Surgical Sciences (Mans), School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI

The increasing popularity of chinchillas as companion animals has led to a concurrent increase in their presentation to vet- erinarians for reduced appetite due to various primary causes. Syringe feeding of critical care formula is often prescribed to treat hyporexic chinchillas, but the administration is stressful for the animal and labor-intensive. Therefore, the admin- istration of appetite-stimulating drugs to increase food intake in hyporexic chinchillas may be beneficial. In this study, two different drugs with proven appetite-stimulating properties in other species, capromorelin and mirtazapine, were systemat- ically evaluated in randomized, blinded, cross-over experiments. Capromorelin was evaluated at 3 and 10 mg/kg PO q24h for 5 doses in normorexic chinchillas and at 10 mg/kg PO q24h for 5 doses in chinchillas with experimentally induced hyporexia (alfaxalone-butorphanol sedation). Mirtazapine was evaluated in normorexic chinchillas at 5 and 20 mg/kg PO q24h for 5 doses and at 20 mg/kg in chinchillas with experimentally induced hyporexia (dexmedetomidine-midazolam sedation). In all experiments, food intake and fecal output were recorded in 24-hour intervals. Capromorelin resulted in a significant increase in food intake regardless of dose in normorexic animals. In addition, at 10 mg/kg, capromorelin did significantly attenuate the sedation-induced reduction in food intake. Mirtazapine’s effect on food intake was less pro- nounced in both normorexic and hyporexic animals. Future studies are needed to evaluate these drugs in clinical trials.

Research Grant: UW-Madison School of Veterinary Medicine, The Association of Exotic Mammal Veterinarians. Student Support: Boehringer Ingelheim.

2021 National Veterinary Scholars Symposium • 155 Phenotypic and functional characterization of mesenchymal stem cells from flamingo, polar bear, and tiger

Nicole Hamlin, Valerie Johnson

Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Mesenchymal stem cells (MSC) are a more widely used therapeutic agent in human and animal medicine with applica- tions in inflammatory diseases and regenerative medicine. Currently, little work has been done to characterize MSC in exotic species. According to the International Society for Stem Cell Research, MSC must be plastic-adherent with fibro- blast-like morphology and express certain surface markers with the absence of others. MSC can also undergo trilineage differentiation and suppress lymphocyte proliferation. We aimed to characterize MSC from Chilean and Lesser flamingos, polar bear, and tiger using the following criteria: plastic-adherence in culture; fibroblast-like morphology; demonstration of the expression of surface markers CD73, CD90, and CD105; lack of expression of CD34 and CD45; ability to undergo trilineage differentiation; and ability to suppress lymphocyte proliferation. Bone marrow-derived (BM-MSC) and adi- pose-derived (Ad-MSC) MSC were cultured and expanded in vitro, and differentiated into adipocytes, chondrocytes, and osteocytes. Ability to suppress T cell proliferation was investigated by co-culturing lymphocytes isolated from peripheral blood with species-matched MSC. Cells were phenotyped using antibodies from species anticipated to cross-react (human, chicken, canine, and feline). MSC negative surface markers were confirmed by positive staining of PBMC from each spe- cies. All MSC grown in culture were plastic-adherent and exhibited fibroblast-like morphology. Under appropriate culture conditions, trilineage differentiation was confirmed via Oil Red O,Alcian Blue, and Alizarin Red staining. Characteriza- tion of surface markers and lymphocyte suppression assays via flow cytometry are ongoing.

Research Grant: Michigan Translational Research & Commercialization (MTRAC) Grant GR-100403 Student Support: NIH Grant 5T35OD016477-19

Optimizing in vitro biochemical assays predictive of drug resistance in Dirofilaria immitis microfilaria

Naomi Hampton, Vicki Smith, Jeba Jesudoss Chelladurai

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS

Within the last decade, reports of macrocyclic lactone resistance in the canine heartworm Dirofilaria immitis has in- creased. The current gold standard test of experimental infections to determine the drug-resistance status of clinical Dirofilaria immitis isolates is expensive, time consuming, and presents ethical dilemmas. There is a need for a quick, simple, and inexpensive test that can be done in the lab to identify if the strain in question is resistant. We hypothesized that resistant and susceptible D. immitis strains show differences that can be detected byin vitro biochemical assays. Having previously shown proof of concept for these assays, we aimed to optimize them and understand their replicability and reproducibility. D. immitis microfilaria from presumed resistant and susceptible strains were isolated and experimental parameters for two spectrophotometric assays were optimized. We present data on microfilarial responses to changes in parameters such as time, microfilarial concentration, exposure to drugs and mode of detection.To understand the molec- ular basis of the assays, we assessed gene expression of selected drug metabolism and elimination associated genes using qPCR. We present gene expression data relative to the housekeeping gene 18S. In conclusion, we have optimized different experimental parameters with five establishedD. immitis strains to improve the replicability and reproducibility of two in vitro biochemical assays and sought to determine their molecular basis.

Research Grant: Start-up funds provided to JJC by KSU CVM Student Support: NIH T35 Training Grant (T35OD029981)

156 • 2021 National Veterinary Scholars Symposium An interactive, equine neurology case simulator to improve problem-solving skills

Anam Hamza, James Moore, Kelsey Hart, Joe Samson and William Stoughton

Department of Health Management (Hamza, Stoughton), Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, CA; Department of Large Animal Medicine (Hart), Educational Resources (Moore, Samson), The University of Georgia College of Veterinary Medicine, University of Georgia, Athens, GA

The challenges presented by the COVID-19 pandemic have illuminated the need and potential for online learning tools for a variety of settings to improve the learning capabilities of students. The development and use of online clinical case simulators in veterinary medicine would allow for connections to be made between concepts presented in traditional courses and real-life patient interactions, in a low-stress/low-risk environment. Consequently, there is a need for engaging, accessible, and realistic simulators for use in veterinary medicine. The objectives of this project are two-fold: 1) to devel- op an engaging, accessible and realistic online means of presenting equine clinical neurology case material to veterinary students in the pre-clinical portion of their education, and 2) to determine the impact of this approach on the students’ sub- jective scores of their level of confidence about their diagnostic skills and ability to problem-solve as they enter clinical rotations. The effectiveness of this approach will be assessed using a pre-test/post-test model, in which students’ perceived level of self-confidence and problem-solving skills will be evaluated before and after use. It is our hope that incorporating case simulators, such as this one, into the pre-clinical veterinary curriculum will positively impact the students’ confidence and problem-solving skills thereby reducing their level of concern about entering their clinical rotations. Ideally, improv- ing these skills will optimize patient care, ultimately improve their welfare, and increase client satisfaction.

Research Grant: For the Love of a Horse Awards Program, University of Georgia Student Support: Atlantic Veterinary College VetSRA, Boehringer Ingelheim Veterinary Scholar Award

The effect of ruminal fluid transfaunation on Cryptosporidium parvum shedding in calves

Michelle Hand, Clemence Chako

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Cryptosporidium parvum is a protozoan parasite affecting the intestinal tract of a variety of species; of the greatest con- cern are cattle, specifically dairy cattle neonates. Diarrhea is the most common symptom and although symptoms are often self-limiting in immunocompetent patients, more severe issues may arise for immunocompromised patients. C. parvum is commonly spread through feces via oocysts. Oocysts may be shed in the feces of neonate cattle even after symptoms subside, resulting in large numbers of infective oocysts persisting in the environment. This and the high number of oocysts shed per defecation greatly increase the rate of infection rate for both cattle and humans, due to its zoonotic potential. For this reason, C. parvum is also alarming for public health because those with compromised immune systems, such as young children, geriatric patients, and those with HIV are at a higher risk for developing more severe symptoms. This study aims to determine the effect of ruminal fluid transfaunation onC. parvum shedding in dairy calves. Ten healthy newborn calves will be split into two groups. Five calves will serve as the no-treatment control group, while the other five will serve as the experimental group. This latter group will receive ruminal fluid on days 3, 7, 14, 21, and 28. The amount of ruminal fluid administered each day will be 150 mL, 200 mL, 300 mL, 400 mL, and 500 mL, respectively. Fecal samples will be collected from all calves on treatment days. Oocysts isolated from these samples will then be enumerated using a hemo- cytometer. The results will be used to determine the effect of ruminal fluid transfaunation onC. parvum shedding in dairy calves.

Research Grant: None Student Support: Federal Work Study

2021 National Veterinary Scholars Symposium • 157 Genetics of supraventricular arrhythmias caused by accessory pathways in Labrador Retrievers

Caitlin N. Hardgrove, Victor N. Rivas, Samantha L. Kovacs, Kathy N. Wright, Joshua A. Stern

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA, USA (Hardgrove, Rivas, Kovacs, Stern), MedVet Medical & Cancer Center for Pets, Cincinnati, OH, USA (Wright)

Supraventricular cardiac arrhythmias caused by accessory pathways (SVAP) result in a high degree of morbidity and mortality in canine patients. This canine arrhythmia is notoriously difficult to manage with medication alone and cardiac ablation requires specialized equipment and training that limits the procedure to only a few centers in North America. Labrador Retrievers (LRs) make up nearly half of all SVAP-affected dogs, representing a strong breed predisposition that suggests heritability of this disease. We hypothesized that SVAPS in LRs represent an autosomal recessive disorder that associates with genetic variants within cardiac ion-channel genes. We aimed to use combined pedigree analysis, heritabil- ity calculations and whole genome sequencing to investigate the genetic origins of SVAPs in LRs. An extended pedigree was constructed which included 27 SVAP-affected LRs. Heritability was calculated using the MCMCglmm package in R. Whole-genome sequencing of 7 SVAP-affected and 13 clinically healthy (n = 20) Labrador Retrievers including 2 fami- ly trios was completed at 30x coverage and mapped to Canfam4. Visual inspection of the constructed extended pedigree is consistent with an autosomal recessive mode of inheritance. Bayesian analysis identified a mean heritability (h2) for SVAP of 0.10 (95% CI 9.0e-12-0.37). Whole genome sequencing was completed with an average of ~96% mapped reads per animal. Results confirm the heritability of this disease in the breed and suggests the most likely pattern of inheritance is autosomal recessive. Bioinformatic analysis of WGS data is pending.

Research Grant: University of California Davis, School of Veterinary Medicine, Center for Companion Animal Health (Stern PI; 2021-10-F) Student Support: Boehringer Ingelheim Veterinary Scholar funds- Students Training in Advanced Research, UC Davis

Defining the strain-specific effects of high-fat diet on intestinal epithelialchitecture ar in FVB mice

Briana Harper, Kieran Koch-Laskowski, Michael Shanahan, and Praveen Sethupathy

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

In both human and mouse models, high-fat diets (HFDs) have been shown to alter the structure and function of the in- testinal epithelium, a heterogeneous cell monolayer with absorptive, secretory, and protective functions. These changes are associated with chronic health complications, including metabolic diseases. However, susceptibility to HFD-induced epithelial biology and physiology changes can vary across mice strains and individuals, highlighting potential differences driven by genetic predisposition. We hypothesize that HFD leads to variable effects on the intestines in two genetically variable strains of mice. To test this hypothesis, we started by conducting a 16-week diet trial on adult FVB mice fed either a balanced chow diet or a 45% HFD. Next, we collected small intestinal sections to perform histomorphomet- ric analysis, including measurements of villus height, crypt depth, and crypt density, to determine changes in epithelial architecture between diet conditions. We also generated alcian blue-stained tissue sections to quantify how diet affects the number of mucus-producing goblet cells. Further directions for this study will involve comparing our results in FVB mice to other strains, such as the C57BL/6 line. Altogether, our cross-strain analyses will highlight differences in genetic predis- position to HFD-induced changes in gut structure and metabolic function, which will contribute towards the development of translatable therapeutic strategies for various metabolic diseases in both animals and humans.

Research Grant: 1-16-ACE-47 (ADA Pathway Award awarded to P.S.) Student Support: Cornell University, College of Veterinary Medicine, Veterinary Investigators Program

158 • 2021 National Veterinary Scholars Symposium Evaluation of aseptic technique, analgesia and anesthesia protocols in video-based articles for rodent surgery

Shanice L. Harris, Jennifer Haupt

College of Veterinary Medicine, Kansas State University, Manhattan, Kansas (Harris), Department of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts (Haupt)

Aseptic surgical technique for rodent survival surgery is mandated by the Guide for the Care and Use of Laboratory Animals and is required in many institutional standards. The welfare of laboratory animal species is dependent on proper training of surgical techniques including aseptic practices and appropriate use of and anesthetics to alleviate pain and distress. With advances in web-based training, many investigators are seeking outside sources for procedure-spe- cific training. Concerns regarding these resources include demonstration of inappropriate sterile surgical techniques and inadequate anesthetic and analgesic regimens. To investigate potential deficits, 100 randomly selected video-based articles on the topic of rodent survival surgery were evaluated by a boarded veterinary surgeon for aseptic surgical principles, appropriate tissue handling and wound closure, analgesic and anesthetic use, and postoperative monitoring. Analysis revealed that asepsis was maintained in only 15% of the videos with 70% and 15% having minor and major breaks in technique, respectively. Analgesic use was described in 52% of the videos, with pre-emptive analgesia in 47% of proce- dures. (immediate and sustained release) predominated (56%) over non-steroidal anti-inflammatory drugs (28%). Similar to analgesics, the use of anesthetics was described in 53% of the videos and anesthetic depth was checked in only 43% of the videos. Common choices of anesthetic included isoflurane (44%) and ketamine/alpha-2 agonist (33%). This study highlights the inadequacy of these video-based articles for surgery training and alerts the laboratory animal community of the video articles by investigators for surgical training purposes.

Research Grant: None Student Support: Dorothy W. Poitras Professorship

Characterizing Anatomy and Predicting Function of the Male Florida Softshell Turtle Phallic Glans

Sydney Harrison, Brandon Moore, Ruth Elsey, Mark Does

Department of Biological Sciences, Stephens College, Columbia, Missouri (Harrison, Moore), Rockefeller Wildlife Ref- uge, Louisiana Department of Wildlife and Fisheries (Elsey), Biomedical Engineering, Vanderbilt, Tennessee (Does)

While male Florida softshell turtles (Apalone ferox) possess an intromittent phallus for internal fertilization, the tissue-lev- el structures and associated copulatory functions are unclear. To that end, two adult male phalli were acquired (Rockefeller Wildlife Refuge, LA). Post necropsy and tissue fixation, we accessed gross anatomy, performed paraffin histology, and produced 3D models from MRI imaging to better understand form and function. Phalli displayed a proximal shaft and distal elaborated glans. The glans was composed of two tissue compartments, a dorsal, dense corpus fibrosum (a mam- malian corpus cavernosum analog) and a ventral, vascular inflatable corpus spongiosum.The dorsal glans is composed of a collagen fiber-rich fibrosum that terminates in a point and is flanked with two pairs of lateral angled appendages. The sulcus spermaticus, the semen-transporting open groove, terminates into a cup-like lumen on the ventral surface of the glans. The inflatable corpus spongiosum flanks the sulcus spermaticus and invests the lateral appendages. Our prelimi- nary results have better defined further questions. We can infer the corpus fibrosum aids intromission and the spongiosum inflates to produce an elaborated glans shape via the lateral appendages. However, the sulcus spermaticus does not travel to terminal glans. Therefore, its direct role in insemination is unclear. Glans inflation most likely serves as a copulatory lock prolonging intromission, but additionally, inflation of the lateral appendages may producemale-to-female stimulation invoking cryptic female choice. Progressing from these results, we will investigate male glans function in vivo and begin an extensive analysis of female cloacal morphology.

Research Grant: N/A Student Support: Stephens College School of Health Sciences Summer Research Grant

2021 National Veterinary Scholars Symposium • 159 Evaluating orthopedic injuries during the Yukon Quest

Jenna Hattendorf, Cristina Hansen

College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO (Hattendorf); Department of Veterinary Medicine, College of Natural Science and Mathematics, University of Alaska, Fairbanks, AK (Hansen)

The Yukon Quest honors the history of sled dogs with a 1,000 mile sled dog race that embraces a historically well-traveled path between Whitehorse and Fairbanks. During the race, mushers are led by a team of up to fourteen sled dogs who are elite marathon runners. Canine athlete health is a top priority and veterinarians are stationed at checkpoints throughout the race to ensure all athletes remain in peak condition and if needed, dogs can be dropped from the team. The aim of this study is to evaluate and classify the reasons for dogs being dropped during the Yukon Quest. Preliminary data shows that 68% of dropped dogs experienced an orthopedic injury, 14% experienced GI illness or weren’t eating, 8% experienced cardiorespiratory illness, and 10% were dropped for another reason. Orthopedic injuries impacted 20% of the total dogs that raced, and two thirds of those dogs were dropped. Orthopedic injuries can be treated along the route with wraps, massage, heat and/or ice. However, the International Federation of Sleddog Sports bans the use of pain medications during competition. If more intensive treatments and/or pain medications such as carprofen are required the athlete must be dropped. Some orthopedic injuries get better as the athlete warms up, but many get worse. If the musher pushes a dog too far, the injury may cause lasting damage. In 2020, of the dogs with orthopedic injuries that finished the race, 82% had a wrist injury. This is an example of data that can help mushers determine whether an injury warrants dropping a dog to receive treatment. This study is significant because it will inform mushers of which injuries are likely to need further treat- ment and which can be treated throughout the race.

Research Grant: None Student Support: CVMBS funded scholar

Evaluation of predictive models to determine outcome based on data known at first treatment for BRD in cattle

Lilli Heinen, Brad J. White, David E. Amrine

Beef Cattle Institute, Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Bovine respiratory disease (BRD) is the costliest health condition encountered on North American feedlots. Early detec- tion of chronic cases has the potential to decrease both costs associated with treatment and overall morbidity in a pen of cattle. The study objective was to evaluate diagnostic ability of predictive models based on data collected at first BRD treatment to determine whether an animal finished its feeding period with the group or had a negative outcome. Data used to train and test algorithms included individual animal, pen, and feedlot level data. The final dataset consisted of 175,784 BRD cases from 14 feedlots of which 14.9% did not finish the feeding period. Five algorithms were trained: advanced perceptron, decision forest, logistic regression, neural network, and boosted decision tree. Models underwent threshold probability adjustment to maximize F1 score to balance sensitivity and positive predictive value. Algorithms were eval- uated based on final accuracy, sensitivity, specificity, positive and negative predictive values, and area under the receiver operating characteristics curve. The boosted decision tree model had the highest AUC value (0.713) making it the model with the highest predictive power. This model had the highest F1 score (0.368) indicating the best balance between PPV and sensitivity. Its low PPV (0.276) indicated a weak ability to predict the negative outcome, but its large NPV (0.908) demonstrated the model’s ability to accurately identify the outcome of finishing the feeding period. In summary, the evalu- ated models can be useful for identifying the positive or negative outcome, but not both. Further investigation is needed to improve the predictive ability of these algorithms.

Research Grant: None Student Support: Elanco Animal Health

160 • 2021 National Veterinary Scholars Symposium Prevalence and pathology of equine parvovirus-hepatitis in New York racehorses

Caitlin Henry, Mason Jager, Megan Fahey, and Gerlinde Van de Walle

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York

Theiler’s disease, a.k.a equine serum hepatitis, is a devastating, highly fatal disease of horses. Equine parvovirus-hepatitis (EqPV-H) was discovered in 2018 and has been identified as the likely cause of this disease. While the incidence of Thei- ler’s disease is low, the prevalence of EqPV-H DNA in horses is up to 17% in some regions, suggesting that subclinical or persistent infection is common. The objective of this study was to determine the prevalence and potential pathogenicity of EqPV-H infection in New York racehorses. DNA was extracted from archived formalin-fixed, paraffin-embedded liver tissues from racehorses submitted for necropsy to the New York Animal Health Diagnostic Center as part of the New York State Gaming Commission-Cornell University postmortem examination program. One hundred and seventy samples from horses 2 to 12 years old were evaluated. Extracted DNA was tested for EqPV-H using PCR and gel electrophoresis. Tissue morphology and detection of viral nucleic acid of positive samples were assessed using in situ hybridization. Thirty-nine of the samples were PCR positive (23%). Of PCR-positive cases, 16 samples had positive hybridization for viral nucle- ic acid in hepatocytes. Specifically, 4 cases had rare individual cells with nuclear hybridization only, 6 had nuclear and cytoplasmic hybridization, and 6 showed hybridization in necrotic hepatocytes associated with inflammatory cells.These findings indicate that EqPV-H presence in the liver and viral-associated hepatitis are prevalent in New York racehorses and thus, warrants additional studies examining potential associations between performance and EqPV-H infection.

Research Grant: Harry M. Zweig Memorial Fund for Equine Research Student Support: Boehringer Ingelheim and Cornell University College of Veterinary Medicine

Development of a novel vaccine for Feline Enteric Coronavirus using recombinant Lactobacillus acidophilus

Mikaela E. Henry, Benjamin E. Curtis, and Gregg A. Dean

College of Veterinary Medicine and Biomedical Sciences (Henry), Department of Microbiology, Immunology, and Pathology (Curtis, Dean), Colorado State University, Fort Collins, CO

Coronaviruses are present throughout the world and affect a wide variety of species. In cats, feline infectious peritonitis virus (FIP) has a nearly 100% mortality rate and to date no effective vaccine has been produced. Because FIP emerg- es when the common and innocuous enteric virus, feline enteric coronavirus (FECV), mutates within an individual, we hypothesize that vaccination against FECV will protect cats from FIP emergence. This a novel approach which we be- lieve will be integral in combating this devastating disease. FECV enters the body through the mucosa of the intestines. Because of this, an orally delivered vaccine which can stimulate the mucosal immune system will be more effective at directly neutralizing the virus. The probiotic gram-positive lactic acid bacteria, Lactobacillus acidophilus, is perfectly suited for traversing the harsh environment of the gastrointestinal tract and delivering vaccine epitopes directly to the mucosal immune system. Using common molecular techniques, we have engineered Lactobacillus acidophilus to display FECV epitopes which we have identified as essential to viral biding and entry with host cells as well as replication and construction of the virion. At this time, we have begun a vaccine trial in young weaned kittens; orally immunizing them for 3 rounds (boosters). The next stages of our work will involve challenging the cats orally with FECV, replicating the conditions of natural exposure. Lastly, we will alternate challenge and screening fecal samples until control cats begin to shed virus. After which, ancillary diagnostics including vaccine titers on both serum IgG and fecal IgA will be collected and analyzed to determine the level of protection established by the vaccine.

Research Grant: Morris Animal Foundation Student Support: Mentor Funded

2021 National Veterinary Scholars Symposium • 161 Evaluation of the oral and tumor microbiome of dogs and cats with oral cancer

Victoria A. Herron, Lindsay L. Donnelly, Aaron C. Ericsson

Department of Veterinary Medicine and Surgery (Donnelly), Department of Veterinary Pathobiology (Ericsson), College of Veterinary Medicine - University of Missouri (Herron), Columbia, Missouri

Oral cancer is common in both humans and dogs, and oral flora have been implicated in the pathogenesis of head and neck cancer in humans. Differences in composition of the oral microbiome exist in people with and without cancer, but the oral microbiome of dogs as it relates to oral cancer has not been studied. The objectives of the present study are to character- ize the oral microbiome of dogs with oral cancer and compare it to that of healthy control dogs. We hypothesize that the cheek microbiome of dogs with cancer will differ from that of healthy controls and that there will be a significant compo- sition difference between tumor and cheek samples within dogs with cancer. Cheek swabs were collected from all dogs and tumor swabs were collected from dogs with oral cancer. DNA was extracted, sequenced, and annotated against a 16S rRNA amplicon library using QIIP2 software. Preliminary results show a significant difference in alpha (Shannon Diver- sity index, p = 0.009) and beta (PERMANOVA, Bray-Curtis, p = 0.0009) diversity of the oral microbiome of dogs with and without cancer. Taxa which appear to contribute to this difference includeGemella spp. (p = 0.000001) and Porphy- romonas gingivalis (p = 0.006), which were more prevalent in the cancer group. No significant difference in alpha or beta diversity was detected between tumor and cheek microbiome composition within dogs with cancer.

Research Grant: None Student Support: Boehringer Ingelheim

Self-Assembly of Complex Polymicrobial Infections

Amber A. Hickey, Lydia-Ann J. Ghuneim, and Robert A. Quinn

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI

Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene, which facilitates the transport of chloride ions across cellular membranes. This mutation results in the thickening of mucosal secretions which in turn cause a variety of health problems. Leading to the decrease in lung function and increased incidence of chronic polymicro- bial infection. This project aims to understand the assembly and progression of the CF lung microbiome when inoculated into a mucus-plugged lung bronchiole microcosm. We will be monitoring two microbiome communities obtained from the sputum of Cystic Fibrosis patients along with a single isolate community (P. aeruginosa) and pure sterile media (as a con- trol) over the course of 48 hours, in order to observe community dynamics. The results will be analyzed using multi-omics techniques which include: 16S rRNA sequencing, qPCR, and metabolomics to identify community members and metab- olites in the subsamples. We expect to see an increase in P. aeruginosa abundance in the beginning of the experiment due to oxygen respiration and then at later time points an increase in anaerobes by the end of the 48-hours. These results will allow for a better understanding of how these microorganisms assemble and interact when grown together, which can lead to future advances in understanding the complexity of these chronic infections as a whole.

Research Grant: NIH Grant R01AI145925 to Robert A. Quinn Student Support: NIH Grant 5R25HL103156-11 to Michigan State University

162 • 2021 National Veterinary Scholars Symposium Identification of novel miRNA isolated from serum-derived exosomes as a diagnostic biomarker of bovine TB

Hannah K. Hipkiss, Srinand Sreevatsan

Department of Pathobiology and Diagnostic Investigation (Sreevatsan), College of Veterinary Medicine, Michigan State University, East Lansing, MI.

Current diagnostic methods for the detection of Mycobacterium tuberculosis biovar bovis, the infectious zoonotic agent of bovine tuberculosis (bTB), are reviewed continuously to improve bTB surveillance and eradication programs im- plemented by TB-reporting countries. In the US, implementation of surveillance programs and milk pasteurization has greatly reduced the zoonotic risk of bTB infection in humans, but current methods of detection are time-insensitive, may lack in sensitivity and specificity, or are not capable of detecting latently-infected cattle. The major cost (80%) of eradi- cation programming is derived from veterinary administration of standardized tuberculin skin tests, but many costs such as market exclusion, consumer mistrust, are rarely quantified. Actiphage , a phage-based assay created by Swift et al., has identifiedM. bovis in circulating peripheral blood monocytes of single comparative cervical intradermal tuberculin (SICCT)-positive cows that frequently do not display granulomatous lesions. This challenges the current dogma that M. bovis bacteremia in cattle is rare. Given this insight, identification of circulatingM. bovis biomarkers would be an attrac- tive option given that it is minimally invasive, time-efficient, and highly specific. miRNAs are 18-22nt non-coding RNAs that modify gene expression and transcription. miRNA can be packaged into exosomes to invade host cells and modify the host transcriptome, or self-regulate mRNA to optimize growth conditions of the organism in limiting environments. This study aims to identify exosomal miRNA specific toM. bovis based upon validated miRNA profiles inMycobacterium tuberculosis biovar tuberculosis.

Research Grant: USDA NIFA, Michigan Alliance for Animal Agriculture (MAAA) Student Support: The Graduate School at Michigan State University

Characterization of antimicrobial peptides expressed by the equine ocular surface and amniotic membrane

Erin A. Hisey, Bianca Martins, Brian C. Leonard

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA

The ocular surface is an important interface with the environment, serving as a physical barrier and through the expres- sion of key effector molecules of innate immunity, antimicrobial peptides (AMPs). There are two main AMP subfamilies, cathelicidins and defensins, which have broad-spectrum antimicrobial activity against many pathogen types, including bacteria, viruses and fungi. This study aimed to define the AMP expression profile of the equine cornea and conjunctiva as well as amnionic membrane, a tissue used widely as a bioscaffold for surgical repair of the cornea as it exhibits anti-in- flammatory, antifibrotic, and antimicrobial properties. Thus, this study represents the initial characterization of the AMP repertoire of the equine amniotic membrane. Due to the gene sequence similarities in the beta-defensins and cathelicidins, orthologous genes were identified in the equine genome for investigation through a targeted qPCR approach. A prelimi- nary interrogation identified expression of DEFB103A (beta-defensin 103A) and eCATH3 (equine cathelicidin 3) in cor- neal epithelium. DEFB103A expression was also identified in equine amniotic membrane. These preliminary data suggest that the equine corneal epithelium and amniotic membrane express AMPs, and further characterization with quantification will be performed to determine the expression of additional AMPs. This characterization of equine AMPs may provide im- portant insights into the predisposition of horses to infectious keratitis, particularly fungal disease. Additionally, defining the AMP expression of the equine amnion can allow for prospective selection of amniotic membrane scaffolds with more potent AMP activity for the repair of significant corneal defects.

Research Grant: Center for Companion Animal Health and startup funds from the University of California, Davis Student Support: UC Davis School of Veterinary Medicine Endowment Fund (STAR program), and MRLT lab funds

2021 National Veterinary Scholars Symposium • 163 Comparing standard urine collection techniques to Peezy midstream device in a canine model

Meghan E. Hoel, Michael W. Wood

University of Wisconsin School of Veterinary Medicine

Collecting urine from a dog safely, non-invasively, and sterilely is technically challenging. Common urine collection techniques include cystocentesis and standard free catch (SFC). Cystocentesis is most sterile but requires the most techni- cal expertise. SFC samples are easiest to obtain but often have bacterial contamination. This prospective cross over study aims to determine whether obtaining urine samples from dogs using a sterile modified Peezy midstream urine collection device (MCD) yields decreased contaminant bacteria compared to SFC collected samples. A secondary aim is determining which technique is most clinically efficient. The hypothesis is that MCD will yield less bacterial contamination than SFC and is more efficient than cystocentesis. 22 healthy client-owned dogs > 15 kg had three urine samples randomly collected using the MCD, SFC, and cystocentesis over one week. Samples were analyzed by urinalysis and aerobic culture. Bacte- rial contamination was measured by comparing bacterial colony forming units per milliliter for SFC and MCD collections to cystocentesis. Efficiency of collection was measured by number of urine collection attempts and staff required. Prelimi- nary data trends indicate that the MCD yields a bacterial contamination rate of 33% and SFC 57% (P = 0.19). The median number of collection attempts for MCD was 1 and cystocentesis 2 attempts (P = 0.87). The median number of people to obtain an MCD and cystocentesis was 1 and 3 technical staff respectively (P < 0.01). While not currently significant, the current trends suggest that true midstream urine collection may yield decreased bacterial contaminants and improved efficiency.

Research Grant: None Student Support: University of Wisconsin School of Veterinary Medicine

Improving detection of digital dermatitis in beef cattle using computer vision and portable devices

Alexandria Hoerth, Srikanthmadhavan Aravamuthan, Juan Francisco Mandujano Reyes, Claiborne Bronkhorst, Montana Lins, Naomi Mathews, Nathan Bollig, Dorte Dopfer

Department of Medical Sciences, School of Veterinary Medicine, UW-Madison, Madison, Wisconsin

Digital Dermatitis (DD) is an infectious claw disease that affects beef and dairy cattle feet and causes lameness and reduced animal welfare worldwide (Dopfer et al., 2012). As DD is infectious, it can spread quickly within a herd and becomes increasingly difficult to manage, especially with possible intermingling exposure of beef cattle in a feedlot (Plummer and Krull, 2017). Computer vision (CV) models can be used for real time detection of DD, but improvements are needed to create more portable devices and increase utility for on farm use. We hypothesized that an upgraded version of a YOLOv4-tiny model can be used to create agreement that quantifies as “moderate” by Cohen’s kappa between the improved CV and human evaluator to quantify DD prevalence in feedlot beef cattle. A Raspberry Pi microcomputer will be used to run the CV model real time with a wireless GoPro Hero 5 camera. A treatment list will be created by combin- ing CV detection with radio frequency cow identification (RFID) signals. 1,832 images were reviewed for accuracy of 3 M-stage lesions (M0, M2, and M4) based on classification from Dopfer et al., 1997 and correctly formatted for training using YOLOv4-tiny and Darknet. Evaluation of the YOLOv4-tiny custom model shows a mAP of 64.20%. Real time object detection (external validation) will occur on-farm to determine accuracy of classification of DD lesions of unknown hooves in real time. Calculating Cohen’s kappa will occur for internal and external validation to measure agreement between the CV and human evaluator. The improved CV model will allow better identification of beef cattle with DD to receive prompt treatment, therefore having the potential to decrease DD prevalence in feedlots.

Research Grant: USDA-NIFA Animal Health Student Support: School of Veterinary Medicine, UW-Madison, Madison, Wisconsin

164 • 2021 National Veterinary Scholars Symposium A Role for EBOV VP40 PPxY Motif in Regulating Tight Junction Complexes of the BBB and Corneal-Epithelium

Justin M. Hoffman, Ronald N. Harty

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA

Ebola virus (EBOV), a hemorrhagic fever virus of the Filoviridae family, continues to cause high morbidity and mortality since its discovery in the 1970s with recent outbreaks in the Democratic Republic of the Congo and Uganda. EBOV is a single-stranded, negative sense RNA virus composed of seven genes coding for numerous proteins including viral matrix protein 40 (eVP40). eVP40 possesses a conserved Late domain Pro-Pro-x-Tyr (PPxY) motif at the N-terminus which has been shown previously to mediate interactions with type I WW-domains of host proteins to drive virion assembly and egress. Recent studies identified several PPxY interactors largely involved in the regulation of cellular tight junctions (TJs) including MAGI3, YAP, and TAZ, which are normally regulated by the PPxY motifs of host angiomotin (AMOT). The aim of the current study is to determine whether eVP40’s PPxY motif disrupts TJs at immune privileged sites includ- ing the blood-brain-barrier (BBB) and corneal epithelium (CE) by competing with homologous PPxY motifs found on AMOT. eVP40-GFP expressing lentivirus particles were generated using a 3rd generation packaging system, and I will use them to transduce D3 cells (BBB) and IHCE cells (CE) to assess TJ permeability via transepithelial electrical resis- tance (TER) assays. Our findings may identify a role for eVP40 in altering barrier permeability in the BBB and CE, which may contribute to regulating EBOV spread and disease progression during both acute and persistent infections.

Research Grant: NIH AI138052, NIH AI139392 Student Support: NIH T35 OD010919

Pharmacokinetics of grapiprant and effects on TNF alpha concentrations following oral administration to horses

Silke L. Hoffmann, Russell W. Baden, Dan S. McKemie, Philip H. Kass, Heather K. Knych

K.L. Maddy Equine Analytical Pharmacology Lab (Hoffmann, Baden, McKemie, Knych), Department of Population Health and Reproduction (Kass), UC Davis School of Veterinary Medicine, University of California, Davis, CA

Prostaglandin E2 (PGE2) is an arachidonic acid pathway product that elicits inflammation, excites sensory neurons and increases vasodilation and vascular permeability. The EP4 receptor is the primary mediator of the PGE2 proinflammatory pathway. Grapiprant, a Non-Steroidal Anti-Inflammatory (NSAID) is a PGE2 EP4 receptor antagonist that is an effective anti-inflammatory in dogs and devoid of some adverse effects seen with traditional NSAIDs. Previous reports have -de scribed the pharmacokinetics of grapiprant in horses when administered at low doses, however pharmacodynamic effects have yet to be described. This study’s objective was to describe the pharmacokinetics and pharmacodynamic effects of grapiprant using an ex vivo inflammatory model. Eight horses received one administration of 15 mg/kg grapiprant. Drug concentration determination blood samples were collected for 96 hours post administration. Plasma concentrations were determined using liquid chromatography-tandem mass spectrometry and non-compartmental analysis used to determine pharmacokinetic parameters. Additional blood samples were collected prior to and post drug administration, simulated a with PGE2 and TNF- concentrations determined using an immunoassay. The maximum grapiprant concentration, time to maximum concentration and area under the curve were 327.5 (188.4-663.0) ng/mL, 1 (0.75-2.0) hr and 831.8 (512.6- 1421.6) h*ng/mL respectively. The terminal half-life (harmonic mean (range)) was 11.1 (8.27-21.2) hr. Stimulation of a grapiprant treated blood with PGE2 resulted in significant inhibition of TNF- for 2 hours post drug administration. These results suggest a short duration of anti-inflammatory effect (2-4 hours) when administered at a dose of 15 mg/kg.

Research Grant: none Student Support: NIH T35 Training Grant 5T35OD010956-22

2021 National Veterinary Scholars Symposium • 165 Erosion of paint-coated lead fishing gear in a simulated loon gizzard

Jillian Hojsak, Dr. Mark Pokras

Loon Preservation Committee, Cummings School of Veterinary Medicine

The primary cause of death for adult common loons (Gavia immer) is lead poisoning from the ingestion of lead fishing tackle. Although some states are beginning to regulate the use of lead jigs, many anglers and agencies believe that a pro- tective coat of paint will prevent lead absorption and toxicity. The hypothesis of this study is that a paint coating on fishing jigs does not prevent the exposure of a lead-content core during the digestive process of the common loon, a piscivorous waterbird. A previous study on the Pekin duck digestive system (Anas platyrhynchos domesticus) reported a 45% erosion of lead shot, with significantly elevated levels of lead in multiple organs. The common loon has a thick, muscular gizzard comparable to an herbivorous bird like the Pekin duck, as well as a highly acidic gizzard characteristic of a carnivorous bird. This study simulates the function of the loon gizzard in order to document the extent to which a jig’s paint coat, hook, and lead core degrade and lose mass within a seven day testing period. Lortone 3A Rotary Rock Tumblers are used to simulate the gizzard’s digestive action. Small quartzite rocks and a HCl acid solution placed in the tumblers simulate the materials that are normally found in loon gizzards. Data is collected on the mass, diameter, percent paint degradation, and qualitative appearance of each jig every 24 hours in the seven day trial. This study investigates to what extent the rocks and acidic environment of the loon gizzard and a simulated contraction of its thick muscular walls will erode the paint within a week of ingestion. Both sale and usage of lead fishing gear need to be prohibited across the common loon range, regardless of a paint coat.

Research Grant: Summer Star Foundation for Nature, Art and Humanity Student Support: Summer Star Foundation for Nature, Art and Humanity

Methionine sulfoxide reductases (MSRs) and Fe-S cluster biogenesis

Alexandra Hommer, Carolyn Sevier

Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York

Iron-sulfur (Fe-S) clusters are protein cofactors that facilitate essential cellular processes. A deficiency in Fe-S proteins or Fe-S cluster biogenesis is linked to several disorders, ranging from cancer to ataxias. Reactive oxygen species (ROS) are highly reactive molecules formed by O2 reduction. Methionine side chains are particularly susceptible to oxidation by ROS. Notably, several Fe-S biogenesis enzymes contain higher than average methionine content. The eukaryote, S. cerevisiae, has been a model for the study of Fe-S biogenesis. Previous work in yeast established that strains deleted for the enzymes that repair oxidized methionines (methionine sulfoxide reductases, MSRs) show an upregulation of genes involved in iron regulation and a decrease in Fe-S protein activity (Sideri et al. 2009 Microbiology 115:612). We hypoth- esize that the mitochondrial Fe-S biogenesis machinery is susceptible to methionine oxidation, which disrupts the early stages of Fe-S cluster biogenesis. To test this model, we aim first to establish a role for the mitochondrial MSR in Fe-S cluster formation. Yeast have two MSR enzymes: Mxr1 and Mxr2. Mxr1 is localized to the cytoplasm, while Mxr2 is dual localized to the cytoplasm and the mitochondria depending on start codon usage. A methionine-to-isoleucine mutation in Mxr2 (Mxr2-M1L) disrupts mitochondrial Mxr2 production but maintains cytoplasmic activity. My goal is to establish if Fe levels and Fe-S protein activities are altered in cells lacking mitochondrial Mxr2 activity. If a disruption in Fe-S bio- genesis can be linked to an inability to repair methionine oxidation, a new focus for the prevention of the damage to Fe-S proteins may emerge.

Research Grant: NIH R01 GM105958 Student Support: NIH T35 OD010941, Cornell University College of Veterinary Medicine

166 • 2021 National Veterinary Scholars Symposium Endocrine responses to mare’s milk in neonatal foals

Julia Horton, Laura Hostnik, Hannah Kinsella, Hailey Snyder, Jamie Summers, and Ramiro Toribio

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

Critical illness in the neonatal foal is debilitating and can lead to loss of the suckle response necessary to acquire enteral nutrition. Clinically, energy dysregulation is common in premature or critically ill foals managed with enteral and paren- teral nutrition. The goal of this study was to evaluate the effect of mare’s milk and suckling on energy homeostasis in the healthy neonatal foal. Healthy Standardbred foals (n = 12), 24-72 hours of age, were allocated into 3 groups: foals nursed from the mare’s teat for 5 minutes (DN; n = 5), foals received mare’s milk via nasogastric tube (NGT) (MM; n = 5), and control foals received Lactated Ringer’s Solution (LRS) enterally (LRS; n = 2). Blood samples were collected frequently for 210 minutes. Blood glucose (BG) was determined by a point-of-care glucometer. Serum insulin and glucagon concen- trations will be determined via immunoassay. BG concentrations were not normally distributed. Non-parametric analysis was used to compare area under the glucose curve (AUCglu) among treatment groups. For DN foals, a gradual rise in BG was observed 1 hour after nursing, while MM foals had an increase in BG at 5 minutes. Robust elevations in BG were observed in all groups after removal of the muzzle at 180 minutes. Our findings suggest that bolus enteral feeding via NGT produces a sharper and less sustained increase in BG compared to nursing, while fasting may lead to exaggerated hormonal responses from the endocrine pancreas after reintroduction of nursing, perhaps as an adaptation to prevent en- ergy dysregulation during the neonatal period. These findings have clinical implications in the nutritional management of hospitalized foals.

Research Grant: OSU CVM Equine Intramural Funding Student Support: Epperson Summer Veterinary Research Scholar

Characterization of CD3+/CD20+ Large Cell Canine Lymphoma

Cory M. Howard, Steffanie Anderson, and Bonnie Harrington

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan.

Introduction: Immunophenotyping of canine non-Hodgkin’s lymphoma for B-cell and T-cell surface antigens is commonly performed to better elucidate the clinical outcome or potential treatment options. Expression of CD3 is associated with T-cell malignancies while CD20 is expressed in B-cells. However, a small subset of canine non-Hodgkin lymphomas express both CD3 and CD20 (CD3+/CD20+). Currently, this form of non-Hodgkin lymphoma remains poorly defined at the molecular level. Objective: In this retrospective study, we aimed to better characterize the histogenesis of CD3+/CD20+ lymphoma. Formalin-fixed-paraffin-embedded tissues from ten cases of +CD3 /CD20+ large cell lymphoma and breed- matched controls of peripheral large T-cell lymphoma and diffuse large B-cell lymphoma were selected from the Michigan State University Veterinary Diagnostic Lab. Results: Using PARR, we identified monoclonal T-cell receptor (TCR) rear- rangements in all biphenotypic cases. In addition, three out of ten cases also exhibited monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of dual lineage rearrangement. There was no significant difference in fre- quency of antigen receptor rearrangement between CD3+/CD20+ and CD3+ cases. In comparison with CD20+ lymphomas, CD3+/CD20+ lymphoma exhibited TCR rearrangement more frequently and IgH rearrangement less frequently (P = 0.0007 and 0.003, respectively). Immunohistochemical staining of the B-cell marker PAX5 was negative in all CD3+/CD20+ cas- es. Conclusions: Cases of canine CD3+/CD20+ lymphoma demonstrate similar antigen receptor rearrangements and PAX5 staining properties when compared with CD3+ lymphomas, suggesting a similar histogenesis of these two subsets.

Research Grant: None Student Support: Morris Animal Foundation and the Graduate School at Michigan State University

2021 National Veterinary Scholars Symposium • 167 First detection of a bat coronavirus in a New England big brown bat

Callista Huang, Kaitlin Sawatzki, Jonathan Runstadler

Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, Tufts University, Grafton, MA

Coronaviruses are common in many species of bats and can be a source of emerging infectious disease in other species. The breadth of coronavirus viral diversity in bat reservoirs in North America is poorly understood and coronaviruses must be further studied to elucidate transmissibility and causes of zoonotic spillover. We report the detection of a coronavirus in a New England bat. The virus was detected in a fecal sample from a big brown bat (Eptesicus fuscus). RNA extraction was conducted on 40 fecal samples of big brown bats from New England and Virginia, collected between July 2020 and April 2021. Two rounds of semi-nested RT-PCR were subsequently conducted on the RNA extracted from the samples using pan-coronavirus primers. The PCR products were run on 1.5% agarose gel to visualize the amplicons. Only 1/40 (2.5%) was positive, suggesting lower than expected prevalence or further need of assay optimization in this population. Ongoing research is being conducted to sequence the coronavirus and identify the genus of the coronavirus.

Research Grant: None Student Support: USDA

Molecular mechanisms of antiviral nanoparticles against SARS-CoV-2

Hanah F. Huber, Juliet Nava-Chavez, Cesar Aparicio-Lopez, Robert Delong

Department of Anatomy & Physiology and Nanotechnology Innovation Center, College of Veterinary Medicine, Kansas State University, Manhattan, KS

Despite the development, distribution, and administration of vaccines against SARS-CoV-2, new variants continue to emerge. There is a critical need for antiviral compounds capable of destroying the virus, both before and after infection of a host. The purpose of this project was to explore the antiviral effects of copper (Cu) and manganese zinc sulfide (MnZnS) nanoparticles in targeting SARS-CoV-2 for destruction and delivery, respectively. The major hypotheses for this proj- ect were 1) Cu nanoparticles degrade viral proteins and RNA, providing the opportunity to enhance the effectiveness of face masks in limiting the spread of the virus between hosts by killing the virus on contact, and 2) MnZnS nanoparticles disrupt binding of SARS-CoV-2 to its receptor hACE2, preventing viral infection of host cells. To test these hypotheses, model proteins and RNA were exposed to Cu nanoparticle-coated face masks and degradation was determined through protein and RNA gel electrophoresis. Protein and RNA interaction with MnZnS nanoparticles are being explored by CD and UV spectroscopy methods. Additionally, a genome wide RNA meta-analysis identified a 99.9% conserved homopu- rine-rich sequence within the SARS-CoV-2 genome which remained unaltered in variants of concern. RNAfold Webserver predicts this sequence forms a stable stem-loop which is currently being characterized and may be a potential target site for nucleic acid delivery using a nanoparticle delivery system.

Research Grant: NSF CBET PD 2029579 and K-State University College of Veterinary Medicine intramural funds (CEZID pilot grant program) Student Support: NIH T35 Training Grant T35OD029981

168 • 2021 National Veterinary Scholars Symposium Myostatin deletion preserves glucose regulation, muscle function, and endothelial function in type 1 diabetes

Denton R. Huff, Emily L. Nunan, Jillian L. Gore, Joshua T. Butcher

Department of Physiological Sciences (Huff, Nunan, Gore, Butcher), College ofVeterinary Medicine, Oklahoma State University, Stillwater, OK

Type 1 Diabetes Mellitus (T1DM) is a disease characterized by the destruction of insulin secreting pancreatic beta cells resulting in hyperglycemia, skeletal muscle muscle wasting, and vascular dysfunction. Animals afflicted with T1DM suffer from increased morbidity and early mortality, likely a result of an inadequate glucose homeostasis long-term. Muscle is the largest glucose sink in the body, thus targeting muscle function and health may prove beneficial in type 1 diabetics. Myostatin (GDF-8) is a negative regulator of muscle growth that is secreted by skeletal muscle and upregulated in type 1 diabetics. My hypothesis is that increased muscle mass via myostatin deletion protects the body from glucose dysreg- ulation and preserves muscle function in streptozotocin (STZ) induced T1DM in mice. Two groups of mice--wildtype control and myostatin gene deletion (knockout)--were used in this study to look at glucose regulation and muscle function. The ability of myostatin deletion to prevent cardiometabolic dysfunction in T1DM mice was assessed using intraperi- toneal glucose tolerance tests, HbA1c, metabolic cages, and pressure myography. Muscle function was assessed in vivo using electrical stimulation of the gastrocnemius muscle with a force and fatigue protocol. Myostatin deletion inhibited STZ-induced increases in plasma glucose, prevented decreases in muscle function, and preserved renal function. Myo- statin deletion also prevented endothelial dysfunction in gracilis arterioles, although endothelial independent dilation was unaltered at 28 days. Taken together, this data suggests that myostatin inhibition may be a target for effective treatment of the cardiometabolic dysfunction that occurs in T1DM.

Research Grant: NIA K01-AG064121 and College of Medicine Intramural Funds Student Support: Summer Research Training Program

Anti-Bovine Serum Albumin IgG antibody production in horses post-vaccination

Morgan Hull, Kaori Knights, Nora Springer, Elizabeth Perry, Katie Delph, and Elizabeth Davis

Department of Diagnostic Medicine/Pathobiology (Hull, Knights, Springer), Department of Clinical Sciences (Perry, Delph, Davis), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Post-vaccination hypersensitivity reactions in horses may be fatal. Bovine serum albumin (BSA), a common vaccine component, may stimulate production of anti-BSA antibodies and prime the immune system for future hypersensitivity re- actions upon booster vaccination. The objective of this study was to measure levels of total and isotype 3/5 anti-BSA IgG antibody pre- and post-vaccination in healthy horses and horses that experienced a vaccine-associated hypersensitivity reaction. We hypothesized that horses would develop anti-BSA IgG post vaccination, and horses with documented hyper- sensitivity reactions would have greater anti-BSA IgG levels compared to non-reactor horses. 74 clinically healthy horses had serum collected pre-vaccination and at 2 weeks, 1, 3, 6, 9, and 12 months post-vaccination. Serum was acquired from 25 horses that had a post-vaccination hypersensitivity reaction. Anti-BSA IgG and IgG 3/5 antibodies were measured via ELISA. Unvaccinated horse serum and commercial hyperimmunized equine plasma served as negative and positive controls, respectively. O.D. values were normalized to the negative control. Both anti-BSA IgG and anti-BSA IgG 3/5 antibodies were significantly increased from pre-vaccination values by two-weeks post-vaccination.This increase persist- ed at three months post-vaccination then returned to baseline 6 months post-vaccination. The 25 horses with a hypersen- sitivity reaction had significantly increased anti-BSA IgG 3/5, but not total IgG, compared to pre-vaccination levels of the clinically healthy samples. The results support that horses develop antibodies to BSA contained in vaccines and there is an association between anti-BSA IgG 3/5, in hypersensitivity reactions to equine vaccines.

Research Grant: Kansas State University College of Veterinary Medicine Mentored Clinical, Applied, or Translational (MCAT) Research Award Student Support: NIH T35 Training Grant T35OD029981

2021 National Veterinary Scholars Symposium • 169 Investigating Sirtuin 5 (SIRT5)-mediated regulation of macrophage polarization in the tumor microenvironment

Clarey Hung, James Mullmann, Irma R. Fernandez, Destiny Van, Robert S. Weiss

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

SIRT5 is an NAD(+)-dependent lysine deacylase that is upregulated in many aggressive types of cancer, including breast cancer, and is a promising therapeutic target. The Weiss lab has shown that SIRT5 loss in breast cancer cells in vitro inhibits proliferation and anchorage independent growth, while targeting Sirt5 in MMTV-PyMT mouse model of breast cancer slows mammary tumor growth and metastasis in vivo. Interestingly, Sirt5 knockout (KO) host mice failed to sup- port the tumorigenic growth and metastasis of transplanted SIRT5 proficient mouse mammary carcinoma cells, suggesting that SIRT5 has key tumor-promoting functions in the tumor microenvironment (TME). Furthermore, RNA sequencing of mammary tumors revealed that SIRT5 loss affects many immune response genes. Others also have shown that SIRT5 blocks IL-1b production in LPS-activated macrophages, inhibiting inflammation.We therefore hypothesized that SIRT5 inhibits a pro-inflammatory macrophage (M1) response and induces the differentiation of anti-inflammatory/pro-tumor (M2) macrophages. To test this hypothesis, we performed immunohistochemistry (IHC) staining for the pan-macrophage marker F4/80 in mammary tumor sections from Sirt5 WT and KO MMTV-PyMT mice. No significant difference in total macrophage tumor infiltration was found between the two groups. Ongoing studies will test whether SIRT5 loss causes a shift from a pro-inflammatory (M1) to a pro-tumor (M2) state by IHC staining for M2 and M1 polarization markers (Argi- nase 1 and iNOS, respectively). Resolving the effects of SIRT5 on macrophage phenotypes may reveal new opportunities to modulate the TME to suppress tumor growth and metastasis.

Research Grant: NIH R01 CA223534 Student Support: NIH T35 OD010941, Cornell University College of Veterinary Medicine

Prevalence of Intestinal Helminths in Dogs and Cats and History of Anthelmintic and Preventive Products Usage

Pauline J. Hunter, Noriko Aoi

Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama

In this study, medical files of the canine and feline patients that visited Veterinary Medical Teaching Hospital at Tuskegee University (TU-VMTH) between January 2020 and June 2021 were reviewed. During this time, 519 fecal flotation tests were performed on samples from 484 patients. There were 467 canine and 52 feline samples, and ages at time of sample collection were categorized as < 6 months (n = 162), 6 to 18 months (n = 53), 18 months to 6 years (n = 166), > 6 years (n = 134) and unknown (n = 4). Genders were intact female (n = 114), intact male (n = 177), spayed female (n = 118), neutered male (n = 105) and litter (5). Body weight was categorized as <25 pounds (n = 296), 25-50 pounds (n = 88), >51 pounds (n = 134), and unknown (n = 1). The reason of the visits was wellness (n = 458), sick (n = 33), and others (n = 28). No ova were found in 412 of 519 fecal samples (79.4%), while ova of Ancylostoma, Trichuris vulpis, Toxocara and/ or other types of intestinal parasites were identified in 107 samples (20.6%). The most common anthelmintics at or prior to the fecal flotation was Drontal® Plus tablets, followed by Panacur® and Nemex-2. The most common heartworm pre- ventive with or without anthelmintic effects used in dogs prior to the visits wasAdvantage Multi®, followed by Sentinel Spectrum®, ProHeart® 6 or 12 and Simparica Trio®. However, only 172 of 353 dogs and cats at > 6 months (48.7%) had history of purchase of heartworm preventive and only 88/172 (51.2%) had confirmed purchase of 12 months’ supply prior to visits. Prevalence of intestinal helminths was discussed and compared against backgrounds and purchase history of the preventive products, months of purchase, and use of anthelmintics. Research Grant: DHHS COE Grant # D34HP00001 Student Support: DHHS COE Grant # D34HP00001

170 • 2021 National Veterinary Scholars Symposium Safety and efficacy of a novel anti-DEK aptamer treatment for osteoarthritis using a canine model

Jessica Hynes, David M. Markovitz, Dalen Agnew, Hailey Maresca-Fichter, Joanna Acosta-Bencosme, Jane M. Manfredi

Department of Pathobiology and Diagnostic Investigation (Hynes, Acosta-Bencosme, Maresca-Fichter, Agnew, Manfredi) College of Veterinary Medicine, Michigan State University, East Lansing, MI; Department of Internal Medicine (Markovitz) University of Michigan, Ann Arbor, MI

Osteoarthritis (OA) is the most common orthopedic disorder in many species, and current treatments are limited to pain management alone. Given the newly recognized inflammatory nature of OA, the objective of this study was to look at the efficacy of intra-articular treatment with a novel anti-inflammatory, anti-DEK aptamer, in a canine OA model. Sixteen bea- gles underwent surgical transection of the cranial cruciate ligament of a randomly chosen leg and were given an injection with either the anti-DEK aptamer or sterile saline; directly post-op and two weeks post-op. Serial data collection included: objective gait and stance analyses, thigh circumference and goniometry. Statistics included repeated measure ANOVAS and mixed effects analyses (P < 0.05). Gait analysis showed, stance time (P < 0.03) and maximum peak pressure (P < 0.009) for the control (C) group were decreased over time, as was the maximum force for the treatment (T) group (P < 0.001) directly after the second injection, and control group after both the first and second injection (P < 0.002). Stance analysis revealed the T group had four instances of significantly decreased weight bearing (P < 0.05) and the C group had eleven (P < 0.04). Thigh circumference for both groups (T-P < 0.03, C-P < 0.05) decreased over time, as did the range of extension for the T group between days 30 and 180 (P < 0.03). Control treated dogs demonstrated more instances of significant decline in leg use and increased lameness post-operatively. These data support the finding that the anti-DEK aptamer had some efficacy in decreasing the inflammation and pain associated with gicallysur induced OA.

Research Grant: MTRAC Life Sciences Student Support: Graduate School at Michigan State University

Social aspects of antimicrobial use, resistance, and regulation in dairy farming in Alberta, Canada

Jennifer A. Ida, Warren M. Wilson, Daryl V. Nydam, S. Craig Gerlach, Susan J. Kutz, Cindy L. Adams, and Herman W. Barkema

Dept. of Production Animal Health (Ida, Barkema), Dept. of Anthropology (Wilson, Gerlach), Dept. of Ecosystem and Public Health (Kutz), Dept. of Veterinary Clinical and Diagnostic Sciences (Adams), University of Calgary, Calgary, AB, Dept. of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY (Nydam)

Antimicrobial resistance (AMR), or the ability of a microbe to withstand treatment with antimicrobials, is an emerging health issue that has been largely attributed to use of antimicrobials. Many of the current research and policy initiatives focus on knowledge translation and behavioral change mechanisms as ways to achieve reductions in antimicrobial use (AMU). However, the current approach fails to address underlying drivers of practice and is narrowly focused on achiev- ing a numeric goal. Given the failure to understand the contextual drivers of decisions made by dairy farmers concerning AMU, this study investigated one community’s understanding of AMU, AMR, and regulation in the dairy industry in Alberta via the use of ethnography. This included participation in on-farm activities and observations of relevant inter- actions on dairy farms for a period of 3.5 months. Interviews were conducted with 25 dairy farmers; nine were analyzed using thematic analysis. This resulted in four key takeaways. The farmers: 1) feel that AMU policies implemented in other contexts would be impractical if implemented in Alberta and would constrain their freedom to make what they perceive to be the best decisions for their animals; 2) believe that their first-hand knowledge is undervalued by consumers; 3) do not believe that the public trusts them to make the correct AMU choices and, consequently, worry that AMU policy will be guided by what they believe are misguided consumer concerns; and 4) are skeptical of a link between AMU in livestock and AMR in humans. Based on these findings, a better understanding of the sociocultural and political-economic infra- structure that supports such perceptions is warranted and should inform future policy.

Research Grant: Canada's Natural Sciences and Engineering Research Council (NSERC) Industrial Research Chair Program Student Support: NIH T35 OD010941; Cornell University College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 171 Assessing bat diversity and abundance in Massachusetts using acoustic monitoring

Vanessa M. Indeglia, Alison H. Robbins, Meera S. Gatlin

Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, North Grafton, Massachusetts

Bats have a major ecological value as seed dispersers, pollinators, and consumers of insects, including agricultural pests. Due to recent substantial decline of bats, there is currently a lack of data on bat populations on a national scale. Bat abundance and diversity can be assessed using ultrasonic recorders to collect bat calls and analyze the data to identify bat species and call frequencies. The goal of this study to complete acoustic surveys of bat populations in MA and contribute our results to the NABat program. With this study, we aim to identify optimal bat habitats for stationary detector place- ments, analyze bat call recordings to identify North American bat species in four MA locations, and promote community engagement through creation of bat walking routes at the Arnold Arboretum in Boston. We hypothesize that there will be higher species diversity and abundance in locations with larger areas of more suitable bat habitats compared to areas with poorer bat habitats. Data collection is ongoing and involves placement of two stationary monitors within 100 km priority grids established by NABat in Westford, Carlisle, Boston, and North Grafton for a period of 10 to 14 days. Initial walking surveying is conducted prior to stationary detector placement. Evening surveying using mobile detectors are being con- ducted several times to identify bat activity at the Arboretum for the walking routes. All acoustic data collected will be analyzed using the Kaleidoscope Pro software and submitted to the NABat program, which we hope will contribute to the evaluation of current bat population trends, threats, and promotion of future conservation efforts.

Research Grant: The Arnold Arboretum at Harvard University Student Support: Cummings School of Veterinary Medicine

Evaluation of lesions associated with condemnation of livers from Missouri cattle

Paige Isensee, Rosalie Ierardi, Pamela Adkins

Department of Food Animal Medicine and Surgery (Isensee, Adkins), Department of Veterinary Pathobiology (Ierardi), College of Veterinary Medicine, University of Missouri, Columbia, Missouri

USDA inspected beef processing facilities condemn livers based on evidence of flukes, abscesses, tapeworms, caroteno- sis, necrosis, telangiectasis, or contamination. Liver abscesses and flukes are the top reasons for condemnation worldwide and both lesions are associated with a negative impact on cattle production and carcass value. There is currently little research on causes of liver condemnation in the United States. The objective of this study was to evaluate the causes of liver condemnation in Missouri cattle. Condemned liver samples were collected from USDA inspected beef processing plants in Missouri. At the time of harvest, liver abnormalities were identified by a USDA inspector. Animal age, breed, and general area of origin were recorded. Samples were stored with unique identification information at 4°C.Within 24 hours of harvest, samples were transported to the University of Missouri on ice. All samples were grossly evaluated. Fluid was aseptically collected from all identified abscesses and cultured aerobically and anaerobically on Columbia BloodAgar at 37°C for 48 hours. Isolated bacteria were identified using MALDI-TOF mass spectrometry. Sample collection is ongo- ing; to date, 16 samples have been collected and grossly evaluated. Based on USDA inspection guidelines, gross lesions that have been identified were classified as evidence of fluke (6/16), telangiectasis (5/16), necrosis (3/16), and abscesses (2/16). No liver flukes have been visualized among the samples.Fusobacterium necrophorum was identified in 1/2 of the abscesses. Overall, understanding the causes of liver condemnation will help veterinarians and producers improve the health and welfare of cattle, ultimately improving carcass quality.

Research Grant: None Student Support: USDA National Institute of Food and Agriculture, Animal Health project 1025111

172 • 2021 National Veterinary Scholars Symposium Clinical use of ursodiol in feline medicine

Emily Jachec, Alexandra Wood, Jenessa Winston

Department of Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

Ursodiol is a naturally occurring bile acid prescribed to cats for a wide range of gallbladder and liver conditions. Ursodiol is known for its ability to expand the bile acid pool by increasing bile flow and displacing hepatotoxic bile acids.Addi - tionally, ursodiol has anti-inflammatory and anti-fibrotic properties. Therefore, ursodiol is used in a variety of feline hepat- ic diseases, including acute/chronic hepatopathies, cholangiohepatitis, and for gallbladder abnormalities. Alongside a vari- ety of clinical uses, there can be a wide range of dosage prescribed and duration of administration. A better understanding of ursodiol’s clinical utility, prescribed dosage and patient outcome can be beneficial to the veterinary community and to feline patients who suffer from various hepatic conditions. Therefore, this retrospective study aimed to determine the clin- ical use, dosage, and duration of therapy of ursodiol administration in feline patients at a teaching hospital over 13 years. Since 2008, 88 feline patients were prescribed ursodiol at the Ohio State University Veterinary Medical Center. Metadata from these patients, including signalment, history, diagnostics (including laboratory data and diagnostic imaging), ursodi- ol dosage/duration, and patient outcomes were recorded. In feline patients, ursodiol was most commonly prescribed for gallbladder sludge, cholestasis and as a hepatoprotectant. The most common prescribed dose was 10.89 mg/kg/day (range 5.2 to 33.9 mg/kg/day). Overall, these findings allow us to understand current clinical administration of ursodiol in feline patients, in order to guide future ursodiol research and pursue novel clinical applications of ursodiol administration.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Student Scholar

Safety and Efficacy of Low-Dose Radiation Therapy as a Modality for the Management of Feline Chronic Rhinitis

Elizabeth Jackson, Lauren Kustasz, Leanne Magestro, Stephan Carey

College of Veterinary Medicine, Michigan State University, East Lansing, MI

Objective 1)To determine whether the use of low-dose radiation therapy as an anti-inflammatory modality causes a reduc- tion in owner-reported clinical signs of chronic rhinitis, and 2) To determine whether low-dose radiation therapy protocols can be administered to cats with chronic rhinitis without the development of short- and long-term radiation therapy-asso- ciated adverse effects.Animals 14 client owned cats presented to Michigan State University with a diagnosis of chronic rhinitis. Diagnosis defined via clinical history, CT, rhinoscopy, and indicative biopsy.Methods Cats included in the study underwent three radiation therapy sessions under general anesthesia (2 Gy fractions once daily over three consecutive days). Clinical efficacy was evaluated via owner-reported assessment of symptoms using a severity of nasal inflammatory disease (SNIFLD) questionnaire modified for use in feline chronic rhinitis.Adverse effects were documented via owner follow-up as well as recheck examinations at 1-, 3-, 6- and 12-month post radiation therapy. Results This clinical trial is still ongoing. Of the three of cats treated thus far, two cats have shown marked improvement of quality of life reported at home and resolution of clinical signs. Final results are currently pending further recheck examinations and treatment of additional cats. Significance The preliminary information gained from this study provides support for the development of a promising treatment modality for cats with refractory feline chronic rhinitis and will have a significant impact on the quality of life of cats with this condition.

Research Grant: Michigan State University College of Veterinary Medicine Endowed Research Funds Student Support: Boehringer Ingelheim Veterinary Scholars Fellowship, Seeger Companion Animal Endowment Fund

2021 National Veterinary Scholars Symposium • 173 Regulation of host gene expression by an emerging paramyxovirus

Jared Jackson, Elizabeth Wrobel, and Biao He

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia

J paramyxovirus (JPV) was first isolated from moribund wild mice inAustralia in 1972. It is classified in the recently pro- posed Jeilongvirus genus, under the family Paramyxoviridae. JPV produces the small hydrophobic (SH) protein. Mouse fibroblasts (L929 cells) infected with rJPVDSH produced significantly more TNF-a and had greater rates of apoptosis versus cells infected with rJPV, suggesting that SH modulates host immune defenses. We used RNA-seq to assess how the SH protein influences host gene expression during early infection. L929 cells were infected with either rJPV or rJPVDSH at a MOI of 5. Mock-infected cells served as a control. RNA was collected at 2hpi, 4hpi, 8hpi, and 12hpi and Illumina sequenced. The reads were analyzed using the Tophat2-Cufflinks protocol. Differentially expressed genes (DEGs) with $ # a log2FC value 1 with a false discovery rate (FDR) 0.05 were considered statistically significant. At 2hpi, very few DEGs were detected, but these numbers dramatically increased as the infection progressed. At 12hpi, 709 DEGs in the rJPV infection and 623 DEGs in rJPVDSH infection were discovered. At 4hpi, 8hpi, and 12hpi, the rJPV-infected cells have more DEGs than the rJPVDSH-infected cells. Many DEGs were unique to only one type of viral infection, indicating that the host transcriptional response differs between the two viruses.A subset of DEGs, including Rtp4, Ifit3, and G0s2, were analyzed by qRT-PCR and validated our RNA-seq results. This is the first study to assess how host gene expression changes in response to infection with a jeilongvirus. In addition, this study provides new insights into the complicated molecular mechanisms underlying the ability of the SH protein to affect host immunity.

Research Grant: Unknown Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

Role of Estradiol in Intestinal Ischemia and Reperfusion Injury

Nathan W. Jackson, Miaomiao Wu, Jennifer Rowe, Sherry D. Fleming

College of Veterinary Medicine (Jackson) and Division of Biology (Wu, Rowe, Fleming), Kansas State University, Manhattan, KS

Stroke, myocardial infarct, gastrointestinal injury, and many more conditions result from ischemic events, but the return of blood flow during treatment leads to reperfusion injury. Ischemia-Reperfusion (I/R) injury is the leading cause of human death and severe injury in first-world nations. In animal health, clinical scenario examples of I/R include equine colic and canine gastric torsion. Animal models increase our understanding of the mechanisms of injury and demonstrate differences in damage between sexes. The post reperfusion role of estradiol in the immunoinflammatory response is not well studied. Estradiol was predicted to modulate the innate immune response and this hypothesis was studied using in vitro and in vivo experiments. As a significant contributor to the innate immune response, macrophages (cell line and bone marrow-de- rived) were treated with or without estradiol prior to being exposed to normoxia and hypoxia. In vivo studies included subjecting homozygote or heterozygote (control) estrogen receptor 2 (ESR2) deficient mice and wildtype or ovariecto- mized mice to intestinal I/R injury. Preliminary studies indicate that intestinal eicosanoid concentrations significantly increase in ESR2 knockout female mice without being significantly affected in male mice.The absence of estradiol signal- ing increases injury in female but not male ESR2 knockout mice when compared to wildtype mice. Overall, it appears that estradiol changes the immunostimulatory mechanism of intestinal I/R induced injury.

Research Grant: DOD W81XWH-18-1-0716, NIH P20 GM103418, JCRC at K-State. Student Support: Boehringer Ingelheim

174 • 2021 National Veterinary Scholars Symposium Correction formula determination for estimated glomerular filtration rate in puppies with and without XLHN

Sarah A. Jacobson, George E. Lees, and Mary B. Nabity

Department of Veterinary Pathobiology (Jacobson, Nabity), Department of Small Animal Clinical Sciences (Lees), College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Iohexol plasma clearance is a common method for estimating glomerular filtration rate (GFR) to assess kidney function in dogs. To most accurately estimate GFR, an 8-point sampling method is required, and in adult dogs, correction formu- las have been developed to permit accurate GFR estimation from more limited sampling schemes. Our objective was to decrease the number of sampling points required to estimate GFR in puppies with and without X-linked hereditary nephropathy (XLHN) by developing a correction formula specific to this population. Previously, GFR was calculated in these puppies based on non-compartmental analysis of the 8-point sampling measurements using WinNonlin, wherein the trapezoidal rule was applied to determine the area under the curve. Using this 8-point GFR as the response variable, multiple potential correction formulas were generated via linear regression models, where the 1, 2, and 6 hour, 1, 4, and 6 hour, 1 and 6 hour, and 2, 3, and 4 hour uncorrected GFR determinations were the explanatory variable. Ongoing analysis will determine which correction formula and sampling times produces the most consistently similar GFRs as compared with the 8-point calculation. We expect this formula to differ from the previously established correction formula for adult dogs in this colony. Use of a correction formula will reduce the number of blood draws from puppies in this colony that will be required to estimate GFR in the future. This study also highlights the need for further investigation to determine a correction formula that would be adequate for most puppies, with and without kidney disease.

Research Grant: None Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Understanding how the food preservative tBHQ can be associated with Th2-meditated food allergies

Madison Janey, Yining Jin, Allison P. Boss, and Cheryl D. Rockwell

BRUSH Summer Research Program, College of Veterinary Medicine (Janey), Department of Pharmacology and Toxicology, College of Human Medicine (Jin, Boss, and Rockwell), Department of Food Science and Human Nutrition (Boss), Michigan State University, Lansing, MI

In the past few decades, the U.S has seen a rapid increase in food allergy cases, but the cause remains unknown. Re- cently, Dr. Rockwell has conducted research finding a potential reason for the basis of allergies. There is a possible link between food preservatives such as tert-butlyhydroquinone (tBHQ) and T Cells within the immune system that could be responsible for the development of allergies. While previous studies to examine the role of tBHQ on food allergies have already been conducted on adult mice, our current experiment focuses on juvenile mice because allergies typically arrive in infants. We hypothesize that by developing and characterizing this model of food allergies in juvenile mice, we can then determine the effect of tBHQ and its immune response to food allergens at an early age. The BALB/c mice were fed with 0.0014% tBHQ or control diet (without tBHQ) and were used to produce pups. The pups were sensitized to ovalbumin (OVA) from day 12. Sensitization was conducted by a tape-stripping method to expose skin to the OVA (10mg/mouse) in a 3-day interval six times. The OVA-sensitized pups were challenged by oral gavage with OVA (10mg/mouse). Rectal temperature will be measured. Symptoms will be scored on a scale from 0 to 5 based on severity. Tests are then run for mast cell degranulation in the gut and measurements for detecting OVA-specific antibodies IgE and IgG1 from the plasma. We expect that the clinical symptoms of the tBHQ group will display a more severe allergic response.

Research Grant: National Institute of Environmental Health Sciences (NIEHS) grants ES030766 and ES024966 Student Support: NIH Grant R25 HL103156

2021 National Veterinary Scholars Symposium • 175 Ultrasound analysis of Holstein calf mammary gland development

Derek Jantzen, Laura L. Hernandez, Joao Dorea, Alysia L. Vang

Summer Scholars Program, University of Wisconsin-Madison, Madison, WI (Jantzen), Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI (Hernandez, Dorea), Endocrinology and Reproductive Physiology Program, University of Wisconsin-Madison, Madison, WI (Vang)

The mammary gland has been an important focus in bovine research, yet not many studies focus on development from pre-weaning though first gestation. During this time is when development of mammary epithelium occurs; milk produc- tion is highly dependent on development of this tissue. For this reason, mammary gland analysis is required to further understand the development and growth, along with the effects of environmental factors such as diet. Holstein calves were pair-fed high (HI; n = 15) and low (LOW; n = 15) nutrition diets for eight weeks. LOW diet contained 0.45 kg of milk replacer per day with 22% crude protein and 15% fat, and HI diet contained 1.12 kg of milk replacer with 27% crude protein and 20% fat. Blood samples were collected weekly for glucose analysis. No differences were detected for glu- cose concentrations in calves on different diets (P > 0.05). Mammary gland ultrasound images were taken twice weekly. The parenchyma (PAR) tissue of 1,414 ultrasound mammary gland images were segmented, and the area was extracted. Images were analyzed using Matlab 2021b and statistical analysis was conducted using a mixed model including the fixed effect of time, diet (HI and LOW) the interaction time*diet, birth weight, and the random effect of animal.There was no statistical difference in PAR circularity, but the PAR perimeter and maximum length was greater in HI (10.1 and 290 mm, respectively) compared to LOW (8.5 and 243 mm, respectively). Mammary biopsies taken at week eight will be used to monitor the histological changes and gene expression. Data suggests that features from mammary gland ultrasound images can be a useful to monitor the development of parenchymal tissue in pre-weaned calves.

Research Grant: USDA 2020-67015-30831 Student Support: Foundation for Agriculture Research

Monocyte-derived dendritic cell signaling response following Cryptosporidium parvum exposure

Jessica Jasenski, Jack Guinan, and Brina Lopez

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Cryptosporidium parvum (CP) is a zoonotic intestinal parasite commonly found in cattle. While CP is deadly to calves, adult cattle remain resistant. CP attacks intestinal epithelial cells and may result in death if a protective immune response does not occur. This study aims to generate adult cattle monocyte derived dendritic cells (MoDC) from magnetically puri- fied CD2- monocytes and culture them in serum-free medium. Following MoDC exposure to CP, the protective immunity developed will be characterized by examining the MoDC metabolic, cytokine, and phenotypic profiles.To ensure MoDC stimulation, metabolic transition to aerobic glycolysis following cellular activation will be measured using an extracellular flux analyzer (Seahorse XFe96). Cellular phenotype will be assessed using fluorescent antibodies specific to CD25, CD50, CD205, and MHC class-II to determine activation status, and then quantified using flow cytometry (Guava easyCyte 12HT Luminex). Proinflammatory cytokines released by MoDCs in response to CP (IFNg, IL-1a, IL-1b, IL-4, IL-8, IL- 10, IL-17A, MIP-1a, and TNFa) will be quantified using a bovine specific cytokine panel (Magpix-Milliplex in conjunc- tion with MilliporeSigma).

Research Grant: Midwestern University Faculty Fund Student Support: Boehringer Ingelheim Summer Scholars Program and Federal Work Study

176 • 2021 National Veterinary Scholars Symposium Contents and efficacy of quick start nitrifying bacteria aquaria supplements

Sirah Javier, Mark Acierno, and Alyssa Scagnelli

Department of Specialty Medicine (Acierno), College of Veterinary Medicine, Midwestern University, Glendale, AZ

Ammonia is toxic to fish and a major contributor to “new tank syndrome.”Aquarium enthusiasts use a variety of quick start nitrifying products to accelerate the conversion of toxic ammonia to less dangerous nitrites and nitrates. These prod- ucts claim to contain a solution of beneficial ammonia-oxidizing bacteria; however, composition of these bacterial cultures is poorly understood. In addition, there are questions concerning the viability and efficacy of these products once the product has been shipped and purchased by local consumers. In this study, we model five different aquaria inoculated with 1 ppm of ammonium hydroxide. Each of the five tanks is inoculated with a different quick start nitrifying product. Levels of ammonia, nitrite, and nitrate are analyzed using colorimetry over a 2-week period. A control aquaria containing only 1 ppm of ammonium hydroxide was also established. Each quick start product was cultured to identify the bacterial content of each product using blood agar plates and ATCC Medium 2265. In addition, we analyzed the contents of each product using PCR. This information will provide valuable insights into the efficacy of these products.

Research Grant: Midwestern University Student Support: Boehringer Ingelheim Veterinary Scholars Program

The role of Complement Receptors (CR) 1 and 2 in Borrelia burgdorferi infection

Daniela Jimenez, Vuvi Tran, Nicole Baumgarth

Veterinary Scientist Training Program (Jimenez) and Department of Pathology, Microbiology and Immunology (Baumgarth) School of Veterinary Medicine, University of California Davis, CA; Center for Immunology and Infectious Diseases, University of California, Davis, CA (Tran, Baumgarth)

Lyme Disease, the most common vector-borne disease in the US, is caused by Borrelia burgdorferi. Infection triggers robust antibody responses by its hosts, which control bacterial burden and inhibit disease, but cannot clear the infection. Mechanisms underlying this lack of antibody effectiveness are unknown. Germinal centers (GC) generate neither long- lived plasma cells nor memory B cells in experimental infection of laboratory mice. B. burgdorferi expresses proteins, such as BBK32, that suppress complement activation and inhibit complement-mediated lysis. Complement also regulates GC responses through interaction of antigen-complement and antibody-antigen immune complexes with complement receptors (CR) 1 and 2 expressed by B cells and Follicular Dendritic Cells. Inhibiting those interactions may suppress effective antibody-mediated immunity toB. burgdorferi. To study this, B-cell specific CR1/2 deficient mixed bone marrow chimeric mice were generated, infected with B. burgdorferi and compared to control chimeras. Immune cell populations were measured 28 days post infection via flow cytometry, bacterial tissue loads were quantified via RT-PCR, and anti-Bor- relia IgG responses were measured by ELISA. No significant changes in the host’s ability to clear the infection or in the anti-Borrelia IgG response were observed. To assess whether complement activation is inhibited so strongly by B. burg- dorferi that removing CR1/2 does not show detrimental effects, we are comparing mice infected with either a BBK32-de- ficient or a supplementedB. burgdorferi strain and will discuss our findings. By studying immune evasion strategies we expect to help the development of future therapeutic interventions that support pathogen clearance.

Research Grant: NIH/NIAID R01AI157007 Student Support: Students Training in Advanced Research (STAR) Program

2021 National Veterinary Scholars Symposium • 177 Efficacy of lidocaine cream vs EMLA (lidocaine-prilocaine) cream for pain-free venipuncture in rabbits

Shannon S. John, Dylan Lucich, João Brandão

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

Over the years exotic animals, like rabbits, have increased in popularity as household pets. Rabbits present a unique risk when handling because of their tendency to cause spinal injury during attempts of escape. Proper pain relief prior to simple procedures such as catheterization can help alleviate handling stress, but knowledge available to veterinarians and caretakers of pain management techniques is limited. However, knowledge available to veterinarians and caretakers of proper pain management techniques is limited. The current recommendation for catheterization in rabbits is application of lidocaine-prilocaine (EMLA) cream one hour before injection. In a clinical setting, this current standard is tedious and is impractical. By catheterizing the marginal ear vein of rabbits this study aims to (i) compare the efficacy of lidocaine cream to EMLA cream as a pain relief option for venipuncture, and (ii) compare different application times of EMLA cream. Pre-catheterization treatment options will include, EMLA cream applied for 10 minutes and 60 minutes, lidocaine cream for 10 minutes, and placebo cream for 60 minutes (control). Treatments are deemed successful only if the catheter is secured and patent to the marginal ear vein at first attempt of catheterization. The study is currently underway and results remain to be evaluated.

Research Grant: None Student Support: Oklahoma State University, CVM, Associate Dean for Research & Graduate Education

Relationship between personality traits and physiological indicators of chronic stress in ring-tailed lemurs

Callan Johnson, Hannah Spitzer, Henrik Stryhn, Lucia Amendola, Rebecca Meagher, Kathryn Seeley, and Kathryn Proudfoot

Department of Health Management, Atlantic Veterinary College (Johnson, Spitzer, Stryhn, Amendola, Proudfoot), University of Prince Edward Island, Charlottetown, PEI; Department of Animal Science and Aquaculture (Meagher), Dalhousie University, Truro, NS; Columbus Zoo and Aquarium (Seeley), Columbus, OH

Personality traits have been linked to chronic stress in humans, which can contribute to poor health outcomes. Less work has been done to understand the relationship between personality traits and chronic stress in non-human primates such as lemurs. The objectives are to 1) describe personality traits in ring-tailed lemurs under human care, 2) determine how reliably human raters can assess these traits, and 3) evaluate the association between personality trait ratings, age, sex and physiological indicators of chronic stress. A total of 22 ring-tailed lemurs housed at the Duke Lemur Center, Durham, NC will be included in the study. To assess personality traits and determine inter-rater reliability, 14 keepers will be given electronic surveys for each lemur and at least 2 keepers will rate each animal. The surveys will include a 54-item Hom- inoid Personality Questionnaire that has been validated for non-human primates. Chronic stress has previously been evalu- ated for each lemur using an “Allostatic Load Index” (0 to 6) calculated using a combination of 6 biomarkers associated with chronic stress (albumin, cortisol, DHEA-S, DNA damage, glucose and PGE-2). The hypotheses are that a range of lemur personality traits will be found, and that the questionnaire will be a reliable method to assess these traits. It is antic- ipated that older animals will rate lower in extraversion, neuroticism and openness, females will rate higher in dominance and that animals that rate higher in neuroticism will have higher indicators of chronic stress. Gaining a better understand- ing of personality traits in ring-tailed lemurs under human care will lead to improved welfare and has the potential to be applied on native range animals.

Research Grant: Columbus Zoo and Aquarium Conservation Fund Student Support: Atlantic Veterinary College Summer Research Award

178 • 2021 National Veterinary Scholars Symposium Cross-protective potential of an Edwardsiella ictaluri vaccine against E. piscicida in Oreochromis niloticus

Davida Johnson, Diem Thu Nguyen, David Wise, Suja Aarattuthodi, Alvin Camus, Matt Griffin, Esteban Soto

University of California, Davis, California (Johnson, Nguyen, Soto); Mississippi State University, Stoneville, Mississippi (Aarattuthodi, Griffin, Wise); University of Georgia, Athens, Georgia (Camus)

Edwardsiella piscicida causes enteric disease in a wide variety of fish species worldwide. Current control measures against E. piscicida infection have been largely ineffective and have contributed to antimicrobial resistance within the Edwardsiella genus. As a result, researchers have focused their efforts on developing adequate prophylactic practices. Edwardsiella ictaluri is a closely related species and a live-attenuated E. ictaluri vaccine has confirmed cross-protective effect againstE. piscicida in catfish. The objective of this study was to determine if thisE. ictaluri vaccine can also mit- igate E. piscicida infection in Nile tilapia (Oreochromis niloticus). To assess cross protective capabilities of this vaccine in Nile tilapia, fingerling were vaccinated via immersion (1x106, 1x107, or 1x108 CFU/ml), monitored for adverse effects, and challenged via intracoelomic injection (~105 CFU/fish) of a wild-type E. piscicida thirty days post-immunization. Al- though the vaccine was safe for Nile tilapia, no protection was conferred by the vaccine in immunized tilapia as survival curves amongst challenged treatments were similar (P > 0.05). Moreover, following the challenge, ten arbitrarily selected surviving fish from each treatment were collected and posterior kidney swabs inoculated in agar media to determine per- sistent E. piscicida infection. Edwardsiella piscicida was isolated from all surviving fish exposed toE. piscicida, regard- less of treatment. Further investigation of the live attenuated E. ictaluri vaccine in non-ictalurid fish species is necessary to better understand the cross protective potential of this vaccine.

Research Grant: This study was supported by The Southern Regional Aquaculture Center Research program. Student Support: Davida Johnson was supported by The Southern Regional Aquaculture Center Research program.

Risk factors associated with short-term perioperative anesthesia-related deaths of dogs and cats in Arizona

Maddison L. Johnson, Kendall A. Lewis, Phillip A. Hairr, and Rachael E. Kreisler

Department of Primary Care, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Several studies analyzing anesthetic-related deaths in small animal clinics have reported death rates ranging from 0.23- 2.2%, which is significantly greater than that in humans (0.02%).Additionally, researchers have identified specific factors associated with increased risk of death (e.g., ASA status, age, intubation). An Arizona statute requiring veterinary clinics to maintain anesthesia recovery logs provided a unique opportunity for a nested case-control analysis. The aim of this study was to investigate mortality rate and risk factors for dogs and cats undergoing anesthesia in Arizona from 2016 to 2019. Small animal clinics were recruited by email, phone, and through notice in the Arizona Veterinary Medical Asso- ciation newsletter. Participating clinics provided anonymized anesthesia recovery logs and medical records for cases of anesthesia-related death and controls. Potential anesthetic risk factors were coded from medical records and multivariable logistic regression was used to calculate odds ratios (OR) for the following potential risk factors: sex, age, weight, ASA status, urgency of procedure, intubation, pulse monitoring, administration of perioperative fluids, induction protocol, duration of procedure, and year. P-values less than 0.1 were considered significant at this exploratory stage. Seven prac- tices provided a total of 39,449 log entries. Nine of 16,865 cats and ten of 22,584 dogs died, resulting in a mortality rate of 0.05% (95%CI: 0.02%, 0.10%) and 0.04% (95% CI: 0.02%, 0.08%), respectively. ASA status greater than 1 had an OR of 4.6 (P = 0.108) and female sex had an OR of 11.7 (P = 0.050). Immediate-term mortality rates were low compared to historic rates covering up to 48 hours postoperatively.

Research Grant: Midwestern University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

2021 National Veterinary Scholars Symposium • 179 Locomotor activity in nutrient restricted primiparous beef cows during the final 72 hours prepartum

Remie M. Johnson, Colby A. Redifer, Lindsey G. Wichman, Abigail R. Rathert, and Allison M. Meyer

Division of Animal Sciences, University of Missouri, Columbia, MO

The objective of this study was to determine whether late gestational nutrient restriction (NR) changes locomotion activ- ity in primiparous heifers in the 72 h prepartum. On d 160 of gestation heifers were moved to 3.7 x 15.8 m pens and fed either 100% (control; CON; n = 11) or 70% (NR; n = 13) of energy and protein requirements for maintenance, pregnancy, and growth. IceQube accelerometers (iceRobotics, Edinburgh, UK) were placed on the left hind fetlock d 265 prepartum to determine motion index (MI), standing time (ST), lying time (LT), step count and number of lying bouts (LB). Hour 0 was denoted as the time of calf expulsion. Data were analyzed by day prepartum (-3 to -1 d), by 6-h periods in the final 24 h, and by hour in the final 6 h with fixed effects of treatment, time prepartum, and the interaction. Standing time increased and LT decreased (P = 0.01) on d -1 compared with -2 and -3 d. In the 72 h prepartum, LT was less and ST greater (P = 0.04) in NR dams compared with CON. The interaction of day x treatment affected P( = 0.01) LB, where LB number increased (P # 0.01) on -1 d compared with -2 and -3 d for both treatments. Laying bout number increased (P < 0.01) in the last 6h prepartum compared with prior 6 h periods. During the last 24 h, NR dams had greater ST and less (P = 0.01) LT than CON dams. The pattern of change in MI during 6 h prepartum was altered (P = 0.005) by NR. Standing time was greater and LT was less (P = 0.04) at -1 h compared with the hours prior except for h -4. Number of LB was greatest (P < 0.001) in the final -1 h prepartum. These data suggest that heifer locomotive behavior changes leading to parturition, with minimal differences caused by late gestational nutrient restriction.

Research Grant: This work is supported by AFRI Competitive Grant no. 2017-67015-26587 from the USDA National Institute of Food and Agriculture. Student Support: Stipend for Remie Johnson is supported by an endowment established by IDEXX-BioAnalytics

Antibiotic resistance profiles ofStaphylococcus spp. isolated from food matrices

Sierra Johnson, Teshome Yehualaeshet

Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama

The developing resistance of Staphylococcus spp. to antibiotics has generated a serious public health concern globally. The purpose of this study is to perform a cross-sectional data collection and analysis of Staphylococcus spp. isolates from various food sample sources. Antimicrobial resistance data was collected from 35 recent sample submissions and analyzed for the species identification and antimicrobial sensitivity of each isolate.Within this experiment, we identified the genus and species of each sample and analyzed the antimicrobial resistance profile of theStaphylococcus species utilizing the MicroScan. From all the submitted isolates (n = 35), ten Staphylococcus species were identified, predominatelyS. hyicus (8), S. aureus (6), and S. epidermidis (6). Based on the food samples, antibiotic resistance profile showcasedTrimetho - prim/Sulfamethoxazole as a particularly effective antibiotic against the most strains ofStaphylococcus species, with Ciprofloxacin following close behind. Erythromycin was shown to be the least effective antibiotic on the panel as a large quantity of the isolates had developed some form of antibiotic resistance. Regarding the level of species, S. hyicus was noted as the most susceptible species to the antibiotics, while S. sciuri was noted as the most resistant. Appropriate actions must be taken on importance of antimicrobial stewardship, maintaining the standard operational procedures of cleaning and sanitation of processing plants and the establishments to monitor the development, and spread of antibiotic resistance in the food system. Further study will be required to keep pace with the continuously growing resistance of Staphylococ- cus species in the food chain.

Research Grant: Boehringer Ingelheim Veterinary Scholars Program (BIVSP) and Tuskegee University, College of Veterinary Medicine (Grant # HRSA #D34HP00001) Student Support: Boehringer Ingelheim (BIVSP) and Tuskegee University, College of Veterinary Medicine

180 • 2021 National Veterinary Scholars Symposium Therapeutic Intervention of SARS-CoV-2: Isolation and Purification of SARS-CoV-2 Nucleocapsid Protein

Dylan Johnston, Mohammad Mir

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA

SARS-CoV-2, the etiological agent responsible for the COVID-19 pandemic, is a highly contagious positive sense RNA virus responsible for the deaths of hundreds of thousands of individuals worldwide. SARS-CoV-2 genome encodes a high- ly conserved nucleocapsid (N) protein that specifically binds to the 5’ untranslated region (5’ UTR) of the viral genomic RNA. N protein-UTR interaction plays critical role in virus replication and is thus a novel therapeutic target to combact SARS-CoV-2 infection. The purpose for this project was to express and purify the C-terminally His-tagged SARS-CoV-2 N protein from E. coli. E. coli cells were transformed with a plasmid expressing C-terminally His-tagged SARS-CoV-2 N protein. The bacterial colonies were screened for N protein expression by growing the colonies. N protein expression was induced by the addition of IPTG when the OD600 nm reached 0.5. The cells were pelleted down and the lysates were examined by SDS-PAGE. The stained gels revealed the expression of N protein with the correct molecular weight of 50 KDa. The highly expressing positive colony was grown in large volume of media for large scale expression of N protein. The bacterial lysate containing N protein was loaded on Ni-NTA column, using the Akta pure protein purification system. The column was washed with buffer containing the 20 mM imidazole, followed by elution with immidazole gradient from 20-500 mM. The peak fractions containing the purified N protein were re-examined by SDS-PAGE analysis, which showed the N protein of correct moleculer weight devoid of any contamination. The purified protein will be used in our ongoing studies focused on the development of a new antiviral therapeutic for SARS-CoV-2 infection.

Research Grant: US Department of Defense, Military Infectious Diseases Program Student Support: Boehringer Ingelheim

Impacts of the COVID-19 pandemic on antimicrobial use in companion animals

Ashlan L Jolley, William J Love, Cristina Lanzas

Department of Population Health and Pathobiology, College of Veterinary Medicine, NC State University, Raleigh, NC

Judicious antimicrobial use (AMU) is important for preventing the evolution of antimicrobial resistance in bacterial pathogens, making subsequent use of these drugs less effective in both human and veterinary medicine.The COVID-19 pandemic required many practices to alter their operations and decrease the number of patients seen. Adding in shifts in pet care seeking behaviors by owners, such changes may have impacted AMU during the pandemic. The goal of this re- search is to quantify potential changes in prescribing practices arising from the pandemic. To do so, a retrospective study was performed using prescribing data collected from the pharmacy at NC State College of Veterinary Medicine’s referral hospital and primary care centers for dogs and cats. This data contained records of all antimicrobial prescriptions from NC State from 2019-2020, and were used to categorize each as occurring before or during the pandemic. Each instance of AMU was counted to generate overall usage totals for individual antimicrobials in the dataset. This study included 32,034 prescriptions, which were classified according to the FDA’s system for ranking drugs of human medical importance. Ordinal logistic regression was used to estimate the relative frequency of each classification type being prescribed during the pandemic compared to beforehand. When adjusted for patient age, species, and hospital service, analysis of the fitted model found patients seen during the pandemic were not significantly more likely to receive more important antimicro- bials than before the pandemic (OR = 1.1, p = 0.13). These results indicate that the pandemic did not significantly impact prescribing practices of antimicrobials for companion animals in this study.

Research Grant: FDA U01FD007057 Student Support: NIH Interdisciplinary Biomedical Research Training Program T35-T35OD011070

2021 National Veterinary Scholars Symposium • 181 Establishing the stability of a MVA viral vector pancoronavirus vaccine motif via serial passaging

Maya Jonas, Cole Knuese, Jaime Lopera Madrid, Daniele Oswaldo Maldonado, Jorge Osorio

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin

Today, one would likely have marked difficulty finding anyone who does not know the significance behind SARS-CoV-2. Most people are also familiar with the various vaccine options available to specifically prevent against SARS-CoV-2. However, as coronaviruses are a relatively quickly mutating family of viruses and this is not the first pandemic caused by a coronavirus, the need for a vaccine that protects against multiple coronaviruses, including SARS-CoV-2 variants, is be- coming increasingly evident. The aim of this project is to create a pancoronavirus vaccine using a MVA viral vector motif and a combination of three or more mosaic Spike proteins. Within this larger project, the goal of my research is to test the stability of this vaccine construct using serial passaging, passing the vaccine motif through cells ten successive times to examine changes in protein expression, stability, and sequence via mutations. Preliminary results are showing a potential shift in protein expression within cells around passage six with cells expressing disproportionate amounts of the three mo- saic Spike proteins. Results of this study will then be utilized to adjust the vaccine construct in order to create an effective pancoronavirus vaccine that can then be tested in an animal model. With the development of a successful pancoronavirus vaccine, future coronavirus pandemics will be mitigated, providing an immense benefit to public health.

Research Grant: Unknown Student Support: MFJ funded by NIH T35OD011078

A Retrospective Study (2008-2018) of Canine Pyometra in Grenada, West Indies

Adrian Jones, Afroza Khanam, Mellisa Walters, Wayne Sylvester, Firdous Khan

Department of Large Animal Medicine and Surgery (Jones, Khanam, Khan), Small Animal Clinic (Walters, Sylvester), School of Veterinary Medicine, St. George’s University, True Blue, Grenada, West Indies

Pyometra is a serious infection of the uterus that primarily affects mature intact bitches.The prevalence of pyometra and the risk factors associated with the condition have been investigated in various countries. This retrospective study aimed to determine the occurrence and risk factors of pyometra in Grenada, West Indies. The subjects were owned bitches pre- sented to the St. George’s University Small Animal Clinic between 2008 and 2018. Data on the age, breed, and gonadecto- my status of the bitches were retrieved from an electronic database (AVImark ). Statistical analyses were conducted using the IBM SPSS program. Of the 3583 bitches presented during the study period, 33 (0.9%) were diagnosed with pyometra. The mean age at diagnosis of pyometra was 5.5 years (60.54, standard error). Affected bitches included 15 purebreds, 9 mixed-breed, 8 Grenadian pothounds, and 1 bitch with missing breed information. Of the affected bitches, 24 were intact and 9 were spayed. However, Chi-squared tests did not indicate a significant influence of breed (P = 0.315) or gonadecto- my status (P = 0.493) on the occurrence of pyometra. Although this study provided useful preliminary information about the occurrence of canine pyometra in Grenada, further research involving larger and broader sample sizes is required to determine the prevalence and risk factors of canine pyometra in the region.

Research Grant: None. Student Support: Boehringer Ingelheim Veterinary Scholars Program

182 • 2021 National Veterinary Scholars Symposium Effects of Dexmedetomidine on Intestinal Epithelial Stem Cell Expression in Equine Intestinal Strangulation

Hannah Jones, Elsa Ludwig, John Freund, Natalia L Rosa Padilla, Kerry O’Donnell, Liara Gonzalez

College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Equine small intestinal strangulation (SIS) occurs in 58-85% of small intestine colics. Ischemia-reperfusion injury (IRI) due to SIS leads to epithelial sloughing and intestinal stem cell (ISC) destruction. The resulting complications from IRI can be fatal and short-term survival rates following SIS range from 60-70%. Intestinal stem cell proliferation serves to regenerate damaged intestinal epithelium, therefore ISC preservation is critical to patient survival. Dexmedetomidine is an alpha-2 adrenergic receptor agonist often used during anesthesia to minimize inhalant concentration. Previous studies have been conducted using dexmedetomidine in laboratory animals to attenuate intestinal IRI, which is likely due to its anti-in- flammatory properties. We hypothesized that dexmedetomidine would be protective to ISCs in clinical equine SIS cases. 16 SIS cases were assessed: 10/16 received dexmedetomidine intraoperatively and 6/16 did not receive dexmedetomidine. Uninjured tissue from 3 horses served as control. ISCs were identified using immunofluorescence (IF) for Ki67, SOX9, and PH3 biomarkers (n = 16). Epithelial cell apoptosis was identified using CC3 (n = 13). Positive cells were quantified and compared using Mann-Whitney test and Welch’s test of mean counts between proximal and distal resection sites (P < 0.05). An increased number of Ki67 cells were found in the proximal resection sites for horses that received dexmedeto- midine compared to the ones that did not (P = 0.045). No significant differences in SOX9, PH3, or CC3 were found. The increased number of Ki67 cells at the proximal resection site may indicate increased proliferation and therefore a potential for regeneration in equine dexmedetomidine recipients.

Research Grant: None Student Support: NC State Veterinary Scholars Program

Wildfires causing poor air quality and its association with infected corneal ulcerations in dogs

Katrina E.V. Jones, Michala de Linde Henriksen, Joshua B. Daniels, Michael R. Lappin

College of Veterinary Medicine and Biomedical Sciences, Colorado State University (Jones, Henriksen, Lappin), Veterinary Diagnostic Laboratory, College of Veterinary Medicine and Biomedical Sciences, Colorado State University (Daniels)

In the fall of 2020, Colorado experienced the two largest wildfires in state history. The smoke blanketed the college town of Fort Collins, Colorado, the location of the Veterinary Teaching Hospital at Colorado State University (CSU-VTH) for approximately three months (August, September, and October). The comparative ophthalmology service at the CSU-VTH noted an influx of canine patients with infected corneal ulcerations during this time.The hypothesis of this study was that there was an increase in infected corneal ulcerations in dogs during the three months of the wildfires compared to previous years. It was also hypothesized that poor air quality (elevated air quality index (AQI)) was associated with infected canine corneal ulcerations. The medical records from canine patients presented to CSU-VTH’s ophthalmology service with in- fected corneal ulcerations in August, September, and October of 2018, 2019, and 2020 were evaluated. The study did not reveal a significant difference between canine patients presented with infected corneal ulcerations during the three months for 2020 (2%,13/639), 2019 (1.1%,7/660), and 2018 (1.6%,9/569) (P = 0.364). A trend toward significant difference was found between bacterial growth versus no growth from ulcerations between 2020 (77%,10/13), 2019 (29%,2/7), and 2018 (44%,4/9) (P = 0.086). The AQI was significantly elevated for dogs presented with infected corneal ulceration in 2020 compared to 2018 and 2019 (P = 0.0034, P < 0.0001, respectively). This study highlights the risk of developing an infect- ed corneal ulceration during the wildfire season. As the duration and frequency of wildfires continues to rise globally the effect of these wildfires on animal health should be investigated further.

Research Grant: None Student Support: USDA Animal Health & Disease Scholar

2021 National Veterinary Scholars Symposium • 183 Off-target effects of small molecule therapeutics of

Hannah J. Jorgensen, Eric W. Ottesen, Natalia N. Singh, Diou Luo, Bailey M. Kaas, Joonbae Seo, and Ravindra N. Singh

Department of Biomedical Sciences, Iowa State University, Ames, IA

Spinal muscular atrophy (SMA) is a genetic disease that causes progressive loss of motor neurons and decreased life expectancy, and it is the leading cause of infant mortality. SMA primarily results from deletion of or mutations in the Sur- vival Motor Neuron 1 (SMN1) gene, which leads to decreased SMN protein. Humans have an additional copy of the gene, SMN2, but its exon 7 is predominantly skipped during pre-mRNA alternative splicing, resulting in an unstable, less func- tional protein. Therefore, SMN2 cannot fully compensate if SMN1 is mutated. However, several small compounds, includ- ing and branaplam, have been discovered that can increase SMN2 exon 7 inclusion, leading to full-length SMN protein. Risdiplam is FDA approved to treat SMA, and branaplam is undergoing clinical trials. The goal of this study was to identify the potential off-target effects of high concentrations of risdiplam and branaplam. RNA-seq analysis of SMA patient cells treated with high concentrations of risdiplam and branaplam revealed numerous off-target effects, including aberrant transcription and splicing. We performed RT-PCR of selected genes to validate the off-target effects captured by RNA-seq. To gain mechanistic insights into the off-target effects of risdiplam and branaplam, we generated minigenes of affected exons and their flanking intronic sequences. Our results revealed the role of diverse sequence motifs in aberrant splicing triggered by high concentrations of risdiplam and branaplam. Our findings are significant for adopting safe dosing regimens as well as for developing future small molecule-based therapies for SMA.

Research Grant: NIH R01 NS055925 Student Support: ISU College of Veterinary Medicine Summer Scholar Research Program

Comparative analyses of adjuvants and immunization routes on antibody induction against the RBD of SARS-CoV-2

Yujin Joung*, Vikram Srivastava*, Michael Cho *These authors contributed equally.

Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA

Despite global efforts to rapidly vaccinate the population to achieve herd immunity against COVID-19, the world is still grappling with a large number of new infections of SARS-CoV-2. The nature of the RNA viruses being prone to mutation is thought to be one of the reasons why this virus is daunting to combat. Recent developments of novel immunogens has shown a marked progress towards our efforts to end this pandemic.We and others have demonstrated that the receptor binding domains is one of the most effective immunogens for vaccine development. RBD-based subunit protein vaccines require adjuvants to elicit more potent immune response. The major goal of the current study is to identify optimal ad- juvants and immunization routes for an RBD-based COVID-19 vaccine. Mice were immunized three times with RBD expressed in 293F cells either subcutaneously or intramuscularly using Alhydrogel, AddaVax, AS03, MPLA or AS04-like (MPLA mixed with Alhydrogel) adjuvants. Mice were bled a day before and two weeks after each immunization. The an- tibody titers were assessed by ELISA against homologous immunogen. The result showed that subcutaneous route failed to induce a strong immune response whereas AS04-like adjuvanted group, immunized intramuscularly induced a strong antibody response. AS03 is a more potent adjuvant than MPLA when administered through subcutaneous route. These findings indicate that adjuvants and immunization routes play an important role in inducing immune responses.We plan to further evaluate immune responses by assessing neutralizing activity and cellular immunity.

Research Grant: Iowa State University Student Support: Van Gelder Foundation

184 • 2021 National Veterinary Scholars Symposium Platelet activation by metabolites of clopidogrel

Brooke T. Kahn, Dawn S. Kusznski. Barbara D. Christian, and D. Adam Lauver

Department of Pharmacology and Toxicology, College of Veterinary Medicine, Michigan State University, East Lansing, MI

Since clopidogrel’s approval by the FDA in 1997, it has been used for both humans and animals to prevent thrombosis, heart attacks, and stroke. Clopidogrel is a prodrug and must be metabolized into the active form by CYP450 enzymes in the liver. The active metabolite (M4) of clopidogrel inhibits platelet activation by preventing ADP from binding to the P2Y12 receptor. However, there are at least 14 additional metabolites which have been assumed to be biologically inert. Our lab developed a conjugate of the M4 active metabolite to reduce interpatient variability. Preliminary studies demon- strated that when the prodrug was administered there was a prolonged bleeding time compared to vehicle and the admin- istration of M4 alone. This suggests that at least one of the additional metabolites disrupts hemostasis. In this study we sought to compare the inhibitory effects of various clopidogrel metabolites on platelet function to explain the differences in bleeding time. To characterize the effects of these metabolites on platelet function, platelet-rich plasma was incubated with the active metabolite (M4) and three additional metabolites that have been assumed to be inactive (M1, M2, and M5) and then stimulated with platelet agonists. We anticipate that M4 will cause inhibition of platelet aggregation while M1 and M5 do not inhibit platelet aggregation. Since M2 can be further metabolized into M4, we may see some inhibition. These results suggest that platelet inhibition is not the mechanism by which the metabolites of clopidogrel increase bleed- ing time. Future studies are necessary to assess the effects of these metabolites on other aspects of the hemostatic system, specifically the effects on vascular physiology.

Research Grant: Lab funding by an internal DFI (discretionary funds initiative) grant at MSU Student Support: Student funding provided by the Graduate School at Michigan State University

Cell specific EphA4 alters immune cell recruitment and vascular remodeling following acute ischemic stroke

Alexandra M. Kaloss, Nathalie A. Groot, and Michelle H. Theus

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA

Stroke is the fifth leading cause of death and a principle cause of long-term disability in the United States. Most stroke cases are ischemic in nature, resulting from a vascular obstruction that reduces cerebral blood flow and ultimately leads to permanent cell death and neurological deficits. Recent studies suggest pial collateral vessels are a critical determinant of stroke outcome. Pial collateral vessels are formed during embryonic development and bridge distal arterioles in the pia mater of the brain. Under normal conditions, these vessels are small and inactive. Following ischemic stroke, pial collaterals remodel and enlarge through a process termed arteriogenesis, thereby allowing increased blood flow back into at-risk tissues. Using conditional endothelial cell-specific EphA4 knockout (EphA4fl/fl/Cdh5::CreERT2; KO) and wild type (EphA4fl/fl; WT) mice, our findings suggest that the EphA4 receptor tyrosine kinase plays a major role in regulating pial collateral growth after permanent middle cerebral artery occlusion (pMCAO). To analyze collateral size and cellular remodeling, we employed vessel painting and immunohistochemistry techniques. We found that, compared to WT con- trols, KO mice displayed a significant reduction in infarct volume which correlated with larger ipsilateral pial collateral vessels as early as 4.5hrs and up to 24hrs post-pMCAO. In parallel to the increase in collateral size observed, a significant increase in the number of CD11b+ immune cells recruited to the collateral vessel was seen in KO mice at 6hrs post-pM- CAO compared to WT mice. These findings demonstrate that EC-specific EphA4 limits collateral growth and size and is a novel therapeutic target for ischemic stroke treatment.

Research Grant: NIH R01 NS112541 Student Support: Virginia-Maryland Regional College of Veterinary Medicine dual degree program

2021 National Veterinary Scholars Symposium • 185 Inflammatory markers of respiratory syncytial virus in the cotton rat

Danyue Kang, Olivia Harder, Stefan Niewiesk

Department of Veterinary Bioscience, College of Veterinary Medicine, Ohio State University, Columbus, OH

Respiratory syncytial virus (RSV) is a common cause of upper and lower respiratory tract infection. We wished to in- vestigate whether RSV infection induces inflammatory proteins as e.g. infection with SARS-COV-2. The inflammatory proteins tested include C reactive protein (CRP), high mobility group box 1 (HMGB1), and prostaglandin D2 (PGD2). We investigated in the cotton rat (Sigmodon hispidus) which inflammatory protein correlated with RSV infection. CRP is of- ten used as a marker of inflammation during bacterial infection, but is typically not highly elevated during viral infection. During RSV infection in adult cotton rats, the level of CRP slightly increased during virus replication. The inflammatory protein HMGB1 triggers TLR4 signaling, which induces COX-2 and subsequently PGD2 expression. Increased PGD2 production can negatively affect the migration of dendritic cells and the activation ofT cells during viral infection. Our results demonstrated that the level of HMGB1 increased during viral growth, peaked during viral clearance, and remained stable for 4 days after complete clearance of virus. For PGD2, there was an initially peak on day 1 after infection, then a secondary, larger peak during viral clearance. Therefore, in adult cotton rats, CRP may be used as a control for inflamma- tory protein during infection. In contrast, the increased expression of both HMGB1 and PGD2 and their role in the patho- genesis of RSV infection need to be investigated further.

Research Grant: Faculty discretionary account through the College of Veterinary Medicine Student Support: OSU Veterinary Student Summer Research Program, Boehringer Ingelheim Veterinary Student Scholar

Assessing the pharmacodynamics of the novel antibiotic, bovine lactoferrin (bLF) in chickens

Leeza Kao, Maisie Dawes

College of Veterinary Medicine, Western University of Health Sciences, Pomona, California

Necrotic enteritis (NE) is a devastating disease that costs the poultry industry $6 billion annually. Although the disease is effectively managed with antibiotics, consumer stigma, legislative restrictions, and concerns of antimicrobial resistance have created a demand for novel therapeutic alternatives. Bovine lactoferrin (bLF) has been shown to possess iron-depen- dent antimicrobial properties, such as biofilm inhibition, as well as enhance the effects of probiotics on enterocyte growth. However, its distribution and pharmacodynamics in chickens is poorly understood. We intend to define bLF localization patterns in abdominal viscera critical to NE pathogenesis, in bLF supplemented chickens. Since bLF is sensitive to pepsin degradation, we hypothesize that tissues from intramuscular (IM) treated birds will have greater relative abundance of the protein, and further anticipate seeing differences in microanatomical structures, such as intestinal villi, between treated and control birds. Twenty-two (22) healthy-specific-pathogen-free White Leghorn chicks were treated with bLF via ga- vage or IM injection. Control cohorts were sham treated with PBS. Sections of duodenum, proximal cecum (CP), and liver were harvested on days 3 and 14. The duodenum is the site most affected by the damage of NE, while the CP contains Peyer’s patches with potential immunologic changes in response to bLF supplementation. In bovine, the liver has been shown to be the site of bLF metabolism. Sections will be analyzed for mucosal changes and bLF localization using H&E staining and immunohistochemistry, respectively. Our findings will guide future investigations into the potential benefits of this novel antimicrobial to the poultry industry.

Research Grant: WesternU College of Veterinary Medicine Office of Research Student Support: Boehringer Ingelheim

186 • 2021 National Veterinary Scholars Symposium Evaluation of echocardiographic findings and cardiac biomarkers in cats eating different types of cat foods

Shelby I. Karp, John E. Rush, Emily T. Karlin, Joey N. LaMastro, Lisa M. Freeman

Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA

Heart disease is common in cats, with a prevalence between 10-15% in the general cat population and even higher rates in certain breeds. Cardiomyopathies are the most common type of heart disease in cats, with hypertrophic cardiomyop- athy (HCM) being the most common form. However, other forms of cardiomyopathy can also be seen in cats, including dilated cardiomyopathy (DCM), restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, and cardiomyopathy with a nonspecific phenotype. While genetic mutations associated with HCM have been identified in two cat breeds, the cause of most feline cardiomyopathies remains unknown. One of the known etiologies of feline DCM is dietary taurine deficien- cy. Dilated cardiomyopathy used to be one of the most common heart diseases affecting cats and, prior to 1987, carried a grave prognosis. In 1987, a landmark paper was published reporting an association between taurine deficiency and DCM in cats, and that taurine supplementation could reverse the myocardial changes. As a result of this association, dietary tau- rine levels in commercial cat diets have increased and taurine deficiency and DCM have become uncommon in cats. Since then, there have been few studies regarding other potential associations between diet and cardiomyopathies in cats. The purpose of our present study is to investigate whether there may be associations between the diets of apparently healthy cats and echocardiographic measurements, cardiac biomarkers (N-terminal B-type natriuretic and high-sensitivity cardiac troponin I), or plasma and whole blood taurine concentrations.

Research Grant: The EveryCat Health Foundation (grant W21-028) Student Support: The Barkley Fund

Investigating target antigen density influence on canine CAR-T functionality

Jo Kawabata, Sho Yoshimoto, Brian T. Kasper, Antonia Rotolo, Nicola J. Mason

Department of Clinical Sciences and Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Chimeric Antigen Receptor T-Cells (CAR-T) are T-cells expressing antibody-based antigen receptors with an affinity to a specific target. In humans, CD19 CAR-T have shown therapeutic success in B-cell leukemias/lymphomas. However, while canine CAR-T have shown high functionality against the CD20+ canine B-cell line, CLBL1, they have shown limited therapeutic effect in dogs with B-cell lymphoma. Our previous work showed CLBL1 expresses remarkably higher levels of CD20 compared to peripheral blood B-cells. Furthermore, other studies showed enhanced CD20 CAR-T func- tionality when targeting cells with high target antigen density (Watanabe, et al., 2015). Therefore, we hypothesize low antigen density on the peripheral blood B-cells failed to activate CAR-T in vivo. To address this, we evaluated CD20 CAR-T effector function against target cells with high or low CD20. We hypothesized to see higher functionality in CD20 CAR-T targeting cells with higher CD20 expression. In preliminary experiments, cryopreserved CD20 CAR-T were cocultured with a canine B-cell line, GL1, expressing high (GL1-20hi) and low (GL1-20lo) CD20; subsequently, expres- sion of CD107b, a marker correlated with cytotoxicity, and production of cytokine, IFNg, was evaluated. We saw higher expression of CD107b against GL1-20hi than against GL1-20lo but no difference in IFNg production. This is indicative that some CAR-T functionality are higher against higher target antigen density. Future experiments are planned in triplicates with fresh CAR-T to evaluate cytotoxicity, cytokine production, and proliferation. This data can be used to screen patients to predict prognoses of CAR-T therapy as well as contribute to further development of both human and canine CAR-T.

Research Grant: PetCo Love Foundation Student Support: NIH T35 OD010919

2021 National Veterinary Scholars Symposium • 187 A point-of-care microbiome-based diagnostic for canine gut health

Luke Kazmierski, Leah Soderman, Mark P. Rondeau, Daniel P. Beiting

Department of Pathobiology (Kazmierski, Beiting), Department of Clinical Sciences and Advanced Medicine (Soderman, Rondeau), School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

A diverse, stable microbiome is necessary for proper health, and dysregulation of the microbiome has been shown to be the culprit of many gut diseases in canines, such as Canine Chronic Enteropathy (CCE). Research has shown that Clos- tridium hiranonis is a potential marker for healthy gut function in dogs, while certain pathobionts, notably Clostridium perfringens, are a marker for dysregulated gut function. Measuring the relative abundance of these two bacteria species in canines directly in the clinic could help clinicians stratify patients and provide a useful metric to gauge therapeutic response in dogs being treated for chronic enteropathies and inflammatory bowel disease.The aim of this study was to develop and test assays that measure C. hiranonis, C. perfringens and total bacterial 16S rRNA rapidly and directly from canine stool samples. To accomplish this goal, we developed a multiplexed quantitative PCR (QPCR) assay that takes advantage of a rapid DNA extraction protocol and portable QPCR device, allowing point-of-care testing. First, individual assays were designed and tested on DNA purified from strains ofC. hiranonis and C. perfringens. To test the ability of this assay to perform using real clinical samples, the triplex assay was then applied to archived canine stool samples from a recent study of CCE. The results showed that the C. hiranonis, C. perfringens, and total bacterial 16S rRNA assays were sensitive and specific as a multiplexed panel applied to purified DNA. Importantly, the assay also detected C. hiranonis and C. perfringens in canine stool subjected to the Biomeme lab-free DNA extraction protocol.

Research Grant: None Student Support: NIH T35 OD010919

Salmonella serovars from Grenadian animals and their antibiotic susceptibility pattern: A retrospective study

Allison M. Kearney, Wayne Sylvester, Victor A. Amadi

Department of Pathobiology (Kearney, Amadi) and Department of Small Animal Clinic (Sylvester), School of Veterinary Medicine, St. George’s University, St. George’s, Grenada, West Indies

Salmonella has been associated with illnesses in humans and animals worldwide, but the role of wildlife in the dissemina- tion has not been well studied. This study compared the prevalence of Salmonella serovars and determined the antibiotic susceptibility pattern of Salmonella strains from Grenadian animals. Data for this study was collected from previously published Salmonella studies (2010-2020) in animals (number of samples): tree boas (45), green iguanas (62), dogs (144), cane toads (58), blue land crabs (65), red-footed tortoises (114), Indian mongooses (156), brown rats (170), and marine turtles (102) from Grenada. The prevalence rates of the serovars observed were compared using Chi-square analysis. Based on the individual animal prevalence of Salmonella, green iguanas (54.8%) showed the highest prevalence rate, while dogs (5.6%) showed the lowest rate. Serovars Rubislaw (18.7%) and Montevideo (16.2%) were the most common serovars observed. The difference in the serovar proportions observed in dogs and Indian mongooses, and cane toads and Indian mongooses were not significant (P > 0.05). However, the difference seen in dogs and cane toads was significant (P = 0.04). Most of the isolates were susceptible to the tested antibiotics except for streptomycin and tetracycline, which showed resistance rates of 21.5% and 11.3%, respectively. This study confirms the previous findings that Grenadian animals harbor different serovars and are presently not main reservoirs for antibiotic resistant strains.This compilation of data from Grenadian animals can be useful to track emerging antibiotic resistant strains and possible transmission route. Larger sample sizes might elucidate the difference in serovar proportions observed.

Research Grant: None. Student Support: Boehringer Ingelheim Veterinary Scholars Program

188 • 2021 National Veterinary Scholars Symposium Cervical inflammatory-associated marker expression in rats following respiratory motor neuron death

Amy N. Keilholz, Nicole L. Nichols

Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Respiratory distress and failure as a result of motor neuron death are seen in neurodegenerative diseases and injuries. However, no effective treatments exist to consistently improve ventilation; therefore, development of strategies to restore respiratory function is crucial. Significant plasticity is noted in the respiratory system as breathing function is initially maintained despite loss of respiratory motor neurons, but the underlying compensatory mechanisms are poorly under- stood. To better understand this respiratory compensation, we developed a novel model using intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) to induce selective respiratory (e.g., phrenic) motor neuron death. We have found that inhibition of COX1/2-induced inflammationvia acute (non-steroidal anti-inflammatory drug) delivery first attenuates and then enhances plasticity and breathing over the course of motor neuron death in CTB-SAP rats vs. controls. This suggests that COX1/2-induced inflammation may be first beneficial and then deleterious following motor neuron death. The goal of this study is to use qRT-PCR to identify potential inflammatory-associated targets e.g.( , TNF-a) in the cervical spinal cord of CTB-SAP rats. We hypothesize that cervical inflammatory-associated marker expression is increased in CTB-SAP rats vs. controls, and that acute ketoprofen delivery (IP) reduces expression back towards control levels. This study will aid in understanding which inflammatory-associated factors may be responsible for the differential effect on plasticity and breathing in CTB-SAP rats, and these factors could ultimately be therapeutic targets to improve ventilatory function in patients with respiratory deficits.

Research Grant: Spinal Cord Injury/Disease Research Program (SCIDRP), NIH K99/R00 HL119606, and University of Missouri College of Veterinary Medicine Committee on Research (COR) grants Student Support: Supported by an endowment established by IDEXX-BioAnalytics

Anti-tick vaccine development for Anaplasma marginale

Stefan H. Keller, Sammuel Shahzad, Kyle J. Cacciatore, Bridgette M. Rogers, Roger W. Stich

Department of Veterinary Pathobiology (Keller, Shahzad, Cacciatore, Rogers, Stich), College of Veterinary Medicine, University of Missouri, Columbia, MO

Livestock are critical for poverty reduction and agricultural development, supporting the livelihoods and food security of 1.3 billion people. Tick-borne diseases of cattle are economic constraints in resource-limited regions, with bovine ana- plasmosis considered one of the most economically impactful of these diseases. Additionally, utilization of the tractable bovine anaplasmosis model system is expected to lead to the development of novel methods to interfere with transmission of Anaplasma marginale, the etiologic agent of bovine anaplasmosis, which in turn is expected to become translatable to closely related pathogens of companion animals and people, and thus is expected to have a positive impact on the lives of both animals and people. Several different tick homogenates were tested for induction of immune responses that interfered with tick-transmission of A. marginale. Different preparations from the same ticks did not block transmission. Currently, our overall objective is to identify tick molecules and epitopes targeted only by antibodies produced by these protected hosts. Thus, the first aim is to define mimetic peptide motifs uniquely bound by antibodies from protected cattle, by using immunoglobulins from protective and non-protective sera for positive and negative selection of a random phage-display library. The second aim is to use these same immunoglobulins for immunoprecipitation of tick molecules by antibodies from protected cattle. This work is expected to identify novel targets for interference with transmission of A. marginale, and possibly other pathogens, to and from tick vectors, which in turn is expected to have a positive impact on the lives of both animals and people.

Research Grant: USDA NIFA 17-67015-26630 Student Support: Foundation for Food and Agricultural Research

2021 National Veterinary Scholars Symposium • 189 Efficacy of pentosan polysulfate for treatment of retinal disease in mucopolysaccharidosis IIIB

Jamillah I. Kelly, Tyler A. Harm, Bethann N. Valentine, Ariel S. Nenninger, N. Matthew Ellinwood, Jodi D. Smith

Department of Veterinary Pathology, College of Veterinary Medicine (Kelly, Harm, Valentine, Nenninger, Smith), and Department of Animal Science, College of Agricultural and Life Sciences (Ellinwood), Iowa State University, Ames, IA. *Dr. Ellinwood’s current address is National MPS Society, Durham, North Carolina

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease that causes central nervous system degeneration due to deficiency of the lysosomal enzyme needed for the degradation of the glycosaminoglycan (GAG), heparan sulfate.There are currently no approved therapies for this disorder. Pentosan polysulfate (PPS) is a drug known for its anti-inflammatory properties. It has been demonstrated to decrease markers of disease, including GAG accumulation, in various lysosomal storage diseases, but has not previously been evaluated as a therapeutic for MPS IIIB. For this study we performed quan- titative immunohistochemistry for lysosomal integral membrane protein 2 (LIMP-2) on the retina of five four-month-old dogs with MPS IIIB: three animals treated every two weeks, beginning shortly after birth with 2.88 mg/kg subcutaneous PPS, and two untreated controls. LIMP-2 is used as an indirect measure of GAG accumulation in the lysosomes. Images where taken of six contiguous, non-overlapping 200x fields in the central retina, and will be analyzed for percent immuno- reactivity using image analysis software. We will compare the amount of lysosomal storage present in the retina in treated versus untreated dogs. We hypothesize that PPS treated dogs will display a decrease in retinal lysosomal storage compared to those that were untreated. This finding has been observed in previous studies done on the brain and spinal cord of these dogs. From studies like this we hope to gain insight into potential treatments that may help slow the progression of neuro- degeneration in MPS IIIB.

Research Grant: National MPS Society Research Grant #1491 Student Support: None

The Stressful Effect of Propranolol on Macrophages

Sarah Kenney, Ali Khammanivong, Derek M. Korpela, Kaylee Schwertfeger, and Erin B. Dickerson

Department of Veterinary Clinical Sciences College of Veterinary Medicine University of Minnesota (Kenney, Khammanivong, Korpela, Dickerson), Saint Paul, MN; Department of Laboratory Medicine and Pathology, University of Minnesota (Schwertfeger), Minneapolis, MN.

Propranolol has been identified as a potential adjunct to chemotherapy for the treatment of canine hemangiosarcoma (HSA), but its mechanism of action is mostly unknown. We previously showed propranolol inhibits endocytosis and autophagy, depriving HSA cells of nutrients and inducing endoplasmic reticulum (ER) stress. Propranolol also increased the expression of fatty acid (FA) and cholesterol synthesis enzymes, suggesting ER stress and lipotoxicity induce HSA cell death. Saturated FAs have been shown to induce ER stress and polarize macrophages toward an anti-tumor pheno- type; thus, propranolol may kill tumor cells while enhancing anti-tumor immune responses. We tested the hypothesis that propranolol polarizes macrophages toward an anti-tumor phenotype by inducing ER stress and lipid synthesis. We treated the murine macrophage cell line, RAW 264.7, with 25 mM propranolol and looked for changes in the expression of genes involved in lipid synthesis and ER stress using qRT-PCR. Cells treated with propranolol were harvested at 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. Propranolol increased the mRNA expression of Hmgcs1 and Fasn genes involved in cholesterol and fatty acid synthesis, respectively, within four hours of treatment; increases were not observed after 24 hours. Furthermore, macrophage polarization was not detected by flow cytometry. Expression of Ern1, which encodes the ER stress sensor Ire- 1a, was induced within two hours and sustained for 72 hours. Because Ire-1a can inhibit transcription, our data suggest Ire-1a blocked transcription of Hmgcs1 and Fasn. We conclude propranolol induces an ER stress response in RAW 264.7 cells that prevents lipid synthesis and may block polarization toward an anti-tumor phenotype.

Research Grant: Chainbreaker GOpher a Cure Award (EBD and KS), Masonic Cancer Center, University of Minnesota; Rein in Sarcoma Foundation (EBD and KS). Student Support: Supported by the Skadron Family fund and the College of Veterinary Medicine, University of MN

190 • 2021 National Veterinary Scholars Symposium Optimization of a Qubit fluorometer assay for the quantification of cell-free DNA in equine plasma

Abby Kepley, Rosemary Bayless, M. Katie Sheats

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Colic is the leading cause of death in horses. Early intervention for severe colic can decrease patient morbidity and mortal- ity; therefore, novel biomarkers to assist with colic diagnosis and prognosis are needed. Cell-free DNA (cfDNA), released during cellular apoptosis, necrosis and NETosis, has been investigated as a biomarker for gastrointestinal disease and inflammation in other species. Previous experiments in our lab using the tabletop Qubit fluorometer have demonstrated that direct and extracted plasma cfDNA are increased in horses with colic, compared to healthy horses. However, we also detected inaccuracies with direct measurement of cfDNA due to suspected matrix effects of equine plasma. The purpose of this study was to use banked equine plasma samples to optimize the QubitTM dsDNA HS (High Sensitivity) Assay Kit (ThermoFisher) for accurate measurement of cfDNA in equine plasma. We hypothesized that dilution of equine plasma would increase the accuracy of Qubit-measured cfDNA in equine plasma. Results from linearity of dilution experiments with the Qubit and NanoDrop OneC were significantly correlated and demonstrated good linearity. However, treatment with DNase I, and spike and recovery experiments, showed that matrix effects persist up to 1:16 dilution.These findings were consistent with standard addition plasma optimization experiments, which indicated that 1:32 or 1:64 plasma dilu- tion is needed to minimize matrix effects. Investigation of clinical applications of the direct measurement of cfDNA as a biomarker in diluted equine plasma is ongoing.

Research Grant: The Foundation for the Horse Graduate Student Research Grant Student Support: NC State Veterinary Scholars Program

Ex vivo evaluation of a novel tibial tuberosity advancement surgical guide for the treatment of canine CrCLD

Brendan Kerlin, Ross H. Palmer

Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

Cranial cruciate ligament disease (CrCLD) is a common, painful, and expensive to correct orthopedic condition in dogs. Tibial tuberosity advancement (TTA) is one of the surgical options currently used to treat CrCLD. A crucial step in performing a successful TTA is an accurate osteotomy; technical errors in the osteotomy are linked to tibial fracture and delayed healing complications. A novel surgical guidance procedure (consisting of an adjustable osteotomy guide plus pa- tient-specific written instructions for performing the TTA) has been developed which may improve osteotomy accuracy in the hands of inexperienced surgeons. Using model tibias, veterinary students performed two TTAs in a random order, one “guided” using the novel procedure and one “unguided” using conventional methods. Osteotomy position was then mea- sured and compared to an ideal osteotomy to determine if there were any significant differences between the two methods. Trials are underway and results will be reported upon study completion.

Research Grant: Center for Companion Animal Studies Young Investigator Grant and OssAbility Limited Student Support: Colorado State University Veterinary Summer Scholar Program

2021 National Veterinary Scholars Symposium • 191 Using epidemiologic tools and methods to assess the role of adverse weather events in PRRS and PED outbreaks

Gabriela Stefany Kiesel, Dennis N. Makau, Igor A.D. Paploski, Kimberly Vanderwaal

Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St Paul, MN

Porcine reproductive and respiratory syndrome (PRRS) and Porcine Epidemic Diarrhea (PED) are two important diseases that affect the U.S. swine industry, causing revenue loss from billions of pounds of lost pork and hundreds of thousands of dollars in outbreak management costs. Studies have documented airborne PRRS transmission over long distances and identified trees acting as windbreaks around barns to reduce the risk of PRRS occurrence. Environmental factors such as land elevation, slope and land use have also been shown to influence disease outbreaks. However, the effects of adverse weather events on disease occurrence are not well understood. The objective of this study is to investigate the associa- tion between adverse weather events and the occurrence of PRRS/PED. We obtained PED and PRRS outbreak data from Morrison Swine Health Monitoring Project (MSHMP) and data on the occurrence of adverse weather events from the National Oceanic and Atmospheric Administration (NOAA) in a swine dense production area in the U.S. from the years 2014 to 2020. We observed 571 tornados, 16 hurricanes, 80 excessive heat events, 2,505 flooding events and 1,698 heavy rain events in the study area within the 6 years. Additionally, 444 cases of PED and 415 cases of PRRS were reported on farms in the same period. Using a case-control study, we will evaluate associations between the occurrence of PRRS and PED cases on farms in different counties and the occurrence of different weather events.This may provide information that could aid the swine industry in disease management, disaster preparedness and recovery.

Research Grant: None Student Support: United States Department of Agriculture sponsored Multicultural Scholars(MSP) program.

Development of a consistent induced lameness model in the horse

Breanna Killian, Megan McCracken

Veterinary Medical Health Center, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Lameness is one of the most common clinical problems in horses. A model that reliably and repeatedly induces a consis- tent lameness is needed to evaluate long term analgesic therapies. The objective of the present study is to test intermittent- ly applied sole pressure using the heart bar shoe model (HBS) as an alternative to continuously applied sole pressure in a 24-hour lameness model. We hypothesize that tightening the HBS to the same torque will repeatedly induce a consistent severity of lameness in the horse as measured by a body-mounted inertial sensor system (BMIS). Eight horses will be shod with heart-bar shoes on both front feet. The HBS will be tightened to induce a moderate lameness at the trot. The amount of torque required will be recorded. The severity of lameness induced will be measured using BMIS. The sole pressure will be removed and then the same amount of torque will be reapplied to the shoe at 1, 4, 8, 12, 18, and 24 hours, and the severity of lameness will be measured using BMIS. The variability between measured lameness severity at differ- ent time points using the same applied torque will be compared. We expect to find lameness to be consistent between time points suggesting that an intermittent sole pressure model reliably induces a consistent amount of lameness and could be used in the evaluation of longer-acting analgesic therapies.

Research Grant: E. Paige Laurie Endowed Program in Equine Lameness Student Support: Stipend for Breanna Killian is supported by a grant from Boehringer Ingelheim

192 • 2021 National Veterinary Scholars Symposium Role of cholecystokinin in immune-exhaustion of mice fed a Western-style diet

Jessica Kim, Krista Pearman, and Jeffrey Norris

Department of Pharmacology, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Lung cancer is the leading cause of cancer-related death, with 85% of cases classified histologically as non-small cell lung cancer (NSCLC). Epidemiological studies provide evidence that NSCLC risk is positively associated with consumption of a Western diet (WD). Work by our laboratory and others have found significant increases in the number of tumors that develop in mouse models of KRAS-driven NSCLC when feeding a WD compared to a standard chow diet (SCD). Feed- ing a WD also increases tumorigenesis in mouse models of pancreatic cancer in association with circulating cholecysto- kinin (CCK) levels. CCK reduces inflammation and immune system function, in part, by decreasing pro-inflammatory cytokines. Direct stimulation of CCK receptors expressed by lymphocytes also decreases the immune response. Based on evidence that CCK receptor inhibition leads to increased levels of CD8+ T cells in a mouse model of pancreatic cancer, blockade of these receptors is a developing treatment strategy. We have observed increased expression of the immunosup- pressive receptor programmed cell death protein-1 (PD-1) by CD4+ T cells in association with feeding a WD in the mu- rine model of NSCLC. We hypothesized that the increased PD-1 expression by immune cells in mice fed a WD is due to CCK-dependent signaling. Here we report a flow cytometric method of immunophenotypingT cells from murine lungs to determine PD-1 expression. This method will be applied to mice treated with the CCK receptor inhibitor proglumide and fed either a WD or SCD. This approach provides a potential avenue for improving the response of patients with NSCLC to immune checkpoint immunotherapy targeting PD-1.

Research Grant: Midwestern University College of Graduate Studies Intramural Funding Student Support: Ingelheim Veterinary Scholars Program and Federal Work Study

Salmonella serotype surveillance in rodent species inhabiting poultry farms

Kyoo Young Kim

Poultry Diagnostic Research Center, College of Veterinary Medicine, University of Georgia, Athens, GA

Poultry products have been identified since the 1980s as a major source ofSalmonella enterica, which is the second leading cause of foodborne bacterial infections in the U.S. While relevant serotypes have shifted over the years, overall incidence of Salmonella infections in humans has not decreased, indicating the need for more effective control measures. Rodents have been recognized as potential vectors that perpetuate Salmonella presence in poultry farms, but a comprehen- sive surveillance study within the United States is needed. The aims of this study were; 1) To determine the prevalence of Salmonella enterica in three rodent species commonly found inhabiting poultry farms and 2) To compare the prevalence and variation in serotypes found in each species. We hypothesized that the incidence of Salmonella between the rodent species would be comparable, but that each species would harbor different serotypes. Rodent collection from poultry farms resulted in one Rattus norwegicus composited sample, two Rattus rattus composited samples, and four Mus muscu- lus composited samples. Rodents were dissected to extract the gastrointestinal system and these samples were enriched by culture for Salmonella. Positive samples were serogrouped by serum agglutination with Salmonella antisera and serotyped by ISR sequencing. The results showed that of seven composite samples, two Mus musculus samples were positive for Salmonella, both in the serogroup C2. One sample was identified by ISR sequencing as serotypeKentucky . Although a small dataset, this finding suggests there are differences inSalmonella prevalence among rodent species. This information can help direct appropriate rodent control during poultry production to reduce Salmonella.

Research Grant: University of Georgia, Dr. Nikki Shariat Student Support: Boehringer Ingelheim, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 193 Maternal microbiota modulates a Fragile X-like Syndrome in offspring mice

Lily J. Kim, Bernard J. Varian, Tony Chavarria, Katherine T. Weber, Sebastian Carrasco, Fuqing Wu, Eric J. Alm, Susan E. Erdman

Division of Comparative Medicine (Kim, Varian, Chavarria, Weber, Carrasco, Erdman) and Biological Engineering (Wu, Alm), Massachusetts Institute of Technology, Cambridge, MA

Maternal microbial dysbiosis has been implicated in a number of adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that offspring of mice fed a westernized diet (NWD) exhibited higher frequencies of hyperactivity and cranial features similar to Fragile X syndrome (FXS) in humans. We hypothesized that diet-induced dysbiosis during pregnancy induced higher levels of inflammation in the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with Lactobacillus reuteri was sufficient to inhibit FXS-like phenotypes in offspring mice. Assaying cytokine profiles in pregnant females, we found that circulating levels of pro-inflammatory cytokines interleukin (Il)-17 and Il-6 were increased relative to matched gravid mice given supplementary L reuteri probiotic. To test prenatal contributions to FXS, we performed Caesarian (C-section) rederivation to eliminate effects of maternal microbiota during vaginal delivery or nursing after birth.We found that fos- ter-reared offspring still displayed a high frequency of FXS-like features, indicating significant in utero contributions. By contrast, matched foster-reared progeny of L reuteri -treated mothers did not exhibit FXS, supporting a key role for micro- biota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment are strongly associated with the incidence of FXS characteristics in progeny, but can be alleviated by addressing gut dysbiosis through probiotic supplementation. My poster will focus on my work in experimental design and data analyses characterizing this novel microbe-induced syndrome in mice.

Research Grant: NIH P30-ES002109 (SEE), NIH U01 CA164337 (SEE) Student Support: Dorothy W. Poitras Professorship

Utilizing Peptide Amphiphile Micelles (PAMs) to improve genetic therapy delivery for healing Corneal Fibrosis

Austin D. Kimes Megan C. Schulte, Suneel Gupta, Bret D. Ulery, Rajiv R. Mohan

College of Veterinary Medicine (Kimes, Gupta, Mohan) and Department of Biological, Biomedical, and Chemical Engineering, College of Engineering (Kimes, Schulte, Ulery) University of Missouri, Columbia MO; Mason Eye Institute, University of Missouri School of Medicine (Gupta and Mohan)

Corneal fibrosis is the third leading cause of blindness in the United States. Our lab has previously demonstrated effective gene therapy delivery with gold-nanoparticles and DNA plasmids to improve fibrotic eyesight damage. The current treat- ment requires multiple doses a day at higher concentrations because a majority of the plasmid is washed away with tears. Arginine (R) rich peptide amphiphiles (PAs) coupled with Lysine (K) and glutamic acid (E) rich PAs can improve plasmid delivery by utilizing R’s ability to act as a cell penetrating peptide and K and E’s ability to electrostatically interact with the plasmid to form complexation with the DNA plasmid and hopefully form micelles. The PA plasmid complex can allow for prolonged delivery of plasmid and allow for future peptide modifications to promote an endogenous healing response. After synthesizing the three PAs (Palm-K-bAla-XXX-Pro-XXX-Pro-XX-Resin, where X is K, R, or E), varying concen- trations of PAs will be assembled with the plasmid in solution, and using, CMC, electron microscopy, and zeta potential complexation will be accessed. Following successful complexation, an in vitro study will be conducted to determine the corneal epithelium cell penetrating ability of the PA micelles (PAMs) compared to the current gold-nanoparticle method to assess the PAMs viability as a drug delivery system.

Research Grant: Ruth M. Kraeuchi Missouri Endowed Chair of Ophthalmology Fund Student Support: Rajiv Mohan's Start-up fund

194 • 2021 National Veterinary Scholars Symposium Prevalence of B. burgdorferi in I.scapularis: comparison between ticks collected from pets and the environment

Lindsey King, Kathryn Duncan, Kellee Sundstrom and Susan E. Little

Department of Veterinary Pathobiology (King, Duncan, Sundstrom, and Little), College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

Tick-borne pathogens, such as Borrelia burgdorferi (BB), the causative agent for Lyme disease, pose a threat to both hu- mans and dogs alike. In the eastern United States (US), BB is transmitted by Ixodes scapularis (IS). Currently, the majority of Lyme disease cases occur in the upper midwestern and eastern US, although occasionally a case is reported elsewhere. This project determined the prevalence of BB in IS (n = 133) collected from pets in 12 states and in field collected IS (n = 33) from north-central Oklahoma and Kansas. Evaluated ticks were identified morphologically, dissected, DNA extracted, and PCR/sequencing performed to identify characteristic flaBgene fragments of BB. Of the ticks evaluated, BB was iden- tified in 14% (n = 19/133) of pet collected ticks from areas where ymeL disease is endemic, while no BB was detected in the field-caught ticks (n = 0/33). Additionally, field-caught ticks were evaluated using PCR/sequencing of16S rRNA gene fragments to classify the ticks based on clade (data pending). This data reconfirms the presence ofBB in IS from the upper midwestern and eastern states, as well as confirming the absence ofBB in IS ticks from Oklahoma and Kansas. Future sur- veillance of IS ticks in states with no history of BB will continue to track the ongoing spread of Lyme disease in the US.

Research Grant: Funding for this research provided by NIH, AI149638-0 and NSF, OIA-1920946 Student Support: Summer Research Training Program, College of Veterinary Medicine, Oklahoma State University

Effects of genetic manipulation of FASTKD proteins on mitochondrial mRNA and function

Mackenzie K Kinkade, Lihui Song, Christopher P Baines

Department of Biomedical Sciences, College of Veterinary Medicine, Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

Mitochondria are key organelles in the cells of animals which have been studied for more than a century. Dysfunction of mitochondria is a major contributor to the development of cardiac disease. Mitochondrial DNA and the resulting mRNA produce proteins involved in the electron transport chain (ETC). The recently identified FASTKD family of proteins (FASTKD1-5) are RNA binding proteins that regulate mitochondrial mRNA processing and translation. However, the effect each isoform has on mitochondrial function has not been examined and ASTKDsF have never been studied in the heart. The objective of the present study is to evaluate the mechanisms and functions of FASTKD proteins on mitochon- drial function and the role of FASTKD1 in cardiac physiology. We hypothesize that FASTKDs have isoform-specific effects on mitochondrial function, ATP synthesis, and in the case of FASTKD1, cardiac function. Each FASTKD isoform will either be overexpressed or depleted in cultured fibroblasts using adenovirus or siRNA, respectively, and changes in mitochondrial mRNA, ETC proteins, mitochondrial potential, and ATP levels assessed. These indices will be determined in cardiac mitochondria from mice with a deletion of FASTKD1. We will also measure heart size, morphology, and cardi- ac function using echocardiography. We expect to find isoform-specific alterations in mitochondrial function and ATP lev- els in the fibroblasts, as well as deleterious changes in cardiac function in the ASTKD1F knockout mice. Such results will provide new insight into the specific functions of FASTKD proteins in mitochondria and maintenance of cardiac function and warrant further study of this protein family and its potential effects on the development of heart disease.

Research Grant: NIH R21 AG067702 Student Support: University of Missouri College of Veterinary Medicine Office of Research

2021 National Veterinary Scholars Symposium • 195 Reduced cardiac fibrosis and improved function in a novel model of HFpEF using the 5’ SL mutant mice

Jessica Klasing, Jacob Russell, Chastidy Bailey, Shawn Bender, and Chandrasekar Bysani

Biomedical Sciences (Klasing, Russell, Bailey, Bender), School of Veterinary Medicine; Dalton Cardiovascular Center (Bender, Bysani), Medicine (Bysani), School of Medicine, University of Missouri, Columbia, MO; Research Service, Harry S. Truman Veterans Memorial Hospital, (Klasing, Russell, Bailey, Bender, Bysani), Columbia, MO

Heart Failure with Preserved Ejection Fraction (HFpEF) is a complex, multifactorial disease with high morbidity and mortality. There are no evidence-based treatments for HFpEF due to lack of a reproducible preclinical model. Our objec- tive is to develop a novel “two-hit” model of HFpEF, in which mice are fed a high fat diet and administered concurrently with aldosterone and salt to induce metabolic dysregulation and hypertensive stress. Since excess collagen I deposition plays a role in adverse cardiovascular remodeling, we hypothesized that in 5’ SL mutant mice, in which collagen I mRNA and LARP6 (La Ribonucleoprotein 6, Translational Regulator) interaction is blocked, will result in reduced collagen I deposition and fibrosis, and improved cardiac function in the novel “two-hit” animal model of HFpEF. Metabolic stress is induced by feeding mice with a high fat diet for 3 months. For the last month, mice are infused continuously with aldo- sterone via osmotic mini pumps and given 1% salt in the drinking water to induce hypertension. The following endpoints will be measured: cardiac function by echocardiography, aortic pulse wave velocity and aortic stiffening by doppler ultrasound, and cardiac fibrosis by picrosirius red staining. We expect that the wild type littermate mice will exhibit left ventricular hypertrophy, preserved ejection fraction, diastolic dysfunction, and pulmonary congestion consistent with HF- pEF. In contrast, mice with the 5’ SL mutation are expected to have reduced fibrosis, reduced pulmonary congestion, and improvement in cardiac dysfunction. If the results support our hypothesis, the 5’SL structure in collagen type I could serve as a potential therapeutic target to delay the progression of HFpEF.

Research Grant: This study was supported by grants from the US Department of Veterans Affairs (VA-I01-BX004220) and Research Career Scientist (IK6BX004016) to BC. SBB is supported by NIH R01 HL136386. Student Support: University of Missouri College of Veterinary Medicine Office of Research

Phenotypic and genotypic characterization of antimicrobial resistance genes in probiotics

Emily R Klatzer, Claire R Burbick, Trevor L Alexander, Bibiana E Granadillo, and Jamie J Kopper

Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA (Klatzer, Kopper, Granadillo). Washington Animal Disease and Diagnostic Laboratory (Burbick, Alexander) and Department of Veterinary Clinical Sciences, College of Veterinary Medicine (Burbick), Washington State University, Pullman, WA

Antimicrobial resistance (AMR) is a global crisis that jeopardizes modern medicine, affecting both humans and animals. Probiotics, defined as live microorganisms that confer a health benefit on the host, are commonly used in animals. How- ever, there is little regulatory oversight of these products. Unlike in Europe, probiotics in the United States are not tested for the presence of transferrable AMR genes. Thus, our objective was to evaluate bacteria in probiotics marketed for use in major food animal species for phenotypic and genotypic AMR. We hypothesized that the probiotics would contain bacteria with phenotypic AMR that would correlate with the presence of transferrable AMR genes. Nine probiotics were selected, bacteria were cultured from the probiotics, and phenotypic resistance of the bacterial isolates was determined using minimum inhibitory concentration (MIC) disk diffusion. Subsequently, isolates were assessed for transferrable AMR genes via polymerase chain reaction (PCR) and gel electrophoresis. Each probiotic had at least one bacterial isolate that demonstrated phenotypic resistance to at least one antibiotic. 81% of observed phenotypic resistance correlated to a transferrable AMR gene. Additionally, each isolate had at least two transferrable AMR genes. Of these, 16% demonstrat- ed phenotypic resistance. These results indicate that phenotypic resistance is commonly associated with the presence of a transferrable AMR gene. Additionally, many isolates contained transferrable AMR genes, but did not have phenotypic resistance based on MIC testing.

Research Grant: USDA National Institute of Food and Agriculture, Animal Health and Disease Research 1024714 Student Support: USDA National Institute of Food and Agriculture, Animal Health and Disease Research 1024714

196 • 2021 National Veterinary Scholars Symposium Preliminary investigation of the role of the EphA2 receptor in the oncogenic fitness of canine osteosarcoma

Maya Kliewer, Evelyn Harris, Jessica Sharpe, Tim Strozen, and Behzad Toosi

Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan

Osteosarcoma (OSA) is the most common primary bone cancer in canines with a high rate of metastasis and associated poor prognosis. Current treatment approaches include amputation and chemotherapy, however, the survival time remains guarded at 1-year post-treatment. Targeted cancer therapy is emerging as a potential treatment option for OSA, but further research is needed to identify suitable molecular targets. The EphA2 receptor is a member of the erythropoietin-producing hepatocellular (Eph) receptors that are involved in the regulation of cell-cell interactions. Upregulation of EphA2 has been associated with the aggressiveness of multiple human cancers but it has been poorly studied in canine malignancies. The aim of this project is to characterize the role of EphA2 in the pathophysiology of canine OSA. Our gene expression anal- yses using droplet digital PCR and Western blotting revealed overexpression of EphA2 in multiple primary canine OSA cells compared to normal canine osteoblasts. To investigate the function of this receptor, EphA2 was silenced in primary canine OSA cells using specific shRNAs. Stable EphA2 silencing decreased cell proliferation, migration and formation of scattered colonies in culture when compared to non-silenced controls. EphA2 silencing also reduced tumor formation and tumor growth rate in murine models of dog OSA. These original findings suggest important roles for EphA2 in the devel- opment and progression of canine OSA and support the development of conceptually new targeted therapies for dog OSA based on the inhibition of EphA2.

Research Grant: Allard Research Chair in Oncology start-up fund, Saskatchewan Health Research Foundation (SHRF) Student Support: Boehringer Ingelheim Veterinary Scholars Program, WCVM Interprovincial Summer Research Program

Evaluation of benefits and challenges of temporary veterinary clinics in emoter northern Canadian communities

Christopher M. Knap, John VanLeeuwen

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada; Veterinarians Without Borders Canada / Veterinaires Sans Frontieres, Ottawa, Ontario, Canada

In 2019 and 2020, Veterinarians Without Borders (VWB) Canada conducted temporary veterinary clinics for companion animals in five remote northern Canadian communities where there is no local access to veterinary medicine. Over this pe- riod, the VWB Canada teams provided some level of care to > 200 companion animals, including physical examinations, vaccinations, dewormings, medical treatments and sterilizations, as well as education to > 120 families on animal health, welfare and One Health. VWB is planning a 5-year expansion of these projects using the experiences of the first two years of operation to enhance program development and delivery. The aim of this study was to survey the participants of these 2019 and 2020 VWB temporary clinics to obtain quantitative and qualitative data regarding demographic information of animals and owners, the satisfaction and perceived benefits of the clinics, and challenges and concerns with the continu- ation of these efforts under the current model. At the time of publishing, there were 18 respondents, and 100% of respon- dents were strongly satisfied with the treatment of their animal(s) and also believed that community dog issues such as free-roaming animals and dog bite incidents had improved since the introduction of VWB temporary clinics. Furthermore, 89% of respondents believed that their pets were healthier because of the veterinary care received and 78% of respondents believed that their families and communities were likewise healthier. These findings suggest that the continuation of VWB temporary clinics provides real and perceived benefits to remote northern Canadian communities, including improved health outcomes, education and accessibility to animal health care programs.

Research Grant: Boehringer Ingelheim Veterinary Scholars Program Student Support: Atlantic Veterinary College Veterinary Summer Research Award

2021 National Veterinary Scholars Symposium • 197 Immunomodulation from epigenetic reprogramming improves health and production efficiency in aquaculture

Hannah Knight, Beth Peterman and Lora Petrie-Hanson

Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi

The aquaculture industry is reliant on efficient production along with quality of the fish produced. In intensive systems, maintaining and monitoring health is challenging. Implementing new methods of protective immunity would be benefi- cial to the industry. Exposure to beta-glucan provides a broad-based immunity to certain pathogens that can be equivalent to vaccination. The functional changes are due to epigenetic reprogramming and are termed trained immunity. To ana- lyze this immunomodulation, we injected rag1-/- mutant zebrafish with beta-glucan, a characterized immune stimulant. Two weeks after injection, innate immune cells were isolated, and nucleic acids extracted. RNA-seq and ChIP-seq were performed. 1267 differentially expressed genes were entered in KEGG pathway analysis. Cytokine to cytokine receptor interaction, oxidative phosphorylation, phagosome and arginine and proline metabolism pathways were highly significant. Our findings suggest that innate immune cells do undergo functional changes resulting in increased metabolic responses and shifts to aerobic glycolysis. This provides effective elimination of pathogens and immune regulation. Further, signif- icant upregulation of epigenetic regulators, such as arginine, suggest specific histone and chromosomal changes related to immunomodulation. Our findings demonstrate the strong and focused effects indicative of trained immunity induction. Using beta-glucan as a food additive in the aquaculture industry can also combat antimicrobial resistance, by replacing antibiotic use, and optimizing overall health of production systems.

Research Grant: Foundation for Food & Agriculture Research Veterinary Fellowship Program Student Support: None

Butyrate supplementation rescues neural deficits in adult offspring in rat model of gestational sleep apnea

Emma P. Kobitter, Carly R. Mickelson, Armand L. Meza, Abigail B. Radcliff, Kelsea K. Hill, Ruolin Song, Jay Mishra, Sathish Kumar, Jyoti J. Watters, Tracy L. Baker

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, Wisconsin

Sleep disordered breathing (SDB) in pregnancy is increasing in parallel with the obesity epidemic, and is linked to ad- verse perinatal outcomes in the offspring. Consequences of maternal SDB during pregnancy on the adult offspring are not well-understood. Preliminary data indicate that adult male, but not female, offspring of dams exposed to intermittent hypoxia during gestation (GIH - gestational intermittent hypoxia), a rat model of SDB during pregnancy, exhibit increased neuroinflammation, an impaired ability to elicit homeostatic plasticity in the neural respiratory control system, and gut dysbiosis. Specifically, adult male GIH offspring suffer from decreased relative abundance of gut bacteria that produce butyrate, a short-chain fatty acid that can act as a potent anti-inflammatory and regulate immune function in the central nervous system. Neuroinflammation undermines respiratory plasticity, and preliminary data indicate that treatment with tributyrin, a butyrate pro-drug, rescues the ability to elicit homeostatic respiratory plasticity in adult male GIH offspring. However, whether reduction of neuroinflammation underlies the ability of tributyrin to rescue respiratory plasticity remains unknown. The aim of these studies is to assess neuroinflammatory gene expression in CNS regions associated with respiratory control in tributyrin- and vehicle-treated adult male GIH offspring to better understand the mechanism by which tributyrin rescues homeostatic plasticity in adult male GIH offspring.

Research Grant: NIH R01HL142752 Student Support: NIH T35 OD011078

198 • 2021 National Veterinary Scholars Symposium A Comparison of COX Inhibition and Soluble Epoxide Hydrolase Inhibition in Murine Osteoarthritis

Landon T. Kobluk, Alonso Guedes

Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota Twin Cities, St. Paul MN

Osteoarthritis (OA) is a degenerative disease characterized by loss of articular cartilage. It is a challenging condition to treat in equine patients due to a lack of effective analgesics and inability to prevent cartilage loss. Soluble epoxide hy- drolase (sEH) is an enzyme the metabolizes anti-inflammatory epoxide fatty acids into proinflammatory dihydroxy-fatty acids. Inhibition of sEH has shown to be an effective treatment for OA.The purpose of this study was to compare effec- tiveness between cyclooxygenase (COX) inhibition and sEH inhibition in murine experimentally induced OA, as well as evaluate the response to dual COX and sEH inhibition. The hypothesis was that COX inhibition would attenuate the negative effects of OA on locomotor activity better than sEH inhibition, and that dual inhibition would show the least change in locomotor activity as OA progressed. OA was induced via an injection of type IV collagenase into the left stifle and locomotor activity data was collected at weekly intervals to evaluate function and response. Preliminary statistics did not shown variance between treatment groups (ANOVA with P > 0.05), but data collection was not complete at the time of analysis. Future results associated with this study will include both Von Frey and Hargreaves hypersensitivity testing, as well as histopathology on the injected stifle to assess for changes to the articular cartilage.

Research Grant: Grayson Jockey Club Research Foundation Student Support: Supported by the NIH training grant T35OD011118

Cost comparison of home-cooked diets vs. commercial therapeutic diets for dogs with gastrointestinal disease

Gabriella R. Kratzer, Megan Shepherd, Sean J. Delaney, Jenessa A. Winston, Adam J. Rudinsky, Valerie J. Parker

Dept. of Vet. Clin. Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH (Kratzer, Winston, Rudinsky, Parker). Dept. of LA Clin. Sciences, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA (Shepherd). Balance IT, A DBA of Davis Veterinary Medical Consulting, Inc., Davis, CA (Delaney)

Dietary modifications (e.g., novel/limited ingredient, low fat, fiber-enhanced) play a crucial role in the successful manage- ment of a variety of chronic enteropathies in dogs. Despite the availability of commercially prepared veterinary therapeu- tic diets, clients still choose to feed home-cooked diets (HCDs), often due to a perceived lower cost. There is a paucity of data directly comparing the cost of commercially prepared diets (CPDs) to HCDs for canine chronic enteropathies. We hypothesize that feeding complete and balanced HCDs is more expensive than feeding nutritionally comparable CPDs. Cost [on a per 1000 calorie (Mcal) basis] of several complete and balanced HCDs formulated by board certified veterinary nutritionists to mimic the various nutritional and ingredient profiles of CPDs formulated for the management of canine chronic enteropathies will be determined. Costs of needed HCD ingredients from the 3 largest grocery stores in the United States from 3 different regions of the country (i.e., East, Midwest, &West) will be collected, as will costs of supplements (e.g., multivitamin/multimineral, amino acid, omega-3 fatty acid). Costs to feed CPDs, obtained from a national online retailer, and the total HCD ingredient costs to meet the calculated daily maintenance energy requirements of 5 kg, 20 kg, and 40 kg dogs using the exponential equation [1.6 x (70 x BWkg0.75)] will be summarized. To date, HCDs have been formulated. Data on CPDs and HCD ingredients are currently being collected for analysis. Upon completion, this study will aid veterinarians in counseling clients on the costs of two different feeding strategies for the nutritional management of canine chronic enteropathies.

Research Grant: None Student Support: Wolfe Veterinary Summer Research Fellow

2021 National Veterinary Scholars Symposium • 199 A novel flow cytometric assay to assess platelet desialylation in canine immune thrombocytopenia

Kyle D. Krellwitz, Michael A. Barchilon, Austin K. Viall, and Dana N. LeVine

Veterinary Clinical Sciences (Krellwitz, Barchilon, LeVine), Veterinary Pathology (Viall), College of Veterinary Medicine, Iowa State University, Ames, Iowa

Immune thrombocytopenia (ITP) is a hemostatic disorder of dogs with significant morbidity and mortality. Immunosup- pressive therapies for ITP cause dangerous side effects so novel, targeted therapy is needed. In ITP, autoantibodies target platelets for clearance by splenic macrophages, but recent human studies demonstrate autoantibodies may also trigger platelet desialylation. Desialylated platelets resemble aged platelets and are prematurely cleared; preventing desialylation could be a new, safe therapy for ITP dogs. Studying platelet desialylation in canine ITP is impeded by lack of a test for platelet desialylation. We aimed to develop a flow cytometric assay to detect desialylation-exposed plateletb -galactose residues by binding of a fluorochrome (FITC)-labeled lectin,Ricinus communis agglutinin (RCA). Ex vivo desialylated platelets were made from platelet rich plasma (PRP) treated with neuraminidase C (positive control) and PRP incubated with a competing sugar served as a negative control. Controls and native platelets were incubated with RCA-FITC and evaluated flow cytometrically to determine an optimal RCA concentration of 6.26m g/ml. RCA MFI remained stable for 24hr post-venipuncture at 258C (0h median MFI 2618 vs 24h MFI 2720; P = 0.21) but not at 48C (0h MFI 2618 vs 24h MFI 3358; P = 0.03). Analytic precision was acceptable with CV = 4.5%. MFI was not different between 200, 100, 50, 20, and 10 x103 platelets/mL (P = 0.16), demonstrating our assay can be used in thrombocytopenic dogs. Preliminary findings show our assay can detect desialylated canine platelets with good analytic performance. Our assay will enable determina- tion of the role of platelet desialylation in canine ITP and set the stage for novel therapeutics.

Research Grant: ISU CVM Veterinary Clinical Sciences Seed Grant. Student Support: Van Gelder Foundation

Effects of Holstein genotype on innate immune response to intramammary administration of E. coli

Hannah L. Krichbaum, Amber A. Brink, Wanda J. Weber, Brian A. Crooker

Department of Veterinary and Biomedical Science, College of Veterinary Medicine (Krichbaum) and, Department of Animal Sciences, College of Food, Agricultural, and Natural Resource Sciences (Brink, Weber, Crooker), University of Minnesota, St. Paul, MN

Selective breeding of dairy cows has focused primarily on milk yield to increase profitability. However, there is concern that the contemporary dairy cow is more susceptible to mastitis than her ancestors. Mastitis is the most expensive health- cost of dairy cattle, making genes that confer mastitis resistance of interest. The University of Minnesota has a unique herd of Holsteins that have not been subjected to genetic selection since 1964. Previous studies have demonstrated the unselected cows have reduced herd health cost, a more robust immune response to systemic LPS, and are able to defend against intramammary administration of E. coli better than the contemporary cows. A long-term goal of this research is to identify polymorphism that increase mastitis resistance. The goal of this study was to continue evaluation of the ef- fects of Holstein genotype on innate immune response to intramammary administration of E. coli. Escherichia coli is a gram-negative pathogen that causes mastitis in cattle. The prototypical E. coli strain P4 was infused into the mammary gland and blood and milk samples collected (days -1, 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 11). Whey and serum were isolated from these samples and analyzed by ELISA for IL-6 and IL-1b content. Results indicated a trend (P = 0.08) for IL-1b response to be greater in CH than UH (1.92 vs 1.44 6 0.18). There was a trend (P = 0.07) for a genotype x day interaction as the CH cows had greater IL-1b concentrations on d2, 3, 4, and 6 compared to UH cows. These findings suggest that selection practices have altered the ability of the innate immune system to respond to E. coli infections, leaving the contemporary Holstein more susceptible to mastitis.

Research Grant: Supported by the Minnesota Agricultural Experiment Station and by the Rapid Agricultural Response Fund program of the Minnesota State Legislature and Minnesota Agricultural Experiment Station Student Support: Boehringer Ingleheim and the University of Minnesota College of Veterinary Medicine

200 • 2021 National Veterinary Scholars Symposium Micromanaging resistance: miRNA expression and chemosensitivity in testicular germ cell tumors

Kelly Kries, Ratnakar Singh, and Michael J. Spinella

Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL

Testicular germ cell tumors (TGCTs) are the most common type of cancer in young men and their prevalence has in- creased over the past 40 years. Although 70% of metastatic TGCT patients can be cured with chemotherapy, the tumors that become refractory to treatment are largely fatal. After failing to find clear genetic drivers of chemotherapy sensitivity and resistance in TGCTs, research focus has shifted to explore the role of epigenetics in maintaining sensitivity to cispla- tin, azacytidine, and similar drugs. Epigenetic mechanisms such as DNA methylation, histone modifications, and miRNAs modify gene expression and protein function. We hypothesize that the response of TGCTs to cisplatin and azacytidine is regulated in part by alterations in miRNA expression. To assess this relationship, we used qPCR to compare the relative expression of ten candidate miRNAs between parental and matching, isogenic drug-resistant TGCT cells. The miR- NAs were chosen based on reports that suggested a role in TGCT biology. Preliminary data demonstrated that the hsa- miR-302/367 cluster was upregulated in cisplatin-resistant TGCT cells compared to parental cells. This cluster is known to act as an oncogene in other tumor types by increasing proliferation and survival through the MAPK/ERK pathway and increasing levels of survivin, an apoptosis inhibitor. Future experiments will use RNA-seq to assess miRNA populations in these TGCT cell lines in an unbiased, genome-wide manner. Understanding the epigenetic landscape of drug sensitivity and resistance in TGCT cells is the first step in developing novel therapeutic strategies to treat resistant TGCTs and possi- bly other cancers that are not curable in the metastatic setting.

Research Grant: NIH, 5R01CA211875-02 Student Support: Office of the Director, NIH, T35 OD011145

Optimizing an immunohistochemistry protocol to characterize the role of tuft cells in porcine enteric diseases

Austin E. Kroeger, Olufemi O. Fasina

Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA

Porcine enteric diseases cause significant economic losses and impact mucosal innate and adaptive immunity, predisposi- tion to chronic diseases, the welfare of individual animals, and the overall health of the herd. Tuft cells are an important and unique component of the enteric mucosal innate and adaptive responses to helminth infections and metabolite alter- ations in the mouse and human gastrointestinal systems. Their roles and functions in swine enteric diseases have not yet been explored thoroughly. It will benefit pork producers, veterinarians, and scientists to elucidate their chemosensory and immunomodulatory roles in preventing and developing therapeutics for porcine gastrointestinal diseases. This study is designed to develop and optimize an immunohistochemistry (IHC) protocol to identify and characterize porcine enteric tuft cells. The expression of the tuft cell-specific markers doublecortin-like kinase 1 (DCLK1), POU class 2 homeobox 3 (POU2F3), and growth factor-independent 1B (GFI1B) are, and will continue to be, evaluated. Despite the conserved DCLK1 epitope sequences in pigs and mice, initial IHC results using antibodies developed to target mouse DCLK1 anti- gens indicated a lack of DCLK1 expression in the swine ileum, jejunum, and colon. We are exploring additional DCLK1 antibodies and other tuft cell-specific biomarkers for IHC optimization with modifications of antigen retrieval and pre- treatment conditions. Additionally, in future studies, we plan to utilize the optimized protocol to analyze tuft cell responses and kinetics in retrospective cases of swine enteric diseases such as postweaning diarrhea, Brachyspira hyodysenteriae infection, and Clostridioides difficile infection.

Research Grant: ISU CVM-USDA Seed Grant Student Support: Iowa State University Summer Scholar Research Program

2021 National Veterinary Scholars Symposium • 201 The effect of YY1 PcG function on hematopoietic stem cell development

Ashley M. Kuehnl, Courtney C. Hong, Tyler R. Alvarez, Xuan Pan

Department of Medical Sciences (Kuehnl, Hong, Alvarez, Pan), School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin; Carbone Cancer Center (Pan), UW-Madison Blood Research Program, University of Wisconsin-Madison, Madison, Wisconsin

Ying Yang 1 (YY1) is a ubiquitous zinc-finger transcription factor with functional roles in early embryo development, DNA repair, apoptosis, and hematopoiesis. YY1 is not only a transcription factor but also has been identified as a poly- comb group (PcG) protein with the ability of sequence-specific DNA binding and recruitment. Specifically, YY1 REPO domain, a small domain of 26 amino acids (amino acid sequence 201-226) is responsible for the YY1 PcG functions of re- pressing epigenetic transcription and recruiting other PcG proteins to specific DNA sites. Our lab demonstrated previously that full length YY1 plays a critical role in the maintenance of hematopoietic stem cell (HSC) long-term self-renewal and quiescence as well as promoting c-Kit surface expression and signaling in HSCs. The aim of our current study is to further examine the role of YY1 PcG function/REPO domain on HSC development. Using a YY1 REPO domain mutant and Cre recombinase technology, endogenous YY1 was deleted from Yy1 f/DREPO mice by means of the inducible Mx1-Cre system and the constitutively active Vav-Cre system. Our results show that loss of the YY1 REPO domain causes an increase in the percentage and absolute number of bone marrow derived HSCs, suggesting this domain is important for the regulation of HSC populations.

Research Grant: NIH R01 HL146540-01 Student Support: NIH T35 Training Grant 2T35OD011078-11

The effects of gestational intermittent hypoxia on breast cancer development in female offspring

Molly D. Kuhn, Tracy Baker, Jyoti Watters, and Lisa M. Arendt

Department of Comparative Bioscience, University of Wisconsin-Madison, School of Veterinary Medicine, Madison, WI

Breast cancer is one of the most prevalent cancers among women worldwide. At least one study has been done in humans that suggests a correlation between sleep apnea during pregnancy, a type of sleep disordered breathing, and increased risk of breast cancer development in offspring. To model sleep apnea, pregnant Sprague-Dawley rats were exposed to 8 hours of intermittent hypoxia during their respective night cycles, consisting of alternating 2-min hypoxic (10.5% O2) and normoxic episodes (21% O2) during the 8-hours sleep cycle starting at day 10 until delivery at day 21 of gestation. To identify changes in the mammary gland that might contribute to breast cancer risk, mammary glands of the offspring were collected at puberty (8 weeks of age) and as adults (20 weeks of age). The hypothesis of this study is that exposing pregnant rat dams to gestational intermittent hypoxia (GIH) will increase the number of epithelial stem cells present in the mammary tissue of female offspring, thereby increasing their risk of developing mammary tumors with age.This was tested through the isolation of the mammary epithelial cells from offspring and control rats and subsequent quantification of stem cell activity through the formation of mammospheres and adherent colonies. There was a statistically significant increase in the number of primary and secondary mammospheres as well as increased colony formation from epithelial cells isolated from the mammary glands of GIH offspring at puberty and adults compared to controls.This suggests that under hypoxic conditions, the number of epithelial stem cells is increased in the mammary gland, potentially increasing the risk of breast cancer development in this population.

Research Grant: P30CA014520-UW Carbone Cancer Center Support Grant (CCSG) Student Support: Stipend provided by NIH T35 OD011078

202 • 2021 National Veterinary Scholars Symposium Impact of incubation regimes on genital differentiation in hatchling American alligators

Amanda Kujiraoka, Kathryn Wayne, Mark Does, Kevin Flaherty, Ben Parrott, Matthew Milnes, Brandon Moore

Stephens College (Kujiraoka, Flaherty, Moore), Columbia, MO; Sewanee: The University of the South (Wayne), Sewanee, TN; Center for Small Animal Imaging, Vanderbilt University (Does), Nashville, TN; Savannah River Ecology Lab, Uni- versity of Georgia (Parrott), Athens, GA; Georgia College (Milnes) Milledgeville, GA

Crocodilians, including American alligators (Alligator mississippiensis), utilize temperature-dependent sex determination, where the nest temperature during egg incubation regulates gonadal differentiation into testis or ovary. This, in turn, drives sexually-dimorphic hormonal milieus that generate secondary sex characteristics, such as genital morphology from their bi-potential anlagen. Alligator egg incubation at a constant 338C results in testis development and subsequent penile for- mation, while incubation at 308C results in ovary development and clitoral morphology. However, the fine-scale details of genital morphological divergence that occur primarily around the time of hatching require further clarifications. Therefore, we compared the morphology of 338C male (n = 15) and 308C female (n = 19) external genitalia at seven days post-hatch- ing. Post-necropsy, phalli were dissected from cloacae and their left lateral faces were digitally photographed to perform Geometric Morphometry, Procrustes Superimposition, PCA, and an ANOVA. Sex was verified with histology of gonad, as well as gene expression for a subset. The ANOVA from the results of the PCA showed a significant difference between male and female morphologies (P = 0.007). Here we present key anatomical sources of male-female genital divergence. Additionally, we noticed pigmentation variation on the ventral aspect of the glans and quantified a sexual dimorphism: fe- males presented a greater amount of pigmentation than males. Moving forward, we plan to measure phalli from hatchlings incubated at intermediate temperatures (31.58C) and under fluctuating temperature regimes to better characterize genital differentiation under more normal nest environments.

Research Grant: Stephens College School of Health Sciences Summer Research Grant Student Support: None

Analysis of microRNA expression in chondrocytes as an indicator for regenerative potential

Caroline E. Kuldell, Bethany E. Liebig, Gerrit J. Bouma, John D. Kisiday

Department of Clinical Sciences (Kuldell, Liebig, Kisiday) and Department of Biomedical Sciences (Bouma), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Neonatal connective tissue cells differ from their adult counterparts in their propensity for rapid healing in a scar-free manner. The focus of our current study is to investigate the differential gene expression underlying the superior healing capability of neonatal connective tissue cells. For this study we are evaluating culture-expanded chondrocytes, which are known to dedifferentiate to a mesenchymal stromal cell (MSC) phenotype when isolated and grown in vitro. Further, we are focusing on expression of microRNAs based on their established role in wound healing. In the first part of our study we evaluated expression of CD146, which is a marker of therapeutic potency for MSCs and is not expressed by primary chondrocytes. Previously in our lab, adult equine chondrocytes required approximately 20 population doublings before CD146 expression increased from near-background to about 40%. Conversely, expression of CD146 by neonatal equine chondrocytes was greater than 90% by approximately 5 population doublings, and was stable with additional time in expansion culture. Ongoing studies are comparing microRNA expression between neonatal and adult chondrocytes after both populations have reached maximal CD146 expression. Based on differential expression of CD146, we anticipate that expression of the microRNAs that promote resolution of inflammation and neo-tissue formation will be higher for neona- tal chondrocytes.

Research Grant: Research was supported by discretionary funds Student Support: CVMBS Dean's Office

2021 National Veterinary Scholars Symposium • 203 Electron microscopic characterization of recombinant Lactobacillus acidophilus strains for vaccine development

Ayush Kumar, Benjamin Swartzwelter, Gregg A. Dean, and Allison C. Vilander

Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada (Kumar); Department of Microbiology, Immunology and Pathology (Swartzwelter, Dean, Vilander), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Lactobacillus acidophilus (LA) is a Gram-positive probiotic lactic acid bacterium that can survive the hostile environ- ment of the gastrointestinal tract and has endogenous immune activating characteristics. We have created orally delivered mucosal vaccines against human rotavirus and SARS-CoV2 by genetically modifying LA to express viral epitopes within the surface layer protein A (slpA) along with surface expression of the immune activating adjuvants E. coli FimH and Sal- monella Typhimurium FliC. Surface expressed epitopes and adjuvants can be detected on the LA surface by flow cytom- etry, but it is unclear how these exogenous proteins alter the physical characteristics and behavior of LA. To study these characteristics, we are using scanning and transmission electron microscopy to evaluate the morphology, stress state, and the bacterial surface morphology at stationary and exponential phases of bacterial growth of multiple vaccine strains. The analysis of these images will help us evaluate structural integrity and better characterize our vaccine candidates. Moving forward, we plan to use immunogold labeling to describe the specific antigen presentation and bacterial surface alterations caused by the modification of the bacterial surface layer proteins.This electron microscopic evaluation will facilitate the development of the LA platform as an effective oral vaccine platform.

Research Grant: NIH NIAID R01 AI141603 Student Support: NIH T35 Training Grant: 5 T35 OD 15130-9

Sex specific LRRK2 modulation of inflammation during neuroinvasive viral infection in transgenic mice

Ayush Kumar, Georgia Stavrakis, Andrea L. Cox, and Andrew H. Karaba

Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada (Kumar); Division of Infectious Diseases (Stavrakis, Cox, Karaba), Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD.

The innate immune system is critical in the defense of viral infections. A component of the innate immune system, the in- flammasome, leads to the production of proinflammatory cytokines, interleukin (IL)-1b and IL-18, which play an import- ant role in the pathogenesis of herpes simplex virus type 1 (HSV-1). Leucine-rich repeat kinase-2 (LRRK2), the protein most often mutated in familial forms of Parkinson’s disease, has recently been recognized as a modulator of the innate im- mune response. At the same time, LRRK2 mutations can have sex specific effects in animal models. The goal of this pilot study was to determine if over expression of LRRK2 would lead to sex specific differences in inflammation after -1HSV infection. Transgenic mice overexpressing human WT LRRK2 were inoculated via corneal scarification with HSV-1 strain McKrae. Virus was quantified in tear films and inflammatory cytokines were measured in periocular skin (POS) and brains five days post-infection. In this pilot study inflammatory cytokines were higher in the POS of mice compared to brains, and females had higher cytokines than males. Regarding inflammasome activation, higher concentrations of IL-1b were found in the POS whereas the concentration of IL-18 was higher in the brain. If confirmed in a larger study, these results suggest LRRK2 expression increases inflammation in female mice more than males after HSV-1 infection, and that tissue specific measurements of inflammasome cytokines need to be considered when assessing this arm of the innate immune response to HSV-1.

Research Grant: K08AI156021 from the National Institute of Allergy and Infectious Disease Student Support: NIH T35 Grant: 5T35OD024982-03-Training Veterinary Students for Careers in Biomedical Research

204 • 2021 National Veterinary Scholars Symposium Cryptosporidium phosphoenolpyruvate carboxylase as a molecular target for novel potent antiparasitic drugs

Rachael Y. Lahar, Shahbaz M. Khan, and William H. Witola

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum is a zoonotic parasite that causes a diarrheal syndrome in neonatal and immunocompromised an- imals and humans worldwide. Currently, there is no fully effective drug nor vaccine againstC. parvum. Moreover, parasite oocysts contaminate the environment and are resistant to most chemical disinfectants. Thus, it is urgent to identify strat- egies for developing efficacious drugs againstC. parvum. The C. parvum genome lacks the tricarboxylic acid cycle and oxidative phosphorylation steps, suggesting that the parasite depends solely on glycolysis for metabolic energy. C. parvum glycolytic enzymes differ from mammalian orthologs, making them ideal targets for anti-cryptosporidial drug develop- ment. In previous work, our laboratory identified inhibitors forC. parvum glycolytic enzymes lactate dehydrogenase and pyruvate kinase that prevented parasite growth and disease in mice. The focus of the present study was another glyco- lytic enzyme, C. parvum phosphoenolpyruvate carboxylase (CpPEPc), which contributes to synthesis of pyruvate that is utilized for generation of metabolic energy. We will develop and validate an in vitro coupled-enzymatic assay involving the sequential catalytic activities of CpPEPc and C. parvum malate dehydrogenase, leading to the synthesis of pyruvate. The assay will be used to screen compound libraries for inhibitors of CpPEPc that will then be analyzed for anti-crypto- sporidial activity both in vitro and in vivo. Ultimately, our goal is to unveil validated lead compounds for developing new effective drugs for treatingC. parvum infection.

Research Grant: Bill and Melinda Gates Foundation (completed), USDA (pending) Student Support: Office of the Director, NIH, T35 OD011145

The value of a leukogram for preoperative patient assessment in shelter dogs

Poyu Lai, Kate S. KuKanich, Nora L. Springer

Department of Clinical Sciences (KuKanich, Lai), and Department of Diagnostic Medicine and Pathobiology (Springer), College of Veterinary Medicine, Kansas State University, Manhattan, KS

The value of the leukogram for evaluating dogs for elective ovariohysterectomy or neuter is unclear. We hypothesized that healthy-appearing dogs from a shelter environment would have higher leukocyte count than adult reference interval, but that even dogs with leukocytosis would be able to safely proceed with elective surgery. Shelter dogs (N = 138) from the Kansas State University junior surgery course in 2019 were enrolled, and data collected included age (by dental exam), sex, history, physical exam, CBC and biochemistry, 4DX, treatment, and outcomes. Leukogram values were compared statistically between age groups. Forty-five percent of dogs had a leukocytosis (range 13.7-28.3 K/uL, reference inter- val (4.3-13.6 K/uL). There was a higher proportion of dogs < 3 months of age (70.6%) with leukocyte counts above the reference interval than dogs > 6 months of age (36.6%) (p = 0.002). Similarly, dogs < 3 months old had a higher propor- tion (41.2%) of lymphocytes above the reference interval than dogs $ 3 to # 6 months old (18.2%) and dogs > 6 months old (12.2%) (P = 0.008). One dog had a stress leukogram, while 39 dogs (28.3%) had a chronic inflammatory leukogram. Surgery was postponed in one dog due to parvovirus and one dog due to anaplasmosis-related thrombocytopenia; 136/138 (99%) dogs underwent successful surgery. Leukocytosis is common prior to elective surgery in healthy-appearing dogs from shelters, and adult reference intervals might not be applicable for juvenile dogs. Evaluation of leukogram should be reserved for dogs with evidence of illness or infection rather than as a screening test.

Research Grant: Kansas State University Department of Clinical Sciences, a private anonymous donor, and a Kansas State University Pet Tribute Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 205 Investigating diagnostic utility of agarose cell blocks prepared from hepatic and splenic aspirates

Jessica Lambert, Adriana Villasenor, Erika Gruber

Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Cytologic evaluation of fine needle aspirates is an inexpensive and minimally invasive diagnostic technique commonly used in small animal practice. Advanced testing (e.g immunocytochemistry) in these samples is hindered by small and un- evenly distributed samples and limited availability of control materials. Cell blocks are prepared by embedding aspirates into a solid matrix, which can then be fixed and processed in the histopathology lab.The aims of this study were to deter- mine whether 1) adequately cellular blocks can be prepared from aspirates of solid organs, and 2) samples can be stored for 24 or 72 hours prior to cell block preparation. Hepatic and splenic aspirates from canine cadavers were either expelled directly into warmed liquid agarose, or into one of five storage media: Hanks buffered saline solution (HBSS), 5% albu- min in HBSS, 10% albumin in HBSS, fetal bovine serum, or canine serum. Samples in storage media were refrigerated for 24 and 72 hrs, and then embedded into agarose, formalin-fixed, paraffin-embedded, and sectioned for H&E staining.We found agarose cell blocks prepared from liver and spleen aspirate can yield high cellularity specimens with intact excellent cellular morphology. Storage in HBSS alone resulted in fragile cells that tended to rupture; intact cellularity improved with the addition of protein - either albumin or serum. These results demonstrate that agarose cell blocks can be prepared from aspirates of solid tissues, and that aspirates can be stored in a protein-containing buffer for up to 72 hours prior to cell block preparation. Together, these findings expand the potential practical utility of cell blocks to include samples col- lected outside of the academic veterinary hospital.

Research Grant: NC State faculty startup funds Student Support: NC State Veterinary Scholars Program and the Boehringer Ingelheim Veterinary Scholars Program

Per- and polyfluoroalkyl substances (PFAS) and animals: A review

Antonia Langfeldt, Heather D. Brake, and Melinda J. Wilkins

Department of Large Animal Clinical Sciences (Langfeldt, Wilkins), College of Veterinary Medicine, Michigan State University, East Lansing, MI; National Center for Environmental Health and Agency for Toxic Substances and Disease Registry (Brake), Centers for Disease Control and Prevention, Atlanta, GA

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals associated with multiple adverse health effects in animals and humans. They are used globally in many manufacturing and industrial processes, and their uses in consumer products are extensive. Though industries are shifting production from long-chain to short-chain PFAS based on evidence that the latter are less likely to bioaccumulate, the long-term health effects of short-chain PFAS are unknown. Many studies have focused on the public health impacts of PFAS, but the One Health nature of this issue makes it important to understand and investigate how PFAS impact animals. As such, the purpose of this review is to summarize what is cur- rently known about PFAS exposures, bioaccumulation, toxicology, and health effects in animals. Many PFAS exposure pathways exist for animals, and the ability of PFAS to bioaccumulate is concerning. They can bioaccumulate in plants and up trophic chains and travel long distances; consumption of PFAS via the diet is likely the most common exposure route for animals. However, other avenues, such as exposures from consumer products, also play a role. Most of what is known regarding PFAS’ health effects and toxicology in animals stems from laboratory animal studies, but more non-laborato- ry research is emerging. PFAS can affect multiple body systems, and toxicokinetic mechanisms are species-specific and dependent on chemical properties of individual PFAS. Future studies must emphasize identifying health effects of PFAS exposure in animals, and knowledge of how PFAS affect pets remains extremely limited. As the public becomes more aware of PFAS, it will be important for veterinarians to be informed about how PFAS affect animals.

Research Grant: None Student Support: Boehringer Ingelheim and the Graduate School at Michigan State University

206 • 2021 National Veterinary Scholars Symposium Osteoinductive properties and toxicity of a chitosan-based hydrogel for spinal fusion application

Julien Lanza, Blake Darkow, Austin Kimes, Bret Ulery

Department of Biomedical, Biological, and Chemical Engineering, University of Missouri, Columbia, MO (Lanza, Darkow, Kimes, Ulery); Oniris, Nantes-Atlantic College of Veterinary Medicine Food Sciences and Engineering, 44307 Nantes Cedex 03, France (Lanza)

Spinal fusion is a common orthopedic technique used to treat a variety of spinal disorders such as intervertebral disc dis- ease and spinal fractures by fusing two adjacent vertebrae using a graft material. Each year, an average of 500,000 spinal fusions are performed in the United States, yet, the non-union rate is approximately 25% meaning new approaches are still needed to improve patient outcomes. We have developed a novel chitosan-based hydrogel containing simple signaling molecules aimed at improving the fusion rate. Before performing spinal fusion in an in vivo rabbit model, we character- ized the hydrogel’s properties using a series of in vitro and in vivo experiments. The hydrogel’s osteogenic differentiation potential was determined in vitro employing murine mesenchymal stem cells. In this study, we focused on quantifying the release of simple signaling molecules via calcium and phosphate colorimetric assays. We expected that the hydrogel releases significant quantities of ions within the previously defined therapeutic window of these molecules. Moreover, we focused on assessing the biocompatibility and inflammatory response to the hydrogel in vivo via intramuscular implanta- tion in mice. Two weeks after implantation, mice were euthanized, and implant site tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. We expected to see a slight inflammatory response at the implant site in mice and no significant toxicity induced by the hydrogel. Such results indicate that the hydrogel can safely be used in spinal fusion, the next step in testing if the hydrogel can improve the fusion rate.

Research Grant: MTF Biologics Research Grant Student Support: The stipend for Julien Lanza is an endowment established by IDEXX-BioAnalytics.

Understanding the interactions of pets and their owners with Saint Kitts and Nevis wildlife

Annie Le, Morgan A. Graham, Elpida Artemiou, Sarah E. Hooper

Department of Pathobiology (Le, Hooper), College of Veterinary Medicine, University of Missouri, Columbia, MO; Department of Biomedical Sciences (Graham, Hooper) and Department of Clinical Sciences (Artemiou), School of Veterinary Medicine, Ross University, Basseterre, Saint Kitts, West Indies

With humans and pets encroaching on wildlife habitats, anthropogenic disturbance has been suggested to contribute to the development of emerging infectious diseases. This study aims to better understand the interactions of wildlife (e.g. bats, monkeys, rodents, birds) and domestic pets on Saint Kitts and Nevis. The hypothesis of this study is that we will see bats, monkeys, feral cats, and other wildlife consume food and water from domestic pets’ outdoor food and water bowls and pet owner’s pools, indicating a potential for disease transmission. A survey was distributed to assess their understanding of the interrelationships between their pets and wildlife and to evaluate people’s views on biodiversity and conservation. Acous- tic detectors, mists nets, and camera traps will be placed in areas where there is known wildlife contact in pet’s food/water bowls and in the rainforest along trails. We expect that wildlife uses food and water sources shared by pets, which serve as a source of disease transmission. Preliminary survey data indicates the most common species caught by pets are reptiles, insects, rodents, and songbirds, but this may change as more pet owners participate in the survey. Most residents agree that cats pose a threat to native wildlife and that pet owners should take steps to reduce the threat posed by their animals to wildlife. These findings may indicate that there is a relationship between wildlife, domestic pets, and their owners that indicates a potential for zoonotic disease transmission.

Research Grant: RUSVM Intramural Grant #43005-202 Student Support: IDEXX-BioAnalytics

2021 National Veterinary Scholars Symposium • 207 Equine neutrophil extracellular trap formation in response to ex vivo antigenic stimulation

Margaret Lemons, Joshua Schiffman, Lisa Abegglen, Londa Berghaus, Aaron Rogers, Gareth Mitchell, Miranda Sharp, Matthew Buccilli, Kelsey Hart

Department of Large Animal Medicine, University of Georgia College of Veterinary Medicine, Athens, GA (Lemons, Berghaus, Hart). Department of Pediatrics, University of Utah, Salt Lake City, UT (Schiffman, Abegglen). Huntsman Cancer Institute, University of Utah, Salt Lake City, UT (Schiffman, Rogers, Mitchell, Sharp, Buccilli)

Bacterial sepsis and related systemic inflammatory response syndrome (SIRS) are associated with substantial morbidity and mortality in horses and foals. Neutrophils phagocytose, degranulate, and release neutrophil extracellular traps (NETs) to effectively eliminate invading pathogens. The formation of NETs (NETosis) is a highly regulated defense mechanism induced by microorganisms, platelets, and inflammatory cytokines. However, dysregulated NETosis contributes to mor- tality in SIRS and sepsis. Recent studies in humans, dogs, and rodents documented increased NETosis associated with increased sepsis severity and organ dysfunction, but the role of NETs in equine sepsis remains unknown. The objective of this study was to induce equine NETosis ex vivo under inflammatory conditions mimicking sepsis. We hypothesized that treating horse neutrophils with various antigens increases ex vivo NET release in a dose-dependent manner. Isolated neutrophils from healthy horses were stimulated with phorbol myristate acetate, lipopolysaccharide, ionomycin, killed whole-cell Staphylococcus aureus (SAA), killed whole-cell Escherichia coli, healthy and septic foal plasma, or media alone. NET formation was visualized using confocal microscopy and quantified using IncuCyte live-cell analysis. Induced ex vivo NETosis was successfully visualized under conditions that induce NETosis in neutrophils from other species. Incu- Cyte quantification suggests that SAA may be the most potent stimulator ofex vivo NETosis in horses. Additional experi- ments are underway, and detailed results will be presented. Further studies in naturally-occurring equine sepsis are needed to determine if NETs represent prognostic markers and targets for clinical intervention.

Research Grant: None Student Support: Morris Animal Foundation Veterinary Student Scholar Program

Acute Effects of Repetitive Traumatic Brain Injury on Neuroimmune Responses

Kayla E. Leverance, Nicole L. Emmitt, Venkatramana D. Krishna, Andrew Grande, Walter C. Low, and Maxim C-J. Cheeran

Veterinary Population Medicine Department (Leverance, Emmitt, Krishna, Cheeran), Comparative and Molecular Biosciences Graduate Program, College of Veterinary Medicine, University of Minnesota (Emmitt), and Department of Neurosurgery, University of Minnesota Medical School (Grande, Low)

Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative disease caused in part by repeated traumatic brain injury (TBI). It is not known how the interval between sequential injuries and ensuing inflammatory responses results in neuro- pathological outcomes. We tested the hypothesis that after a mild TBI, a second moderate TBI generates an exaggerated neuroinflammation, which is greater after a 3-day interval between successive injuries compared to a 7-day interval.An open controlled cortical impact (CCI) mouse model was used to compare repeat-injured brains with sham-injured and single injury controls. Two craniotomies, on either side of the skull, were centered over the primary and secondary motor cortices. A mild TBI was delivered on the right side with a 2 mm impactor tip at 4 m/sec velocity, 100 ms dwell time, and 1 mm depth. After a 3d or 7d interval, a moderate TBI was delivered on the left side using a 6 m/sec velocity impact. Flow cytometry analysis of immune cell phenotypes and PCR analysis of cytokine gene expression in the brain was assessed 3d post second injury. These outcome measures give insight into the quality and quantity of inflammatory responses generat- ed by a second TBI. Motor deficits due to TBI were assessed by a beam walk assay 2 days after the first and second injury and were compared to baseline data obtained prior to injury. Findings from these present studies will shed light on how neuroinflammation resulting from one TBI could exacerbate the proinflammatory response to a second injury leading to long term behavioral deficits. Understanding the pathogenesis of sequentialTBIs could help identify points of intervention to prevent the neurodegenerative outcomes of repetitive brain injuries.

Research Grant: MN SCI & TBI Research Grants (#128515, #159891), MN Office of Higher Education, State of MN, the NRSA T32 DA007097, NIDA, NIH, and CVM Resident & Graduate Student Research Grants, University of MN Student Support: Supported by the NIH training grant T35OD011118

208 • 2021 National Veterinary Scholars Symposium Deleterious health effects of harmful algal blooms on fishing communities in LakeVictoria, Kenya

Alexander A. Levitskiy, Harriet Okronipa, Kathryn J. Fiorella

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

Lake Victoria’s freshwater fishery supports roughly 30 million people. Within fishing communities, fish is an essential -ani mal-source food, contributing omega-3 fatty acids and micronutrients. However, a paradox exists regarding fish consump- tion: although fish are highly nutritious, they can expose consumers to harmful algal bloom (HAB) toxins.A byproduct of anthropogenic activities, HABs arise from hazardous blooms of that produce toxins like microcystin (MC), a potent hepatotoxin to animals and humans. Beyond the risks associated with drinking contaminated water and direct der- mal contact, individuals may face unrecognized threats from MC accumulation in the fish they eat.To better understand health risks associated with fish consumption, we aim to investigate MC levels in LakeVictoria fish at multiple trophic levels. Variations in fish nutritional profiles are increasingly recognized, and we expect differences in MC concentration to similarly vary across species. Analysis of cross-sectional survey data (n = 90) collected in March 2020 examines fish con- sumption patterns to situate these risks. The proportion of households that reported eating fish within the week prior was 75.6% for dagaa (R. argentea), 65.6% for Nile perch (L. niloticus), 38.9% for Nile tilapia (O. niloticus), and 15.6% for cichlids (Cichlidae). Differences in how fish are consumed may also impact exposure riski.e. ( , dagga are consumed whole with MC likely to accumulate in the hepatopancreas). Further, persistent ingestion of MCs, even below the WHO tolerable daily intake of MC-LR at 0.4 mg/kg, likely poses chronic health threats. A clearer picture of fish consumption patterns will be critical to better categorizing risks.

Research Grant: An Atkinson Venture Fund Award to KJF supports this work. Student Support: AAL is supported by NIH T35 OD010941 and the Cornell University College of Veterinary Medicine.

Clinicopathologic findings in dogs diagnosed with coccidioidomycosis

Samuel G. Levy, Todd D. Carter, Daniel S. Foy, and Stephanie L. Shaver

Department of Specialty Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Coccidioidomycosis is a disease caused by a dimorphic fungus endemic to the Southwest United States and may present with various clinical signs including cough, lethargy, and fever. The non-specific presentation of this disease can make its diagnosis challenging; often a clinical diagnosis is reached using a combination of clinical signs and serology. This retrospective study aimed to evaluate clinicopathologic abnormalities in dogs with coccidioidomycosis at the time of diagnosis and throughout treatment, and to compare these abnormalities with those of healthy dogs. Medical records at the Midwestern University Companion Animal Clinic were searched for all cases of coccidioidomycosis diagnosed between January 2015 and June 2021. Of the cases identified, 115/431 (26.7%) met the inclusion criteria. Fifty-eight dogs were male and 57 were female, with a mean age of 6.2 years. IgG titers were positive in all cases, with a median titer of 1:16 (range: 1:2-1:256). Hyperglobulinemia was identified in 62.6% of dogs, with a median value of 3.9 g/dL (range: 2.2-6.9 g/ dL) at diagnosis. This value decreased with treatment and was significantly lower at 61-150 days (3.2 g/dL; P < 0.0001), 151-240 days (3.2 g/dL; P < 0.0001), and 241-360 days (3.0 g/dL; P = 0.0001) post-diagnosis. Monocytosis was identified in 56.5% of dogs, with a median value of 875 cells/mL (range: 210-3647 cells/mL) at diagnosis. This value also decreased with treatment and was significantly lower at 61-150 days (576 cells/ml; P < 0.0001), 151-240 days (539 cells/ml; P < 0.0001), and 241-360 days (377 cells/ml; P = 0.0001) post-diagnosis. Further data is being collected to compare these variables with those of a healthy dog cohort evaluated during the same time period.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

2021 National Veterinary Scholars Symposium • 209 Mortality rate and risk factors for anesthesia-related deaths of rabbits in an Arizona animal shelter

Kendall A. Lewis, Maddison L. Johnson, Nancy Bradley, Melissa Thompson, and Rachael E. Kreisler

Department of Primary Care, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Mortality rates for healthy rabbits undergoing elective procedures in small animal clinics have been reported to range from 0.73-4.8% and are greater than that of dogs and cats (0.23-2.2%). Researchers have identified factors associated with increased risk of death (e.g., age, ASA status, duration of procedure, and induction protocol). However, these studies have not included animal shelters, which may use different anesthetic protocols.The aim of this study was to perform a nested case-control study on rabbit spays and neuters performed at a shelter in Arizona between 2015-2021. Rabbits with an out- come of death within one week of surgery were considered cases. Four controls were randomly selected per case. Poten- tial risk factors (sex, age, weight, ASA status, year of surgery, anesthetic and analgesic drugs, and duration of procedure) were coded from medical records. There were 1,125 rabbit surgeries with a 48-hour mortality rate of 1.6% (n = 19; 95% CI [1.0, 2.6]), and a one-week mortality rate of 2.2% (n = 25; 95% CI [1.4, 3.3]). Most mortality occurred postoperatively, either same day (60%), next day (4%) or later (24%), or intraoperatively (12%). None died preoperatively. The median weight was 2.4 kg (IQR 1.7, 3.6), median neuter duration 11 min (IQR 6, 14), and median spay duration 18 min (IQR 10, 22). Cases had a median age of 18 months (IQR 8, 24) and 84% were ASA status I. Controls had a median age of 24 months (IQR 12, 36) and 95% were ASA status I. Potential risk factors were evaluated using multivariable logistic regres- sion. Age had an odds ratios (OR) of 0.95 (P = 0.025) and ASA status of II had an OR of 6.4 (P = 0.022). Rabbit mortality rates were similar to historically reported rates from private practice.

Research Grant: Midwestern University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Determining the pharmacokinetic properties of meloxicam in barred owls (Strix varia) undergoing rehabilitation

Zoey Lex, Mark Mitchell, Kelsey Rayment, Laken Russell, Corinne Mayer, and Kimberly Boykin

Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Anthropogenic activities have negatively affected the lives of many birds, including owls.These interactions have led to a rise in cases being admitted to wildlife rehabilitation facilities, especially with traumatic injuries. For example, the Wildlife Hospital of Louisiana has had a greater than two-fold increase in barred owls (Strix varia) being presented over the past five years (2020, 95; 2014, 42). Because most of these animals present with traumatic injuries, it is essential that analgesics are included as a component of their treatment plan. To date, there have been no studies evaluating analgesics in barred owls. It is important to pursue evidence-based research to guide clinicians and ensure best treatment practices so that these animals can be successfully rehabilitated for release. The goals of this study are to determine the harmonic means, times to maximum concentration, and elimination half-lives for single doses of 1.0 mg/kg and 2.0 mg/kg meloxi- cam per os. Barred owls admitted to the Wildlife Hospital of Louisiana for traumatic injuries will be used for the study, as meloxicam is commonly incorporated into our treatment plans. Dosing will be randomly assigned, and cases matched to minimize treatment bias. Blood samples will be collected 15 minutes prior to meloxicam administration and at 30-min, 45- min, and 1-, 2-, 4-, 6-, and 12- hours post-administration. Samples will be processed accordingly and analyzed using high performance liquid chromatography. Our hypotheses are that both doses will be safe and provide adequate plasma concentrations of meloxicam, but that there will be significant differences in the pharmacokinetics between doses.

Research Grant: Boehringer Ingelheim Student Support: School of Veterinary Medicine, Louisiana State University

210 • 2021 National Veterinary Scholars Symposium Novel diagnostics for sepsis: optimizing Fountain FlowTM Cytometry and testing the RapidBacTM Vet immunoassay

Emma Li, Samantha Evans

Department of Veterinary Biosciences, College of Veterinary Medicine, the Ohio State University, Columbus, Ohio

Bacterial sepsis is a life-threatening condition in which rapid diagnosis is critical for survival. The current primary di- agnostic tools for sepsis detection from cavity effusions are bacterial culture (slow turnaround time) and fluid cytology (suffers from low sensitivity). Fountain FlowTM Cytometry (FFC) was designed to detect waterborne microbes at very low concentrations in municipal and commercial settings. FFC may be capable of offering a rapid, portable, and adaptable means of sepsis detection in the clinical setting. A preliminary FFC diagnostic trial conducted by our laboratory on thorac- ic and abdominal fluids from 60 septic mammalian patients demonstrated a sensitivity and specificity of 60% and 94.4%, respectively. The RapidBacTM Vet (RBV) test is an immunoassay designed for point-of-care detection of bacteria in canine and feline urine. The aim of this study was to both optimize the use of the FFC and to assess the capabilities of the RBV test for detection of septic effusions. Sterile cavity effusions from small animal patients will be spiked with bacteria of dif- ferent morphologies, necrotic debris, and commercial lipids. We expect these factors to impact the accuracy of FFC, and that alterations in sample preparation will subsequently improve sensitivity. 20 cavity effusions from mammalian patients will be tested using the RBV test in a case-control study. Preliminary results indicate that false positive results may be problematic for the use of RBV to detect septic effusions. FFC offers multiple advantages over currently used methods of septic effusion detection, and the RBV test offers the unique capability of point-of-care testing for bacterial sepsis. Thus, further testing of both tools is warranted.

Research Grant: None Student Support: NIH T35 Training Grant T35OD010977

An exploratory research of the Common Loon gut microbiome

Jingyi (Jenny) Li, Dr. Mark Pokras

Cummings School of Veterinary Medicine, Tufts University, Grafton, Massachusetts

The microbiome consists of the genetic material of all microbes that live inside the host body. The gastrointestinal micro- biome plays a vital role in host health by helping normal metabolism and protection against pathogen infections. Com- position of the microbiome varies with species, sex, diet, social interaction, and environmental factors. Despite the large amount of data available on the gut microbiome in vertebrates, especially mammals, knowledge on avian microbiology is limited. Studies of animals in the wild are needed if we want to understand how hosts and microbes interact in a natu- ral environment. Common loons (Gavia immer) are threatened in several states in the U.S.. They are also susceptible to several pathogens and sensitive to environmental changes, including exposure to heavy metals and toxins, which may alter host-associated microbial communities. To our knowledge, no previous studies have focused on the loon gut microbial ecology, data which will be key for better understanding the benefits of microbes on host health. The primary goal of this study is to characterize the microbiome diversity and composition of common loons in the Northeastern U.S. In addition, we aim to determine to what extent microbiome changes postmortem in order to make use of archived specimens collect- ed over a broader time scale. Cloacal samples were collected from both live and dead loons; whole genomic DNA is being extracted using a DNeasy PowerLyzer PowerSoil kit (Qiagen Inc., Germantown, MD); and high throughput sequencing will be performed to characterize the bacterial community (microbiome). At last, statistical data analysis will be completed.

Research Grant: Summer Star Foundation for Nature, Art and Humanity Student Support: Summer Star Foundation for Nature, Art and Humanity

2021 National Veterinary Scholars Symposium • 211 De novo alpaca reference genome assembly and genomics of minute chromosome syndrome

Shumin Li, Caitlin Castañeda, Terje Raudsepp, Brian W Davis

College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA(Li), Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA (Castañeda, Raudsepp, Davis)

Minute Chromosome Syndrome (MCS) has been reported to be related to abnormal sexual development and infertility in female alpacas and cytogenetically manifested as dramatic size difference between the homologs of chromosome 36. Prevalence of MCS among alpacas is poorly explored due to lack of cost-effective genetic testing, but may be as high as 16-30% among infertile females, thus significantly contributing to reproductive failure in this species. Our goal is to estab- lish a high-quality reference genome for the alpaca species and identify likely molecular cause(s) of MCS. Animals with MCS were identified by clinical symptoms and cytogenetic analysis. Long read DNA sequencing paired with Hi-C chro- matin conformation and Bionano optical mapping to generate the first chromosome-scale genome for alpaca. Case-control whole genome sequencing was conducted and aligned to the new reference genome. We observed an increased nucleolus organizer region (NOR) on chromosome 36 by fluorescence in situ hybridization (FISH) and are designing a real-time polymerase chain reaction test. Allele frequency difference and copy number variation associated with chromosome 36 between control and case groups was estimated to identify specific breakpoints associated with MCS structural rearrange- ments. These findings provide simple diagnostic approaches of MCS for the alpaca community and will be used to investi- gate a much larger cohort of wild and tamed alpacas.

Research Grant: Morris Animal Foundation and Alpaca Research Foundation Student Support: Boehringer Ingelheim VRSP and Texas A&M College of Veterinary Medicine & Biomedical Sciences

Circumscribing laser cuts attenuate chronic focal cortical seizure propagation in mice

Seth Lieberman, Daniel A. Rivera, Ryan Morton, Amrit Hingorani, Teresa Southard, Lynn Johnson, Jennifer Reufauf, Ryan E. Radwanski, Mingrui Zhao, Oliver Bracko, Theodore H. Schwartz, and Chris B. Schaffer

Meinig School of Biomedical Engineering (Lieberman, Rivera, Morton, Hingorani, Reufauf, Radwanski, Bracko, Schaffer) College of Veterinary Medicine (Lieberman, Southard), Statistical Consulting Unit (Johnson), Cornell University, Ithaca, NY Department of Neurological Surgery (Zhao, Schwartz), Weill Cornell Medicine, New York, NY

Focal epilepsy is characterized by seizures that initiate in a small region of the brain and propagate out. Medical manage- ment fails in about 45% of these patients and resective surgery remains the only alternative, but often leaves patients with severe neurologic deficits.In-vivo imaging of acute seizure propagation has shown that seizures primarily propagate along lateral connections in cortical layers II-III. Therefore, it has been hypothesized that making minimally invasive incisions to severe these lateral connections while maintaining vertical connections would preserve much of normal brain function while blocking seizure propagation. Tissue ablation by tightly-focused femtosecond laser pulses provides a “laser scalpel” that can make subsurface microtransections without damaging surrounding tissue. Here we tested the long-term efficacy of laser cuts in the layers II/III of the cortex in interfering with the propagation of chronic focal seizures, and we examine the impact of the cuts on normal cortical structure and function. Chronic focal seizures were induced by microinjection of iron chloride. In mice with laser cuts that encircled the seizure focus, we observed an 80% reduction in seizure propagation over 3 months. Chronic imaging of ablated regions showed no significant reduction in blood flow to the encircled region and histology after one month showed a small scar. Cuts in forelimb motor cortex exhibited only a minor acute deficit when compared with sham animals in a complex reaching task. In conclusion, laser cuts in layers II/III of the cortex around a seizure focus reduced seizure propagation while maintaining normal structure and function suggesting it maybe a promising new neurosurgical technique.

Research Grant: Financial support from NIH, grant number MH119880 Student Support: None

212 • 2021 National Veterinary Scholars Symposium Investigating the mechanism behind a phenotypic drift in lupus-prone mice

Amy Lin, Xavier Cabana-Puig, Ran Lu, Jing Zhu, S. Ansar Ahmed, Xin M. Luo

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA

Lupus-prone mice are known to have phenotypic variations among different animal facilities.We noticed decreased kid- ney-to-body weight and increased spleen-to-body weight ratios in our in-house colony compared to newly purchased mice from The Jackson Laboratory (JAX). The goal of this study is to characterize the mechanisms behind this phenotypic drift between in-house and JAX mice, specifically in the development of lupus nephritis and splenomegaly. We hypothesize that JAX mice vary in the splenic immunologic composition and immune complex deposition in comparison to in-house mice. To study this, kidney samples were stained for complement C3 and IgG2a then photographed with a confocal micro- scope. The fluorescent intensities of about 30-60 glomeruli per kidney were measured with ImageJ software then analyzed statistically, revealing a trend of increased C3 and IgG2a deposition in the glomeruli of JAX mice consistent with higher kidney-to-body weight ratios. Splenocytes were stained to mark germinal center (GC) B cells then run through flow cy- tometry. Analysis with FlowJo software revealed no differences in the frequency of GC B cells between in-house and JAX colonies. Although results of both experiments were not significant between the two groups, other mechanisms underlying the phenotypic drift should still be explored to preserve the MRL/lpr phenotype for future studies. This project highlights that environmental factors may impact the MRL/lpr phenotype over time and therefore might impact progression of lupus.

Research Grant: Virginia Maryland College of Veterinary Medicine Student Support: NIH Training Grant T35OD011887D

Extracellular vesicles and their role in promoting cell differentiation

Yimei Lin, Yun Ha Hur, Fangyu Wang, Marc Antonyak, and Richard Cerione

Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY

Extracellular vesicles (EVs), including the major sub-classes microvesicles and exosomes, mediate cell-cell communi- cation and have been shown to play important roles in promoting cellular processes that underlie various biologies and pathologies. For example, the Cerione group found that cancer cell-derived EVs can stimulate the growth and survival of cancer cells, while more recently they discovered that the EVs from embryonic stem cells are capable of maintaining pluripotency. Here, I set out to build upon these exciting findings by determining whether the EVs produced by special- ized (i.e. differentiated) cells could enhance cellular differentiation.The microvesicles and exosomes released by fibro- blasts were isolated and then used to treat embryonic stem cells. The changes in the pluripotency and differentiation of the EV-treated stem cells are currently being examined. This study will shed new light on how a unique form of intercellular communication impacts cell fate, and potentially offer new opportunities for regenerative medicine.

Research Grant: Work supported by R35 GM122575/GM/NIGMS and R01 CA201402/CA/NCI to R.C. Student Support: Student supported by NIH T35 AI007227.

2021 National Veterinary Scholars Symposium • 213 Investigating the effects of storage methods on rabbit corneas

Joshua Liu, Bianca Martins

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA

Keratoplasty, or corneal transplantation, has significant implications in veterinary medicine due to the prevalence of corneal disease and injury. Despite multiple clinical trials reporting positive results, keratoplasty is infrequently used in veterinary medicine, as most clinics lack the ability to readily obtain fresh tissue, and extensive research investigating effective methods of cryopreservation has yet to be conducted. While previous research has found that freezing solely the cornea in antibiotic solution leads to significant degradation, is proposed that storing a cornea as a whole globe may result in a decrease in fluid uptake. This study aims to evaluate the changes in histology, transparency, and thickness of corneal tissue when stored as whole globe compared to sole cornea storage. Globes from 20 rabbits were harvested, decontami- nated, and randomly sorted into 4 groups: whole globe storage (G1- n = 6), whole cornea storage (G2 - n = 6), half cornea storage (G3- n = 6), and control (G4 - n = 3 - fresh corneas). Samples from G1-G3 were stored for 6 weeks at -20°C with an antibiotic solution (Ofloxacin). Following storage time, samples were thawed at room temperature and assessed for cor- neal transparency and thickness and submitted to histologic evaluation. Whole globes (G1) had transparency and thickness comparable to fresh corneas (G4), while corneas stored either as whole (G2) or halves (G3) exhibited notable reduction in transparency and increased thickness. Overall, the findings suggest that corneal banks should store whole globes instead of whole or sectioned corneas, as whole globes provide a better-preserved tissue.

Research Grant: UC Davis School of Veterinary Medicine Student Support: STAR Program/Boehringer Ingelheim Veterinary Scholar Funds

Efficacy of diagnostics for reducing inappropriate antimicrobial use in simple canine urinary tract infections

Patrick Liu, Theresa Alando Oduol, Patrick Carney, Casey Cazer

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca NY (Liu, Cazer), Department of Medicine, Brigham and Women’s Hospital, Boston, MA (Oduol), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY (Carney)

The growing importance of the One Health initiative highlights how combating antimicrobial misuse lies at an intersection between animal and human health. Evidence-based guidelines for antimicrobial use among companion animals are one tool to reduce inappropriate antimicrobial prescription and minimize antimicrobial resistance (AMR). The International Society for Companion Animal Infectious Diseases (ISCAID) publishes guidelines for clinicians to diagnose and treat ca- nine urinary tract infections (UTI). This study aims to review how well clinicians adhere to these guidelines and determine the relationship between diagnostic tests and compliance with these guidelines. We reviewed Cornell University’s Com- panion Animal Hospital and Small Animal Community Practices’ medical records for cases of simple canine UTIs seen from June 27th, 2011, to February 1st 2019. Cases diagnosed with uncomplicated UTIs in dogs of any sex and at least 6 months of age were included. Dogs with a recurring history of UTIs, predisposing comorbidities, and antimicrobial use within the 21 days prior to their visit were excluded. The diagnostic approaches were categorized as urinalysis (UA) only, urine culture (UC) + antimicrobial susceptibility test (AST) only, UA + UC + AST, or clinical signs only. We compared antimicrobial prescription adherence to ISCAID guidelines across diagnostic categories with a Fischer’s Exact Test. We hypothesize that higher levels of diagnostics (e.g., UC and AST) will be associated with better adherence to the ISCAID guidelines. If proven true, it will be important to distribute the knowledge to clinicians and clients that urine cultures are associated with reducing antimicrobial misuse, and therefore minimize AMR.

Research Grant: Research Grant: None Student Support: Student Support: NIH T35 OD010941, Cornell College of Veterinary Medicine.

214 • 2021 National Veterinary Scholars Symposium Susceptibility of mosquito and bird cells to Usutu virus

Emma Loessberg, Sarah Kuchinsky, Nisha Duggal

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia

Usutu virus (USUV; flavivirus) is a zoonotic virus, phylogenetically related to West Nile virus (WNV). Similarly to WNV, USUV is maintained within a mosquito-bird enzootic cycle, with ornithophilic mosquitoes of the Culex genus acting as vectors and primarily Passeriformes and Strigiformes acting as reservoir hosts. Disease in humans causes symptoms ranging from fever and skin rashes to encephalitis and meningoencephalitis. USUV was first isolated in Africa in 1959 and spread to Europe in the late 1990s, with concern for the virus growing when a mass blackbird die-off related to the virus was documented in Austria in 2001. The species of mosquitos and birds that are important for the maintenance of USUV has yet to be determined. The aim of this study is to assess the growth kinetics of the Netherlands 2016 and Uganda 2012 strains of USUV in Cx. quinquefasciatus, Cx. tarsalis, and primary song sparrow cell lines. We hypothesize that susceptibility to USUV will vary between species, with higher titers seen in the cell lines inoculated with the Uganda 2012 strain due to the high levels of viremia found when it was previously inoculated in a murine model. Growth curves were performed to examine the growth kinetics of the virus within each cell line. Cells were inoculated at MOI 0.01, and time points were collected daily for five days. Titers were measured via plaque assay in Vero cells. InCx. tarsalis cells, the Ugandan strain reached a significantly higher titer than the Netherland strain.This will be repeated with Cx. quinque- fasciatus and primary song sparrow cell lines. Our results suggest that there may be differences between USUV strains in replication in natural hosts, which will be assessed in vivo in the future.

Research Grant: NIH NIAID R21 AI156322 Student Support: T35OD011887D

Use of the rOmpB gene to detect strain-level variation in in the Arizona region

Mary Griffin Logue, Jeffrey Mutterperl, Alec Oliva, Nicolette Roe, Kayla Allwardt, Charles Schaefer, Jose Hernandez, Rachael Kreisler, Jung Keun Lee, Michael Quinlan, and John VandenBrooks

College of Veterinary Medicine (Logue, Roe, Allwardt, Kreisler, Lee), College of Graduate Studies (Schaefer, Hernandez, Lee, Quinlan, VandenBrooks), Arizona College of Osteopathic Medicine (Mutterperl, Oliva), Midwestern University, Glendale, AZ

Rocky Mountain spotted fever (RMSF) is a zoonotic bacterial disease vectored by ticks and is a threat to public health both domestically and internationally. In 2003, there was an initial outbreak of RMSF in Arizona. Since then, there have been persistent cases from year-to-year, with case fatality rates as high as 80% if not treated within the first five days. However, the main tick vectors for the causative agent, Rickettsia rickettsii, are not found in Arizona. Instead, the recent outbreaks in Arizona and surrounding areas have been linked to a novel vector, Rhipicephalus sanguineus, the brown dog tick. Currently, the brown dog tick has not been identified as a vector outside of the Arizona region, although it is present in every state. This leads to the question of why the brown dog tick can act as a vector in this region, but not elsewhere. While there are many potential contributing factors, we hypothesized that strain level variation in the rickettsial causative agent may make it more suitable to be vectored by the brown dog tick in Arizona. Here, we tested tick samples collected from Arizona, New Mexico, and Mexico for R. rickettsii by amplifying the rickettsial gene encoding a highly conserved outer membrane protein, rOmpA. Due to its conserved nature, rOmpA is not ideal for the detection of strain-level vari- ation. Thus, positive R. rickettsii samples underwent further analysis to amplify the gene that encodes rOmpB. Since rOmpB is the most abundant rickettsial outer membrane protein, and previous studies have detected variation in this gene, it makes an ideal target to detect strain-level variation in R. rickettsii. Strain level variation was then correlated with geo- graphic distribution across the region.

Research Grant: Midwestern University Student One Health Research Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 215 Effects of communication training on shared decision-making in companion animal practice

Sarah Jane E. Lokaisingh, Natasha Janke, Jason B. Coe, and Jane R. Shaw

Department of Population Medicine (Lokaisingh, Coe), Ontario Veterinary College, University of Guelph, Guelph, Ontario; Department of Clinical Sciences (Janke, Shaw), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Communication in veterinary medicine impacts the quality-of-care patients receive. Veterinarians are responsible for shar- ing knowledge and making recommendations to help clients make informed decisions about their pet’s healthcare options. Shared decision-making is the process of clinicians and clients working together to create client-centered healthcare plans. Decisions are made by exploring treatment options together and integrating client preferences. The objective of the re- search project was to assess veterinary professionals’ level of shared decision-making before and after in-practice commu- nication training. A 10-month communication training program was conducted between April 2018 to January 2019, and twenty skills from the Calgary Cambridge Guide were taught to improve relationship centered communication. This re- search project involved the analysis of veterinarian and veterinary technician-client interactions from 4 companion animal practices in Austin, Texas. A total of 170 audio-recorded wellness and problem appointments were collected. OPTION5, an instrument that measures the extent of shared decision-making between clinicians and patients in human medicine, was adapted to score the audio recordings. Preliminary results will be presented, and the findings will inform if skills-based communication training enhances shared decision-making during veterinary problem and wellness appointments.

Research Grant: Zoetis Student Support: Boehringer Ingelheim Veterinary Scholars Program

Anti-inflammatory/anabolic factors IL1Ra and IGF-I are enriched in equine bone marrow concentrate versus PRP

Sidney Long, Grigorios Maleas, Sushmitha Durgam

Department of Veterinary Clinical Sciences (Long, Durgam), College of Veterinary Medicine, The Ohio State University, Columbus, Ohio; Tierklinik in Lusche GmbH (Maleas), Bakum, Germany

Orthobiologics such as bone marrow concentrate (BMAC) and platelet-rich plasma (PRP) are gaining traction for treating equine articular and tendon/ligament injuries due to their anti-inflammatory properties and autologous nature.At present, their clinical use is incumbent on clinician preference/experience, diagnosis, clinical signs and feasibility. Injury-specific treatment protocols are lacking and mandate delineating their comparative biological characteristics. This study assesses the cellular, growth factor, and cytokine compositions of matched BMAC and PRP from 27 horses. BMAC and PRP were processed from the buffy coat of bone marrow and whole blood via centrifugation methods, respectively. Cellular compo- sition, growth factors (TGFb1, IGF-I, PDGF, VEGF), and cytokines (TNFa, IL1b, IL6, IL1 receptor antagonist protein; IL1Ra) are quantified using standard automated hemoanalyzer and ELISA methods, respectively, to test the hypothesis that PRP and BMAC compositions are equivalent. From a cellular composition standpoint, monocyte (8-fold), lymphocyte (3-fold), neutrophil (14-fold), and leukocyte (6-fold) numbers are significantly (P < 0.0001) increased in BMAC compared to matched PRP samples. In contrast, thrombocyte numbers are 5-fold (P < 0.0001) increased in PRP relative to BMAC. Anti-inflammatory protein, IL1Ra (107-fold), tissue anabolic growth factor, IGF-I (433-fold), and angiogenic factor, VEGF (259-fold) are significantly increased (P < 0.0001) in BMAC compared to PRP. Delineating the cellular, growth factor and cytokine compositions of equine BMAC and PRP is a necessary first step to outline injury-specific indications of these common orthobiologic therapies in equine practice.

Research Grant: OSU Faculty Research Startup Funds Student Support: T-35 NIH Training Grant T35OD010977

216 • 2021 National Veterinary Scholars Symposium A porcine gracilis myocutaneous flap model for Vascularized Composite Allotransplantation: technique and tips

Catherine Lopez, Fatih Zor, Yalcin Kulahci, Vijay Gorantla, Susan Appt, Heather DeLoid

Department of Pathology, Wake Forest School of Medicine (Lopez, Appt, DeLoid), Wake Forest Institute for Regenerative Medicine (Zor, Kulahci, Gorantla), Wake Forest Baptist Health, Winston-Salem, NC

Vascularized Composite Allotransplantation (VCA) is a procedure utilizing multiple tissue types transferred between individuals as a single functional unit and used to reconstruct complex tissue defects including face and hand transplants. Immunosuppression is required for transplant survival but side effects include reduced life expectancy, diabetes, severe infections, and cancer. VCA improves quality of life but is not “life-saving” and therefore localized treatments to reduce rejection of allografts with less systemic side effects are needed. Here we present refinements of a translational animal model, porcine gracilis myocutaneous flap (GMCF) allotransplantation.We describe the versatility of GMCF allotrans- plantation, surgical technique to optimize surgery, postoperative follow up and standardized clinical grading of the al- lograft rejection. Using GMCF model, cross transplantation between 2 pigs was performed with each pig serving as donor to each other. A portion of gracilis muscle and overlying skin was harvested with the size of the flap pedicle dependent on femoral vessels. Harvested flaps were transplanted to the recipient’s neck with jugular vein and carotid artery microsurgi- cal vessel anastomoses. Post-operative follow-up of the animals (tissue and blood sampling, dressing changes, and wound care), as well as clinical grading of the flap rejection, was documented.This study provided novel technical refinement of preoperative, intraoperative, and postoperative course of GMCF allotransplantation model and standardized clinical grading for porcine allograft rejection. These observations will provide insight to researchers, veterinarians, and veterinary technicians for future VCA investigations.

Research Grant: Unknown Student Support: NIH T35 OD010946

Comparing temperatures at various rectal depths to alternative sites in New Zealand white rabbits

Dylan Lucich, Shannon John, João Brandão

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

Rabbits are increasingly kept as pets and body temperature has been shown to be a prognostic indicator of survival. Although not yet shown in rabbits, in chinchillas, rectal thermometer depth has a significant impact on the temperature readings (TRs). The hypotheses of this study are that temperature readings (8F) will be significantly greater at a 2cm than a 1cm depth, and less invasive collection sites are as accurate as rectal temperature. For this study, TRs were collected at: rectal depths at 1 and 2 cm (Rc1 and Rc2), axillary (Ax), inguinal (Ig), and corneal thermal imaging (Cn) regions, and a subcutaneous microchip (SMc, IPTT-300, Plexx BV). For each round of TRs, the order of collection at these sites were randomized. Preliminary results have shown that rectal TRs taken at 1 cm averaged 3.428 (P-value < 0.001) lower than those taken at 2cms. When using the TRs of the SMc to verify the accuracy at the other sites, it was found that the Cn re- gion average deviation was 5.388 further from SMc than Rc2 (P-value < 0.001), the Ig region average deviation was 0.538 closer to the SMc than Rc2 (P-value < 0.001), and the Ax region average deviation was 0.588 closer to the SMc than Rc2 (P-value < 0.001). In conclusion, these results show there’s a significant difference in TRs taken between rectal depths of 1 and 2cm, meaning clinically, the thermometer should be inserted at least 2 cm when taking rectal temperatures. The re- sults also show that it wouldn’t be advised to use the Cn region to collect temperature in rabbits, but the Ax and Ig region temperatures are significantly closer to a rabbit’s body temperature based on an SMc than the rectal readings, and could be used as a less invasive means to collect a rabbit’s temperature.

Research Grant: Oklahoma State University Dr. Kristie Plunkett Exotic Animal Fund. Student Support: Oklahoma State University College of Veterinary Medicine Summer Research Training Program.

2021 National Veterinary Scholars Symposium • 217 Targeting the MARCKS/AXL axis to combat pro-tumor macrophage polarization in cancer progression

Madison E. Luker, Chih-Wei Chu, Ching-Hsien Chen

School of Veterinary Medicine (Luker) and Department of Internal Medicine (Chu, Chen), University of California, Davis, CA

The Gas6/AXL signaling pathway has been demonstrated to be upregulated in numerous cancer types, contributing towards an immunosuppressive tumor microenvironment through inducing M2 macrophage polarization. Although the role of AXL in tumor growth and immune regulation has been extensively studied, how AXL activity is regulated re- mains enigmatic. We previously demonstrated that a PIP2-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), is highly expressed in aggressive lung cancer and forms a molecular complex with AXL. This study is aimed at elucidating the mechanism of MARCKS in modulating AXL activity. MARCKS in human and canine tumors has been well documented as their phosphorylation site domain (PSD) is homologous. Given the similarity of the MARCKS PSD in humans and dogs, our current work has utilized human lung cancer cell lines to model canine cancer. Herein, we found that phospho-AXL level is upregulated in response to MARCKS knockdown. Data from immunofluorescence further confirmed co-localization of AXL and PIP2 in MARCKS-knockdown cancer cells, indicating a novel molecular signaling pathway through which MARCKS interferes with AXL activity via regulating PIP2 availability. Treatment with MPS, targeting the MARCKS PSD, was demonstrated to downregulate activity of AXL and its downstream pathways. Notably, a decreased proportion of pro-tumor M2 macrophages was observed upon co-culturing macrophages with MPS-treated cancer cells. Our results suggest the contribution of the MARCKS/AXL complex in regulating immune responses and provides a unique strategy for inhibiting AXL activity through promoting MARCKS sequestration of PIP2, serving as a potential therapeutic strategy for advanced cancer.

Research Grant: None Student Support: UC Davis SVM Students Training in Advanced Research Program and NIH grant: 5T35OD010956-22

DifferentiatingStaphylococcus schleiferi subspecies with MALDI-TOF, biochemical testing and susceptibility

Rohini Lumba, Shannara Welch, Sindi Flores, Logan Ardrey, Jing Wu, Sara D. Lawhon

Department of Clinical Microbiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas

Staphylococcus schleiferi is a potentially zoonotic pathogen that colonizes the skin and mucosal surfaces of animals. Cur- rently there are two subspecies of S. schleiferi; S. schleiferi subsp schleiferi and S. schleiferi subsp coagulans. The sub- species are genetically similar, but some studies suggest there are two separate species. Even with the grey area over the official classification S.of schleiferi, there is methicillin resistance present in some S. schleiferi. The aim of this study was to differS. schleiferi subsp schleiferi and S. schleiferi subsp coagulans using MALDI-TOF and biochemical testing, and to determine the prevalence of mupirocin resistance in isolates from dogs presented to a veterinary teaching hospital. Canine S. schleiferi isolates were collected from Texas A&M Veterinary Medical Teaching Hospital from June 1, 2009 to June 1, 2020 and identified using MALDI-TOF and biochemical tests. Isolates were tested for mupirocin resistance and antimi- crobial susceptibility. A total of 100 isolates of S. schleiferi were identified but MALDI-TOF could not differ between the subspecies. The coagulase test is expected to show a majority of the 100 isolates are coagulase positive. The biochemical tests are not expected to provide definitive identification, as the two subspecies have variable reactivity. Two out of 100 S. schleiferi isolates were positive for mupirocin resistance. Overall, the findings demonstrated that additional studies such as whole genome sequencing or gene targeted qt-PCR are needed for differentiating the subspecies. This study also identified mupirocin resistance in S. schleiferi, which has clinical and potential zoonotic implications for antimicrobial treatment going forward.

Research Grant: This work was supported by funds from FDA grant U18FD006171, the Texas A&M Clinical Science and Translational Research (CSTR) Institute, and departmental funds to S. D. Lawhon. Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine and Biomedical Sciences

218 • 2021 National Veterinary Scholars Symposium Effect of chicken IgY on Staphylococcus aureus growth in vitro

Dominique Lyles, Jatna Rivas, Adu-Addai Benjamin

Department of Biomedical Science, College of Veterinary Medicine, Tuskegee University, Tuskegee Institute, Alabama

Mastitis is one of the most common health problems affecting the animal industry. A major causative agent, Staphylococ- cus aureus (SA) is often treated with antibiotics. Staphylococci rapidly develop antimicrobial resistance. Recent studies have shown IgY as a potential alternative to antibiotics to mitigate emerging antimicrobial resistance. This study aims at determining the effect of IgY concentration on SA inhibition in relation to the duration of incubation. A 2-fold serial dilution of SA was made in nutrient broth. IgY from Rhode Island Red (RIR) chicken eggs was purified using the water dilution method, and standardized to a concentration of 1 mg/mL in PBS; commercial chicken anti-SpA IgY from Exalpha (EA) was used for comparison. Two tubes per replication each containing SA (7.5 x 10^3 CFU) and nutrient broth were used. The IgY treatments were given as follows: no IgY, 5 IgY mg/mL, 25 IgY mg/mL, and 125 IgY mg/mL. Ten mL of inoculum were used in plating. The plates were set in duplicates and incubated at 378C for 24 hours, and then CFU was determined. Significant inhibition of SA was noted in EA IgY concentration of 5m g/ml, 25 IgY mg/mL, and 125 IgY mg/ mL. Significance in RIR IgY was seen in concentration of 5 IgY mg/mL and 25 IgY mg/mL. To determine the inhibition in relation to duration of incubation, the above-mentioned tubes were incubated for 4, 8 and 12 hours, respectively after which were plated on tryptic soy agar for 24 hours. The results indicated both EA and RIR IgY can significantly inhibit SA growth in vitro. Overall, IgY inhibits SA; however further studies are required to research into duration and concentration interaction.

Research Grant: The Health and Human Services Center of Excellence (HHS COE) Veterinary Scholar Program (COE-VSP) Student Support: Tuskegee Veterinary Scholars Program

Evaluation of Protein X as a potential urine biomarker for MPGN in dogs with proteinuric CKD

Crystal X. Ma, Jessica A. Hokamp

Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio

Membranoproliferative glomerulonephritis (MPGN), an immune complex-mediated glomerulopathy, is a common cause of proteinuric chronic kidney disease in dogs. Currently, renal biopsy is required for definitive diagnosis of MPGN to di- rect immunosuppressive therapy. Considering that renal biopsy is surgically invasive and costly, noninvasive and econom- ical methods of MPGN diagnosis are needed. Preliminary data from liquid chromatography tandem mass spectrometry (LC-MS/MS) of canine urine supernatant revealed significantly increased peptides of Protein X from dogs with MPGN/ mixed MPGN (n = 10) versus non-MPGN (n = 15) (p < 0.05). The aim of this retrospective, case-control study is to eval- uate urine Protein X as a potential biomarker for diagnosis of MPGN in proteinuric dogs using quantitative ELISAs and qualitative Western Blot (WB) in correlation with LC-MS/MS data. Human Protein X Antibody was optimized for WB detection of canine urine Protein X. Two commercially available ELISAs (specific for human or canine Protein X) were evaluated to determine their suitability for quantification of canine urine Protein X by correlating results with LC-MS/MS and WB. Urine supernatant samples assessed by the above methods were from proteinuric dogs received by the Interna- tional Veterinary Renal Pathology Service. WB results showed that presence of Protein X in urine of MPGN dogs (60%, n = 10) was significantly higher than that of non-MPGN, proteinuric dogs (13%, n = 15) (Fisher’s exact test, P = 0.028). Further evaluation of WB with a larger sample size is underway to establish higher certainty of results. An ELISA specific for human Protein X did not cross-react with canine urine samples; evaluation of a canine-specific ELISA is forthcoming.

Research Grant: Renal Matched Samples Repository Funds Student Support: NIH T35 Training Grant T35OD010977

2021 National Veterinary Scholars Symposium • 219 Assessment of repeatability of preoperative planning for the tibial tuberosity advancement procedure

Zoe MacKenzie, James Dundas

Department of Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island

Cranial cruciate ligament (CrCL) tears are the most common reason for hindlimb lameness and pain in canine patients. Many procedures have been developed to address this problem, including the Tibial Tuberosity Advancement (TTA) pro- cedure. Preoperative planning for the TTA procedure relies largely on the observer’s ability to identify specific radiograph- ic landmarks and make measurements, which may vary among individual observers. Our pilot study aims to investigate the learning curve required to develop competency in making these preoperative measurements. Radiographs underwent preoperative planning measurements by two individuals of different levels of education (second-year veterinary student and a board-certified veterinary surgeon). After the first round of measurements, the student had another training session, and the process was repeated. The results were analyzed for inter- and intraobserver differences. The concordance cor- relation coefficient increased from 0.79 to 0.97, indicating improved agreement between the two observers during round two. Further, the mean coefficient of variation was close to zero and improved during round two, indicating less variation among the observers. Overall, preliminary results show that the student’s ability to recognize radiographic landmarks and make preoperative measurements significantly improved following the second training session.

Research Grant: Veterinary Student Research Award, Boehringer Ingelheim Veterinary Scholar Student Support: None

Antimicrobial Resistance of Pathogens in Lactating Dairy Cattle

Lauren E. MacNeil, Mariana Fonseca, Javier Sanchez, and Luke C. Heider

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island

Antimicrobial resistance (AMR) has increased in recent years and is posing a concern to human, animal, and environ- mental health. Mastitis is one of the main reasons for antimicrobial use (AMU) and is the most significant contributor to reduced milk quality in the Canadian dairy industry. We hypothesize that farms with more AMU and farms with free-stall management systems will have higher proportions of AMR in milk pathogens. Therefore, the objective of this study was to describe the relative proportion of pathogens resistant to frequently used antimicrobials (AM) during the first month after calving. This was attained by determining AMR in pathogens isolated from composite milk samples collected during the first month of lactation. AMU was acquired using veterinary clinic dispensing records and articulated as antimicrobi- al drug use rate (ADUR), expressed as doses per 100 cows per year as described by Saini et al. (2012). Bacterial strains were identified with Columbia agar with 5% sheep blood culture plates, Gram stains, and MALDI-ToF mass spectrom- etry. Antimicrobial susceptibility was determined using the Sensititre microdilution system and mastitis plate format CMV1AMAF. From July to December 2020, a total of 133 milk samples were obtained from 28 farms in Prince Edward Island (18 free-stall; 10 tie-stall) within two weeks after calving. The average parity was 2.6 (range: 1-8). Farms used on average 38.5 daily doses per 100 cows per year and ranged from 3.02 to 76.8. A total of 62 samples reported a positive culture including: E. coli (3.23%), Staph. aureus (4.46%), and coagulase-negative staphylococci (CNS)(67.75%). Propor- tions of resistant bacteria will be compared across AMU categories and farm characteristics.

Research Grant: Research Grant: Dairy Farmers of Canada (Dairy Research Cluster 3); University of Prince Edward Island; Public Health Agency of Canada Student Support: Student Support: Atlantic Veterinary College Summer Research and Leadership Program

220 • 2021 National Veterinary Scholars Symposium The effect of reproductive status on in vitro embryo production in bison

Ella G. Macquisten, Miranda L. Zwiefelhofer, Eric M. Zwiefelhofer, Gregg P. Adams

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan

The threatened status of bison has resulted in the creation of the bison genome biobank. Previously, cumulus-oocyte-com- plexes (COC) have been collected from mature, non-pregnant bison for the purpose of in vitro embryo production (IVP). The objective was to determine the feasibility of COC collection for IVP from bison of different reproductive statuses. Transvaginal ultrasound-guided follicle aspiration was done on non-pregnant, pregnant, and pre-pubertal bison (n = 4/ group) at a random stage of the follicular wave and again five days later (synchronized stage) in two replicates 30 days apart (~90 and 120 days gestation). COC were matured in vitro for 25-28 h, then co-incubated with sperm (IVF). Cleav- age and embryo development was monitored at 3 and 7-10 days after IVF. Data were analyzed by ANOVA and Glimmix. Compared to random collection, the synchronized collection resulted in a greater number of follicles aspirated (20.863.9, 13.062.7; P = 0.05), greater cleavage rates (109/152 [72%], 54/108 [50%]; P = 0.002) and embryo production (11/152 [7%], 1/108 [1%]; P = 0.05). The number of $3 mm follicles available was similar among pre-pubertal, pregnant and non-pregnant groups (replicates combined: 37.364.8, 26.062.7, 22.964.7; P = 0.08), but a greater number of COC were recovered from pre-pubertal bison (22.063.3, 7.361.3, 12.463.1; P = 0.008). The follicular aspiration rate was lower in the pregnant group than in the non-pregnant and pre-pubertal groups (81/182 [45%], 159/183 [87%], 278/298 [93%]; P < 0.0001). Embryo production rate was similar among pregnant, non-pregnant and pre-pubertal groups (5/162 [3%], 3/46 [7%], 8/90 [9%]; P = 0.17). In conclusion, COC collection and IVP is feasible in bison of different reproductive statuses.

Research Grant: Natural Sciences and Engineering Research Council of Canada Student Support: Boehringer Ingelheim Veterinary Scholarship and Western College of Veterinary Medicine

Evaluation of chitosan alginate nanoparticles for camptothecin delivery to colorectal cancer cells

Elsie Madison, Dilip Reddy Gunturu, and Temesgen Samuel

Department of Biomedical Sciences (Madison, Gunturu); Pathobiology (Samuel), College of Veterinary Medicine, Tuske- gee University, AL

Colorectal cancer is a cancer that affects the colon and rectum. It is also known as bowel cancer, colon cancer, or rectal cancer. In men, colorectal cancer is the third most prevalent cancer, while in women, it is the second most common cancer. Currently, there are anticancer drugs called topoisomerase inhibitors that induces apoptosis and used in many cancers in- cluding colon cancer. Camptothecin (CPT) is the topoisomerase inhibitor that will be used in this experiment. Historically, anticancer drugs used alone are not always sufficient, because the body can elicit harsh side effects. Using the nanoparticle drug delivery system has become the new era of cancer research. From different studies, it is evident that the formula- tion of anticancer drugs into nanoparticles is known to increase the effectiveness.We have synthesized chitosan-alginate nanoparticles loaded with CPT to study on colorectal cancer cells. The benefits of using chitosan-alginate nanoparticles include biodegradability, improved drug bioavailability in the system, muco-adhesive properties, and enhanced safety. The nanoparticles will be characterized for morphological and drug-loading properties. Furthermore, the nanoparticles will be evaluated in vitro in colorectal cancer cells. Future studies will be performed based on the results obtained from present studies.

Research Grant: HRSA #D34HP00001, TU-CBR/RCMI #U54MD007585, and U54CA118623 Student Support: HRSA #D34HP00001

2021 National Veterinary Scholars Symposium • 221 Dermatologic and respiratory health among pets from in inner-city homes with human asthma

Jesse M. Maestas, Kaitlin B. Waite, Meghan F. Davis

1. College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA; 2. Department of Environmental Health and Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 3. Department of Molecular and Comparative Pathobiology, Johns Hopkins Bloomberg School of Medicine, Baltimore, MD

Like inner city children, pets may have a higher disease burden of dermatologic and respiratory conditions due to allergen exposure and/or reduced access to veterinary care. This study aimed to quantify these potential disease factors. Pets were enrolled from two studies of inner-city people with asthma (INHALE, n = 18 adults, n = 8 dogs, n = 9 cats; and ECATCh, n = 129 children, n = 61 dogs, n = 76 cats) with follow up visits every 3 months for 3- and 6-months, respectively. In ECATCh, households were randomized to allergen-reduction intervention after the 1st visit. Owners answered questions and pets received visual health exams at each visit. Symptoms typical of dermatologic (e.g. rash, deep pyoderma) or respi- ratory (e.g. nasal discharge) diseases were grouped to calculate incidence rates and compare odds of symptom prevalence between dogs and cats. Prevalence and incidence of veterinary care was also quantified for the prior year and during the study. Prevalence of any dermatologic disease in dogs and cats at baseline was 6.12% and 0%, with incidence rates of 0.003/dog-month and 0.009/cat-month. Prevalence of any respiratory symptoms in dogs and cats at baseline was 10.20% and 5.26%, with incidence rates of 0.002/dog-month and 0.009/cat-month. Dogs were more likely than cats to have prev- alent dermatologic disease at baseline (OR = 7.37 95%CI = 0.00-150.88, P = 0.28). Prevalence of any vet visit in the prior year was 64% for dogs and 24% for cats, with incidence rates for vet visits at 0.004/dog-month and 0.003/cat-month. Of the animals that presented with disease at baseline, < 7% had been to the vet in the past year. Characterizing the burden of these diseases informs veterinary care delivery in an often-underrepresented community.

Research Grant: American Kennel Club's Canine Health Foundation (CHF02241); NIH NIEHS (R01ES023447 to E.C.M. and P50ES018176 to M.C.M.); NIH NIAID (K24AI114769 to E.C.M.); and NIH OD (K01OD019918 to M.F.D.). Student Support: 5T35OD024982-03- Training Veterinary Students for Careers in Biomedical Research

Determining sub-clinical effects of lead exposure on southern California turkey vultures (Cathartes aura) Jesse M. Maestas, Miguel D. Saggese College of Veterinary Medicine, Western University of Health Sciences, Pomona, California Lead toxicity is a severe health problem affecting humans and animals alike. Lead toxicity has had substantial impacts on populations of waterfowl and the California condor (Gymnogyps californianus) was nearly driven to extinction and is facing substantial recovery challenges in part due to the ingestion of lead ammunition in animal carcasses. Vulture species around the world are also similarly negatively impacted via lead ingestion. Turkey vultures (Cathartes aura) have been the subject of research for lead toxicity and as sentinels for the presence of lead spent ammunition in Southern California. The pathophysiological mechanisms of lead toxicity are relatively well conserved across humans and animals resulting in similar clinical signs when exposed to high lead levels. However, research into the subclinical effects of lead exposure in turkey vultures are lacking. Assessing the sub-clinical effects of lead poisoning may reveal previously unknown levels of immune depression with increasing parasite burden. These effects may impact vulture’s social behavior, reproduction, and other aspects of their ecology. Therefore, in this study we hypothesize that Turkey Vultures exposed to lead would show changes in their hematology, clinical biochemistry and hemoparasite burden. Our study plans to trap wild turkey vultures, conduct physical exams, and obtain blood samples. Blood samples will be analyzed and quantified for multiple parameters including lead, to determine whether it has an influence on hematological, clinical biochemical and/or hemoparasite prev- alence. Our study will provide the starting point to understand the sublethal effects of lead in this species. Research Grant: Partially covered by a Wild Bird Health Grant of the Association of Avian Veterinarians and CVM Matching Funds Student Support: Western University of Health Sciences Veterinary Research Scholars Program

222 • 2021 National Veterinary Scholars Symposium Generation and characterization of a knock out Cyp2d gene cluster rat model

Maria J. Maggio, Daniel J. Davis, Elizabeth C. Bryda (sponsor), Kristin N. Grimsrud

School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (Maggio); Animal Modeling Core (Davis, Bryda), Rat Resource and Research Center (Bryda), College of Veterinary Medicine, University of Missouri, Columbia, Missouri; School of Medicine, University of California, Davis, California (Grimsrud)

Each year in the United States, there are 20,000 patients with significant burns involving at least 25% of their body surface. Opioids are the primary pain management drugs for these patients, even though opioid pharmacokinetic (PK) alterations leading to pain management failures may occur due to burn hypermetabolism and genetic polymorphisms. Cytochrome P450 (CYP), a major class of liver enzymes, are known to be responsible for the metabolism of opioid drugs. Our long term goal is to facilitate studies to better understand the relationship between the various human CYP genes and opioid metabolism in the context of burn treatment. As a first step, we have generated aCyp2d knock out rat model. Using CRISPR/Cas9 genome editing technology, the entire rat Cyp2d1-5 gene cluster was excised. These rat genes are orthol- ogous to several human CYP genes that are important for opioid metabolism. Genetic characterization was performed to confirm the loss of the Cyp2d gene cluster and the stock has been expanded to generate animals homozygous for the gene cluster knock out. With this new model, future studies will test how the efficacy of fentanyl, a commonly used opiate for burn pain management is altered between wild type and homozygous knock out rats using observational sedation scores based on activity level, toe pinch reflex, and respiratory rates, and pain scores measured by a digital paw pressure instru- ment. In addition, this new rat model will serve as the genetic background for future creation of humanized rat models carrying selected human CYP genes to better understand the importance of these genes and their polymorphic variants on opioid metabolism and burn pain management.

Research Grant: NIH 5K01OD026608-02 (Grimsrud); NIH P40 OD011062 (Bryda) Student Support: American Society of Laboratory Animal Practitioners Foundation and IDEXX-BioAnalytics

Assessing PACAP as an EGC stimulant in teleosts

Daniel A. G. Maguire, Shona K. Whyte, Sara L. Purcell, Mark D. Fast

Department of pathology and microbiology, Atlantic veterinary college, University of Prince Edward Island, Charlottetown, PEI

As the scale of aquaculture production continues to increase, the threat of antimicrobial resistance follows a similar trend. In less developed countries with warmer climates, high levels of antibiotic use have left Tilapia particularly vulnerable. One means of combating this is through the use of antimicrobial peptides as a replacement intervention. The neuropeptide pituitary adenylate cyclase-activating polypeptide, or PACAP, exists across species with a multitude of effects within the hosts system. Within teleost species, PACAP serves a role in fighting against bacterial pathogens via the stimulation of the host immune system as well as direct interaction with the bacteria. Host immune stimulation occurs through PACAP inciting expression of proinflammatory cytokines IL-1b, TNFa, and IL-6, as well as upregulating the expression of the PA- CAP receptor VPAC1. One cell that has been seen in high numbers following PACAP immunostimulation are eosinophilic granular cells (ECGs). ECG’s play a similar role to mammalian mast cells in inflammation via degranulation and release of products. For this study, we used duplicate tanks for each treatment, with groups receiving (1) PACAP flush over the gills, (2) receiving PACAP flush through the naris, (3) receiving a PBS flush over the gills, and finally (4) receiving a PBS flush through the naris. Fish were then be sampled over 6, 24, 72 hours and 7 days.At each sampling event, samples for histological and gene expression analysis were collected. The impact of treatment on EGC accumulation and degranula- tion were assessed and will be discussed. Future work will evaluate the molecular signals associated with PACAP admin- istration over short and long-term time frames.

Research Grant: International Development Research Centre- Innovetamr. Titled, 'Developing pituitary adenylate cyclase-activating polypeptide into a treatment for microbial infections in aquaculture.' Student Support: AVC Veterinary Summer Research Award, Boehringer Ingelheim Veterinary Scholar

2021 National Veterinary Scholars Symposium • 223 Interrogating the role of Src inhibition in DMD-mutant osteosarcoma

Maria Maguire, Heather Gardner

Tufts Cummings School of Veterinary Medicine

Large deletions in DMD, the gene that encodes for dystrophin, are present in 50% of canine primary osteosarcoma (OS). Preliminary evidence indicates that CRISPR-Cas9 dystrophin deletion increases phosphorylation of the dystrophin binding partner, b-dystroglycan. The Src kinase facilitates phosphorylation (activation) of b-dystroglycan. It is therefore hypothesized that truncated dystrophin isoforms will show increased sensitivity to Src inhibitors. The Abrams, OSCA-40 and D17 cell lines were used for all experiments. Cells were plated in triplicate in 96-well plates and treated with increas- ing concentrations of the Src inhibitor (AZM475271) for 72 hours. Cells were fixed and stained with sulforhodamine B (SRB). Proliferation was calculated as a percent of untreated controls. Protein was isolated from cell lines using complete lysis buffer and quantified using a Bradford assay. Western blotting was performed using standard techniques. Membranes were probed for Dystrophin (Leica NCLDYS2). Expression of the truncated Dp71 dystrophin isoform is present in canine OS cell lines, as determined by western blotting. Preliminary data indicates that exposure of canine OS cells to increasing concentrations of AZM475271 did not significantly alter cell proliferation. Evaluation of AZM475271 activity in cells in which dystrophin is deleted is underway. In conclusion, AZM475271 does not alter cell proliferation in canine OS cell lines. Confirmation of Src inhibition and evaluation of cellular invasion in cells exposed toAZM475271 is ongoing.

Research Grant: American Kennel Club Canine Health Fund Student Support: Tufts Cummings School of Veterinary Medicine Clinical Trials Office

A new 3-D musculoskeletal model of the chimpanzee forearm, wrist, and hand for studies of knuckle-walking

Nicole Mahmood, Matthew O’Neill

College of Veterinary Medicine (Mahmood), Department of Anatomy, College of Graduate Studies (O’Neill), Midwestern University, Glendale, AZ

Wild chimpanzees use knuckle-walking as their most common form of overground travel, but the musculotendon me- chanics and skeletal joint loading associated with this unique form of quadrupedalism are still not well understood. A three-dimensional musculoskeletal model of the chimpanzee wrist and hand would permit integration of existing 3-D motion, force, and muscle activation data, facilitating a more comprehensive understanding of knuckle-walking. Herein, we created a generic 3-D musculoskeletal model of the arm and hand of an adult chimpanzee using SIMM software. To mobilize the forearm, wrist, and hand, an axis for pronation-supination was defined; flexion-extension and abduction-ad- duction axes were added to the wrist and metacarpophalangeal (MCP) joints of digits 2-5, while the proximal interphalan- geal joints (PIP) were mobilized for flexion-extension. For digit 1, abduction-adduction was included at the carpometacar- pal joint, with flexion-extension at the MCP joint. Nineteen muscle-tendon units that move the forearm, wrist, and digits were represented in the model by twenty-nine elements. Muscle moment arms will be compared to cadaveric moment arm measurements to assess the robustness of the model construction. This 3-D model will be used to facilitate detailed com- parisons of knuckle-walking wrist and hand biomechanics.

Research Grant: NSF BCS 0935327 and 2018436 Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

224 • 2021 National Veterinary Scholars Symposium Evolution of Staphylococcal antibiotic resistance systems across gram-positive bacteria

Elliot Majlessi, Vignesh Sridhar, Neal Hammer, Janani Ravi

Lyman Briggs College, Michigan State University (Majlessi); Microbiology & Molecular Genetics, College of Natural Science, Michigan State University (Hammer); Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI (Majlessi, Sridhar, Ravi)

Bacteria continuously evolve and adapt to their environment - when pathogens encounter antibiotics administered to the host, they evolve further, giving rise to antibiotic resistance. The emergence of antibiotic resistance is a multi-disciplinary issue with several interconnected human, animal, and environmental factors as the major driving force. Excessive/im- proper use of antibiotics leads to the global spread of resistant bacteria, emphasizing the role of a “One Health” approach to better understand and limit the threat of antimicrobial resistance. Here, we apply a computational approach to uncover the evolution of Staphylococcal antibiotic resistance systems across Gram-positive bacteria. This approach is based on a novel computational framework for characterizing bacterial genomes and operons using comparative genomics, molecular evolution, and phylogeny. Specifically, we are studying the conservation and modularity of Staphylococcal envelope stress response systems (ESRs) involved in antibiotic resistance by mapping the constituent domains in terms of their i) ances- try, ii) lineage- and environment-specific variations, and iii) diverse specialized protein/operon functions. Our findings will establish the nature and course of evolution of Staphylococcal antibiotic resistance systems within key Gram-positive lineages.

Research Grant: NSF-funded BEACON Center for the Study of Evolution in Action. Student Support: BRUSH Summer Research Program, Office of Undergraduate Research, Michigan State University

Comparison of moisture analysis methods using canned dog food

Seryna N. Mamane, Adam W. Stern

Department of Comparative, Diagnostic, and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL

Water is arguably the most essential nutrient required to maintain an organism. Food consumption is one-way animals can contribute to their water intake. Moisture, the measured water content in animal food, is variable and is stated on the nu- trition label in the form of a guaranteed maximum percent composition of the food. Accurate reported moisture levels are necessary to ensure other nutrient components in an animal’s diet are sufficient and balanced. This study aims to compare multiple loss-on-drying methods for determining moisture content in canned dog food products and compare the measured moisture percentage to the label’s guaranteed moisture maximum. Twenty different canned dog food products were ana- lyzed using a convectional lab oven (CLO), infrared moisture analyzer (IMA) and microwave moisture analyzer (MMA). The results indicated that 90% of the trials showed no significant difference between the IMA and CLO and between the MMA and CLO. There was no significant difference between all three methods in 55% of the trials and 65% of the trials showed no significant difference between the IMA and MMA. When a significant difference was detected, the differences were less than 1%. On average, the moisture content measured by the CLO method was 3.11% less than the reported guar- anteed moisture maximum. Overall, there was good agreement between the CLO and both the IMA and MMA methods supporting their use for the measurement of moisture with canned dog food.

Research Grant: UF Foundation Richard R. Weiss Fund Student Support: Florida Veterinary Medical Association

2021 National Veterinary Scholars Symposium • 225 Dynamic insulin response to acclimation of a high carbohydrate diet in normal and insulin dysregulated horses

Hailey Maresca-Fichter, Stephanie Valberg, Deborah Velez-Irizarry, Callum George Donnelly, Carrie Finno, Jane Manfredi

Department of Pathobiology and Diagnostic Investigation (Maresca-Fichter, Manfredi), Large Animal Clinical Sciences (Valberg, Velez-Irizarry), College of Veterinary Medicine, Michigan State University, East Lansing, MI; Center for Equine Health (Donnelly, Finno) University of California, Davis, CA

Determining the relationship between acclimation to high carbohydrate diets and insulin dynamics in horses is clinically relevant due to the prevalence of metabolic disorders and frequency of dietary supplementation with carbohydrate rich grains. While it is recommended to generally avoid high starch diets, some previous work has shown that acclimating to carbohydrate rich diets improves insulin sensitivity. The aim of this study was to determine if there is an association be- tween acclimation over time to high dietary carbohydrate load and improved insulin dynamics in normal and insulin dys- regulated (ID) horses. Seventeen adult Quarter Horse mares were determined to be ID (n = 9) or metabolically normal (n = 8) by a prior (0.15 ml/kg BW Karo Light Syrup) oral sugar test (OST). Horses were then fed a hay and sweet feed grain diet (8 gm/kg BW of 25% NSC concentrate) for 4 weeks. A combined glucose insulin test (CGIT) (150 mg/kg glucose IV immediately followed by 0.1 IU/kg insulin IV) was performed followed by a high dose (0.45 ml/kg BW Karo Light Syrup) OST after a 4 day washout period to evaluate tissue level insulin sensitivity and incretin hormone responses. Data was analyzed using McNemar’s Chi-squared test (significant at P < 0.05). After dietary acclimation, 10/17 (58.8%) horses were ID based on CGIT results and 9/17 (52.9%) were ID based on OST results. Overall after acclimation, 5 (29.5%) remained metabolically normal on both tests while 12 (68.5%) were ID on one or more. These findings show no insulin sensitizing benefit to horses already afflicted with ID and do not support acclimation to high carbohydrate diets as doing so can potentially induce ID in previously metabolically normal horses.

Research Grant: CVM Endowed Research Funds, Michigan State University Student Support: NIH Grant 5T35OD016477-19 to Michigan State University

Phylogenomic assessment of feline herpesvirus type 1 (FHV-1) in cheetahs (Acinonyx jubatus)

Morgan E. Marino, Melanie A. Mironovich, Nikole E. Ineck, Renee T. Carter, Andrew C. Lewin

Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Feline herpesvirus type 1 (FHV-1) is endemic in captive cheetahs and can cause devastating disease. Modified live vac- cines (MLV) for FHV-1, which are intended for use in domestic cats, are commonly used as a preventative measure in cheetah populations. The MLV has been anecdotally linked to FHV-like disease when used in cheetahs; however, there has been limited prior investigation into this relationship. Ten FHV-1 isolates were collected from MLV vaccinated and un- vaccinated captive cheetahs throughout the USA and one isolate was collected from a MLV commonly used in cheetahs. Viral DNA was extracted for full genome sequencing by Illumina MiSeq. The sequences along with data from previously sequenced domestic cat FHV-1 isolates underwent phylogenomic and recombinational analysis using Geneious Prime, RDP, IQ-Tree, Splitstree and MEGAX. The results showed that FHV-1 shed by MLV vaccinated cheetahs are almost identical to the MLV, with few variants in the coding regions between genomes. Eight cheetah isolates and the MLV were grouped in a clade with other isolates previously sequenced from domestic cats in the USA. The remaining two cheetah isolates (MM-5; MM-6) were not associated with an established clade but were genetically similar to Australian domestic cat FHV-1 isolates. The likely ancestral origin of these two isolates is recombination events involving both Australian do- mestic cat isolates and isolates obtained from other cheetah host animals. The data strongly suggests that the MLV causes disease and viral shedding in certain cheetahs when used outside of its intended host. The results also represent evidence of horizontal and possible trans-boundary transmission of virus between domestic and wild cat species.

Research Grant: None Student Support: Morris Animal Foundation and Louisiana State University School of Veterinary Medicine

226 • 2021 National Veterinary Scholars Symposium Inflammation induced by gram-positive peptidoglycan may increase apnea frequency in newborn rats

Nicholas Marino, Tracy Baker, Jyoti Watters, Stephen Johnson

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI

Infections in dogs and cats, particularly bacterial, are the second most important cause of newborn mortality after compli- cations during parturition. With risk of infection highest in the first week of life, such inflammation is especially impactful in newborns because neonates are less able to fight off infections. Inflammation can cause life-threatening disruption of breathing and neurodevelopmental disabilities. For example, exposure of neonatal rats to the Gram-negative inflammatory mimetic lipopolysaccharide decreases breathing frequency and is thought to increase breath irregularity and apneas, most likely due to production of inflammatory cytokines and prostaglandins. However, little is known regarding the impact of neonatal inflammation due to a Gram-positive bacterial infection on breathing.We addressed this question by using pleth- ysmography in awake, freely moving neonatal rats (P6-P8) to quantify breathing following injection with saline or the Gram-positive inflammatory mimetic peptidoglycan (10 mg/kg). Preliminary results suggest that peptidoglycan increases apnea frequency as well as breath irregularity. Thus, gram-positive infections would seem to mimic gram-negative infec- tions in their impact on the respiratory system. Therefore, it appears an expanded range of infections induces respiratory deficits, with these breathing disruptions altering blood gas homeostasis and potentially causing neurodevelopmental disorders. Consequently, prompt control of inflammation in response to newborn infections is warranted.

Research Grant: NIH R01 HL142752 Student Support: NIH T35 OD011078

Molecular components of oocyst formation in Cryptosporidium parvum

Ixzacil Marquez, Derek Pinto, Josh A. Lain, Maria G. Nava, and Sumiti Vinayak

College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum is a protozoan parasite that causes watery diarrhea and mortality in agricultural animals and young children following ingestion of oocysts. The thick wall of the C. parvum oocyst allows it to survive harsh environ- ments and resist chemical disinfectants. The wall consists of an outer glycocalyx, rigid lipid bilayer, and an inner layer. The glycocalyx is made of glycoproteins while the innermost layer is composed of Cryptosporidium oocyst wall proteins (COWPs). Electron microscopy and mass spectrometry showed that sporozoites within the oocyst tether to the inner layer and that these tethers are composed of polysaccharides. However, the molecular machinery that synthesizes these polysac- charides has not been characterized. We identified two genes in theC. parvum genome that encode for enzymes required for the synthesis of these polysaccharides and hypothesize they provide structural stability to the oocyst wall for produc- tive transmission of infection. To understand the localization of these proteins and determine their essentiality for parasite survival, we utilized CRISPR/Cas9 genome editing and the C. parvum immunocompromised mouse infection model. We generated targeted Cas9/guide RNA constructs and linear repair DNA templates consisting of a luciferase reporter and a drug selection marker to select for resistant transgenic parasites in the animal model. Experiments are focused on creating these transgenic parasite strains and determining localization as well as essentiality of these enzymes for viable oocyst formation and transmission. Understanding oocyst structure provides unique targets for the development of anti-crypto- sporidial drugs.

Research Grant: University of Illinois College of Veterinary Medicine Student Support: Office of the Director, NIH, T35 OD011145

2021 National Veterinary Scholars Symposium • 227 Diet alters systemic biomarkers associated with the obese equine phenotype: diagnostic and therapeutic value

Jordan M. Marsh, Giovana Di Donato Catandi, and Elaine M. Carnevale

Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado

Equine obesity is increasingly prevalent in the United States, and health risks associated with adiposity are often under- estimated by owners, especially as related to insulin resistance and laminitis. The aims of this study are to elucidate the relationship between phenotypic measures of adiposity and previously studied systemic biomarkers for inflammation and oxidative stress, as well as for novel cellular function assays and to determine the extent that diet supplements can affect cellular functions. We hypothesize that increasing adiposity will result in changes in observable measurements that corre- spond with altered systemic biomarkers; furthermore, dietary supplementation with complex nutrients, antioxidants, and targeted metabolic support will mitigate some of the systemic effects of adiposity. Our study will include three groups of mares: normal weight (n = 6), obese (n = 8), and obese (n = 8) fed dietary supplements that have been shown to lower sys- temic fatty acids, reduce inflammatory markers, and increase muscle metabolic activity in normal-weight mares (unpub- lished data). Observable indications of obesity, including body condition score, cresty neck score, and subcutaneous fat measurements, will be obtained. Initially, the observations will be compared with proxies for insulin resistance. Additional samples will be collected before and after mares have been on their respective diets for 6 to 8 weeks. Cellular biomarkers will include substrate preferences, reactive oxygen species production, and metabolic function. Findings from this study will improve our ability to diagnose systemic alterations in metabolism based on observational criteria and to assess the therapeutic use of nutrients for the obese mare.

Research Grant: Cecil and Irene Hylton Foundation, USDA-NIFA Student Support: USDA-NIFA Animal Health and Disease Fellow

Functional anatomy of the snow leopard (Panthera uncia) tail

Shelby Marsh, Kelsey Reames, Heather F. Smith, K. E. Beth Townsend, Brent Adrian, and Ari Grossman

Department of Anatomy (Smith, Townsend, Adrian, Grossman), College of Veterinary Medicine (Marsh, Reames, Grossman), Midwestern University, Glendale, AZ

The snow leopard (Panthera uncia) inhabits cold, mountainous environments, with sheer cliff faces often steeped in snow. As part of a larger project, we dissected the tail of the snow leopard in order to describe its anatomy and detail adaptations to climate, locomotion, and posture. Particularly, we sought to understand how P. uncia utilizes its tail for balance and warmth. Fat thickness was greatest in the proximal third of the tail, which may aid to keep P. uncia warm in their frigid environment. On the dorsal surface, the superficial tendons ofmm. sacrocaudalis dorsalis lateralis (SDL) and sacrocau- dalis medialis (SDM) were covered by a thin fascial retinaculum oriented perpendicular to the long axis of the tendons. A superficial retinaculum held the main tendons together, and deeper formed an aponeurosis. This created a layered effect within the grouping, with superficial, intermediate, and deep tendons coursing over the tail. SDL tendons coursed togeth- er within their respective layered grouping of the deep caudal fascia to eventually join the aponeuroses, then separately dove deep to attach to bone. SDM does not have its own set of robust tendons; tendons of SDL integrated themselves into SDM. This same pattern repeated ventrally within the ventral lateral muscles. The tendons spanned eight caudal verte- brae from the formation point of the tendon to the insertion site onto bone, crossing medially and laterally to imbed into adjacent muscles. This extensive tendinous arrangement permits more fluid motor control of the tail thatP. uncia needs to stabilize itself. Taken together, the muscular adaptations of the tail reveal a complex mechanism of balance and leverage for navigating the diverse alpine environment.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

228 • 2021 National Veterinary Scholars Symposium Incidence of palmar process fractures in the distal phalanx of Arabian foals

Nicole Marter, Fanglong Dong, Babak Faramarzi

College of Veterinary Medicine (Marter, Faramarzi), Graduate College of Biomedical Sciences (Dong), Western University of Health Sciences, Pomona, California

Palmar process fractures are the most common fracture of the distal phalanx in foals under a year of age. Previous studies reporting the frequency of these fractures are not in agreement, ranging from 75-100%. Trends suggest breed and confor- mation may be associated with variability of reported fracture frequencies. The first aim of this study was to determine the incidence of palmar process fractures in Arabian foals under a year of age. The second aim of this study was to examine relationships that may exist between fracture occurrence and foot anatomy. Digital radiographs and photographs of the forefeet of nine Arabian foals were taken every two months. Data collection occurred when foals were approximately two days to twelve months of age. These images were analyzed to determine palmar process fracture incidence and identify 60 anatomical parameters that could be associated with fracture incidence. Repeated measures ANOVA tests were used to assess the effects of predictors such as fractures on the measured parameters. P-values less than 0.05 were considered to be statistically significant. In total, palmar process fractures were diagnosed in six foals. Incidence was found to be 66%. Increased dorsal length of the distal phalanx, distal width of the middle phalanx, medial sesamoid width, and toe angle were significantly associated with fracture occurrence. Incidence of palmar process fractures in this cohort ofArabian foals was lower than those reported in previous studies with foals of other breeds.

Research Grant: Western University of Health Sciences, Agricultural Research Institute at Cal Poly Pomona Student Support: WesternU Part-Time Fellowship

Changes in antigen, antibody, and microfilaria in dogs during Dirofilaria immitis infection and treatment

Brady Martin, Rachel Beam, Kellee Sundstrom, Megan Lineberry, Susan Little

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma

Canine heartworm disease is a prolific, potentially fatal condition in dogs caused byDirofilaria immitis. Diagnosis of heartworm infection in dogs relies on detection of antigen and microfilaria; heartworm antibody tests are not commercial- ly available for dogs. Immune complexes can block detection of antigen, particularly during early infection and treatment; immune complex disruption (ICD) reverses the block. The purpose of this study was to evaluate the degree to which antigen detection may be blocked in dogs during infection with and treatment for D. immitis. Antibody and microfilaria levels were also assessed. Eight dogs were infected with D. immitis and monitored for 15 months. Six dogs were treated with doxycycline, prednisone, and transdermal moxidectin/imidacloprid; three of the dogs also received melarsomine (3-dose protocol). Two dogs remained as controls. Blood and serum samples were collected from each dog every 3-4 days and tested for microfilaria, antigen - both before and after ICD, and antibody. Antigen was first detected in dogs with ICD at 147 DPI (Days Post Infection) and without ICD in all dogs at 161-217 DPI. Antigen levels did not significantly change within 60 DPT-S (Days Post Treatment-Start). Microfilaria first appeared in 7 of 8 dogs at 182-189 DPI and were not detected in treated dogs by 31-59 DPT-S. Antibody to D. immitis was detected in 5 of 8 dogs by 84 DPI, and all dogs were antibody positive by 140 DPI. These results indicate that detection of D. immitis antibody in the dog shows promise as an early indicator of heartworm infection and as a useful diagnostic adjunct for dogs that are antigen positive- but microfilar- ia negative.

Research Grant: Krull-Ewing Endowment Student Support: Oklahoma State University, College of Veterinary Medicine, Office of the Dean

2021 National Veterinary Scholars Symposium • 229 A biomaterial-based delivery system for immunotherapeutic cytokines

Hannah L. Martin, William L. Murphy

Department of Comparative Biomedical Sciences (Martin), Department of Biomedical Engineering (Murphy), Department of Orthopedics and Rehabilitation (Murphy), University of Wisconsin-Madison, Madison, WI

Cytokines have important roles in cell-to-cell signaling pathways, including stimulating an anti-tumor immune response. Clinical utility of cytokine therapy is limited by short half life of the proteins and significant off-target effects. Develop- ment of a delivery system that could localize and prolong the activity of immune-activating cytokines might enhance the safety and efficacy of cytokine therapy as a cancer treatment. Our lab developed mineral-coated microparticles (MCMs) with a unique surface topography that binds proteins and sustains release over time. Here, we applied MCM technology to the delivery of interleukin 15 (IL-15), a pro-inflammatory cytokine with roles inT lymphocyte and natural killer cell ac- tivation. MCMs were prepared by rotating core particles in simulated body fluid containing high amounts of calcium and phosphate. To bind cargo, MCMs were rotated in solutions containing varying concentrations of IL-15. Cytokine-loaded MCMs were either incubated in media to assess IL-15 release over time, or directly added to T cell culture to measure effects on cell proliferation. We demonstrated a prolonged release profile over at least two weeks in simulated body fluid. There was also notable T lymphocyte proliferation on day six after MCM-based delivery of IL-15 in culture. The ability of MCMs to bind and release bioactive IL-15 makes them a possible drug delivery option for future in vivo tumor studies. This could possibly mitigate current issues of rapid clearance and off-target side effects associated with cytokine therapy.

Research Grant: The Shannon Family Partnership Student Support: NIH award TL1TR002375, UW-Madison SVM Dean’s Office and DVM/PhD Program

A genomics approach to characterize white coat color genes in American bison, Bison bison

Carly Martone, Sam Stroupe, and James Derr

Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

The American bison is one of the most unique species on the planet considering its status as an important wildlife species in National and State Parks, an economically viable livestock species, and a powerful religious symbol for many Na- tive American tribes. The white coat color is particularly sought after due to its economic, aesthetic, and spiritual value. While scientific investigations of the species have included complete genome sequencing, the bison genome has yet to be annotated as has been completed with domestic cattle. The aim of this study is to provide proof-of-concept of a con- current annotation project via white coat color gene(s) identification including those conferring albinism, leucism, dilute color, or other coat color related phenotypes. Genes including PMEL, TYR, KITLG, MITF, SLC45A, MLPH, and MC1R were identified as primary candidate genes as they have been found to cause a white coat color phenotype in various other mammals. White bison have been sequenced and compared to wild-type bison to identify unique alleles in order to determine their potential cause of a white coat color. Anticipated results include identification of potential SNPs, indels, or other mutant variants within one or more of the candidate genes. Overall, identification of the causal allele(s) conferring white coat color in American bison is significant in its application to conservation management, livestock production, and the relationship between bison and Native American people with an immediate benefit of the project to provide proof-of- concept for the concurrent bison genome annotation.

Research Grant: Department of Interior, National Park Service grant (1907362) Student Support: Boehringer Ingelheim Vet Scholars Program, Texas A&M College of Vet Med and Biomed Sciences

230 • 2021 National Veterinary Scholars Symposium Evaluating the effects of telmisartan in healthy dogs as a preclinical model for Shar-Pei fever

Kara Maslyn, Jennifer Hawley, Craig Webb, Tracy Webb, Michael Lappin

Center for Companion Animal Studies, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Chinese Shar-Pei dogs commonly develop Shar-Pei fever, a pro-inflammatory disease characterized by a fever of un- known origin, renal amyloidosis, peritonitis, synovitis, and proteinuria. While traditional therapies help to alleviate clinical signs, optimal therapies remain unknown. Telmisartan is an angiotensin II receptor blocker used primarily to help control hypertension and proteinuria in dogs, but other effects have been described in other species.The aim of this study was to evaluate the immunomodulatory properties and antioxidant effects of telmisartan in healthy dogs. Eight healthy, purpose-bred research beagles of mixed sex were used for this study. Dogs were administered 10 mg of telmisartan once daily for 28 days. A complete blood count (CBC), serum cytokine/chemokine panel, and the following antioxidant as- says were performed multiple times over the course of the study; antioxidant levels of total antioxidant capacity (TAC), CuZn-superoxide dismutase (SOD), catalase (CAT), plasma levels of glutathione reductase (GR), and glutathione perox- idase (GPx). Group mean values after starting telmisartan administration were compared to baseline values by ANOVA with significance defined as P < 0.05. While significant changes in CBC or cytokines/chemokines were not detected, -tel misartan significantly upregulated the activity of serum TAC (P < 0.007), serum SOD (P < 1.78E-5), and plasma GPx (P < 0.001) over the 28 day trial. Telmisartan has antioxidant effects in healthy dogs which suggests an additional indication for the use of this drug in the management of Shar Pei fever. A clinical trial will now be designed to evaluate the therapeutic effects of telmisartan in dogs affected by Shar-Pei fever.

Research Grant: The Shumi SPAID Project and the Center for Companion Animal Studies, Colorado State University, Fort Collins, CO Student Support: The Shumi SPAID Project and the Center for Companion Animal Studies, Colorado State University

Examining CD8+ T cell localization in estrogen receptor positive and negative obese breast cancer metastasis

Nick Massaro, Abbey E. Williams, and Lisa M. Arendt

Doctor of Veterinary Medicine Program (Massaro, Arendt) and Comparative Biomedical Sciences Graduate Program (Williams), University of Wisconsin - Madison, Madison, WI

Obesity is correlated with poorer prognosis and more metastasis in breast cancer patients. Paradoxically, obesity is asso- ciated with improved patient outcomes from multiple cancer types following treatment with immunotherapy. To examine how obesity impacts immune cell populations in estrogen receptor (ER) positive and negative breast cancer subtypes, three-week old FVB/N female mice were fed either a control diet or a high diet fat diet for 16 weeks to induce obesity. Met-1 (ER-) and TC2 (ER+) tumor cells were transplanted into the mammary glands to generate tumors or injected into tail veins to model metastases in both lean and obese mice. Tumors were allowed to grow for 8 weeks to ensure that lung metastases formed, then mice were euthanized, and tissue was collected and formalin-fixed.TC2 tumors, TC2 lungs, and Met-1 lungs were sectioned and stained for markers to detect CD8+ T cells and macrophages using immunohistochemis- try. Consistent with increased macrophage-driven inflammation in obese adipose tissue, we observed increased numbers of macrophages within ER+ TC2 tumors of obese mice. Further, we did not observe a difference in T cell number between Met-1 obese and lean lungs. Future directions for these experiments include evaluating T cell and macrophage function in obese metastatic lungs. Results from this project will be used to evaluate the potential effects of immunotherapy directed at macrophages and CD8+ T cells to treat metastases under conditions of obesity.

Research Grant: NIH/NCI, R01 CA227542 Student Support: School of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 231 Staphylococcus pseudintermedius as a source of hospital infections

Shelby C. Matsuoka, Jenna R. Beilby, Luiz E. Bermudez

Carlson College of Veterinary Medicine (Matsuoka, Beilby, Bermudez), Oregon State University, Corvallis, OR

A retrospective analysis of the Diagnostic laboratory submissions from 2016 to 2020 was performed to assess the patterns of resistance/susceptibility of S. pseudintermedius and determine which location in the hospital might potentially be acting as a reservoir for the bacteria. Data was obtained regarding the site of infection, period of stay in the hospital, antibiotic treatment and outcome. In addition, samples were taken of the hospital environment by swabbing areas in the intensive care unit and anesthesia preparation room in the VTH at OSU. The colonies were isolated and DNA was obtained and submitted for PCR to determine their species. Virulent strains were then tested to look for differences in resistance/suscep- tibility to antibiotics, and how biofilm formation impacts their response to antibiotics.

Research Grant: Boehringer Veterinary Scholar Program (BIVSP) Student Support: Boehringer Veterinary Scholar Program (BIVSP)

Efficacy and safety of long-term imepitoin treatment for canine storm anxiety with individual dose titration

Jennifer Maulini, Ana Clara Muñoz, Carrie Emke, Margaret E. Gruen

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Storm anxiety significantly impacts the welfare of many companion dogs and is typically considered a form of noise aver- sion. However, many dogs with storm anxiety show signs of fear well before storms begin, making this condition more complex compared to other noise aversions. Current pharmaceutical protocols are ill-equipped to treat dogs with storm anxiety, due to storm unpredictability. Pexion (imepitoin) has been found to significantly reduce storm anxiety in dogs, although mild adverse clinical signs occur at the dosage approved for noise aversion (30mg/kg BID). The objective of this pilot study is to use a dose-titration approach to determine the optimal efficacious and safe dose range. By individualizing the dosages per dog, we hypothesize that the range between 10-30mg/kg BID will be sufficient in effect with few adverse reactions. Thirty dogs with storm anxiety will be enrolled in this open-label trial. Over 12 weeks, dogs will complete a 2-week baseline period, 6-week dose titration period, and 4-week stable dosing period. Owners will complete weekly surveys and storm logs to score their dogs using the Canine Anxiety Scale (CAS). These scores will be used to analyze change from baseline at each dosage. All dogs will begin at 10 mg/kg BID; every two weeks, owner global scores will be used to determine whether a dose increase is needed. Adverse events will be recorded throughout. The findings from this study will determine the optimal dosage range for use for storm anxiety. Future studies may be done to determine efficacy of this dosage against a placebo, or in combination with other behavioral treatments.

Research Grant: Ingelheim am Rhein, Boehringer-Ingelheim Vetmedica GmbHm Ludwigsshafen am Rhein, Germany Student Support: Veterinary Scholars Program of North Carolina State University, College of Veterinary Medicine

232 • 2021 National Veterinary Scholars Symposium Fecal bacterial microbiota and antibiotic-induced changes of the microbiome in ferrets (Mustela putorius furo)

Tiera S. McAdam, Barbara A. Wolfe, Michael J. Wilkins, and Kaela K. Amundson

Department of Clinical Sciences (McAdam, Wolfe), College of Veterinary Medicine and Biomedical Sciences, and Department of Soil and Crop Sciences (Wilkins, Amundson), College of Agricultural Sciences, Colorado State University, Fort Collins, CO

Research has shown that the gastrointestinal (GI) microbiome plays an important role in the immune response to patho- gens. Commensal bacteria form a physical and chemical barrier to the gut. They promote differentiation of T-helper (Th17) cells which produce IL-17, a potent pro-inflammatory cytokine which contributes to antibacterial defense, along with several other host defense factors such as TNFα and IFNγ. Domestic ferrets have been shown to harbor Clostrid- ia-related species in their gut microbiome which plays a large role in cell-mediated immunity. Antibiotics are used exten- sively to treat a wide variety of GI bacterial infections. These antibiotics are typically broad-spectrum antibiotics which have been shown to alter the commensal microbiome in animals. Based on previous studies, which show that use of broad-spectrum antibiotics can alter gut microbial community in minks, we hypothesize that antibiotic use in domestic ferrets (Mustela putorius furo) will show a similar trajectory of change. To address this hypothesis, we will conduct both 16s rRNA as well as metagenomic analyses on fecal samples to determine microbial richness and functional profiling of microbial genes. Healthy juveniles with no history of antibiotic use, as well as adults, both before and after antibiotic use, will be analyzed. These studies will help identify the composition and function of the gut microbiome of domestic ferrets and the impacts that antibiotics can have on those factors. With the knowledge of how certain commensal bacterial species contribute to the immune response towards pathogens, this will give us insight into how the immune response may be affected due to perturbations of the gut microbiome.

Research Grant: Smithsonian Institution Burch Fellowship CSU CRC program Student Support: NIH T35 Training Grant

Identification of protective antigens from African Swine Fever Virus proteome

Jayden McCall, Michelle Zajac, Neha Sangewar, Shehnaz Lokhandwala, Waithaka Mwangi

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS

African Swine Fever Virus (ASFV) poses a serious threat to the pork industry, but there is no safe vaccine or treatment available. Development of a protective subunit vaccine requires empirical identification of cognate antigens.The ASFV pp220 polyprotein, encoded by a 7.4 kb gene, is critical for virus production. This polyprotein is processed to generate p14, p34, p37, and p150 individual proteins with the last one being the largest subunit. To identify T cell epitopes, pools of predicted SLA-I binding 9-mer peptides were screened by IFN-g ELISpot assay using PBMCs and splenocytes from pigs immunized with a cocktail of adenoviruses expressing the proteins. Individual peptides from positive pools were then evaluated for ability to induce IFN-g+ PBMC and splenocyte recall responses. Four peptides, namely p34161-169, p37859-867, p1501363-1371, and p1501463-1471, recalled strong IFN-g+ PBMC and splenocyte responses. Peptide p34161-169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859-867 and p1501363-1371 stimulated recall IFN-g+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463-1471 recalled respons- es in PBMCs isolated from 7/10 pigs and splenocytes isolated from 9/10 pigs, respectively. The results show that pp220 polyprotein contains multiple T cell epitopes that induced robust IFN-g+ responses in commercial pigs. Notably, these epi- topes are conserved among different ASFV genotypes and were predicted to bind differentSLA -I alleles. These outcomes and our previous demonstration that pp220 induces strong IgG and CTL responses suggests that pp220 is a promising candidate for inclusion in a prototype vaccine.

Research Grant: This study was supported by the Agriculture and Food Research Initiative Competitive Grant no. 2016- 67015-25041 from the USDA National Institute of Food and Agriculture. Student Support: We acknowledge the Foundation for Food & Agriculture Research for scholar and research support

2021 National Veterinary Scholars Symposium • 233 Pyometra management in the private practice setting

Ashlee J. McCallin, Jennifer W.-C. Turner, and Rachael E. Kreisler

Department of Primary Care, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Current management practices of pyometra in the private practice (PP) setting are not well documented. This study’s goal was to understand pyometra management in the PP setting and the current relationship PPs have with high quality, high volume, spay-neuter (HQHVSN) clinics regarding referral for pyometra surgery. An anonymous electronic survey was piloted and distributed using SurveyMonkey via email, social media, and mailing lists targeting veterinarians who see companion animals. A total of 153 responses were received from June-July 2021 with respondents distributed throughout the United States. Practices performed a median of 10 spay and neuters per week with the median spay cost being $350 (IQR 250-500) for dogs and $250 (IQR 180-350) for cats. The median cost of a pyometra surgery was $1000 (IQR 700- 1475) for dogs and $725 (IQR 400-1000) for cats. All practices treated pyometras at least sometimes, despite the level of disruption (major 19%, moderate 48%, mild 29%, none 4%) to their day. All pyometra presentations were accepted for treatment at least sometimes: BAR/QAR (97%), depressed or non-ambulatory (87%), and obtunded or lateral (61%), with mean estimated survival of 96%, 74%, and 31%, respectively. A majority of respondents viewed HQHVSN clinics as a referral resource either all of the time (20%) or some of the time (35%). However, 45% of respondents did not consider HQHVSN clinics for referral. Private practices will do what they can to treat most pyometras that present at their clinic within a timely manner before referring elsewhere. There is currently inconsistent utilization of HQHVSN clinics as a potential referral resource due to location or appointment availability.

Research Grant: Research Grant: None Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Evaluation of antibodies against SARS-CoV2 Spike proteins in dogs: an analysis of exposure over the pandemic

Sierra McClain, Margaret Kruger, Annabelle Honet, Carine Holz, Brian Petroff, Jennifer Thomas, Srinand Sreevatsan

Michigan State University College of Veterinary Medicine (McClain), Pathobiology and Diagnostic Investigation, (Kruger, Honet, Holz, Petroff, Thomas, Sreevatsan), East Lansing, MI

SARS-CoV2 the virus causing the COVID-19 pandemic continues to spread globally. Despite development of effective vaccines, the virus has endured via the emergence of major new variants globally. At east five highl successful variants of spike are now well-established in human populations. The emergence of new variants are primarily due to mutations, lo- cated in the viral spike (S) protein. This, combined with evidence of documented interspecies transmission to mink, dogs, cats, ferrets, tigers, hamsters, macaques etc., establish that this virus has a broad host range and transmission to diverse hosts provide an opportunity for further mutations and emergence of new strains. With the heavy burden of infection in human populations, the magnitude SARS-CoV2 infection of pet dogs in households is not well-understood. We hypoth- esize that pets are exposed to the virus via contact with their owners. An enzyme-linked immunosorbent assay (ELISA) was developed using Nobivac Canine 1-Cv (control antigen) and the five SARS-CoV2 spike variants (test antigens) iden- tified over the course of the pandemic to detect antibodies against SARS-CoV2. Dog sera (n = 250) collected during the pandemic period (December 2019 - June 2021) and seventy dog sera collected prior to the pandemic era (prior to 2019) will be screened for evidence of SARS-CoV2 exposure in early, mid and late-pandemic era as compared with pre-pan- demic times. The modulation of seroprevalence of antibodies against the five reported spike variants over the pandemic period will be evaluated.

Research Grant: National institutes of Health: 2R25HL103156-11 Student Support: BRUSH Summer Research Program

234 • 2021 National Veterinary Scholars Symposium Elimination kinetics of subcutaneously administered eprinomectin in plasma and milk in dry-off dairy cattle

Simone McCluney, Ranee Miller, Ronald Baynes

Center for Chemical Toxicology Research and Pharmacokinetics, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Eprinomectin is a derivative compound of abamectin belonging to the macrocyclic lactone family. Historically, eprinomectin has been administered topically for treatment against endo- and ectoparasites, and has been shown to be effective for approximately 30 days. The injectable formulation of eprinomectin has been increasingly utilized due to its significantly longer efficacy of around 150 days. While there is zero milk withdrawal time for topical eprinomectin, more research is necessary to establish the residues present in milk following subcutaneous administration. The hypothesis of this project is that dairy cattle given a label dose of 1 mg/kg of injectable eprinomectin (LongRange) at the start of their dry off period, 60 days prior to calving, will have milk residues of eprinomectin below the U.S. milk tolerance level of 12 ppb. 13 mature, pregnant dairy cattle were subjects of which plasma was collected daily for 7 days following eprinomec- tin administration, then at regular intervals for a period of 90 days. Once each cow calved, samples of colostrum and milk were collected daily for compartmental and noncompartmental pharmacokinetic analysis. This analysis is in progress and results will be published when acquired.

Research Grant: Center for Chemical Toxicology and Research and Pharmacokinetics, USDA (FARAD) Student Support: Veterinary Scholars Program

Impact of formalin baths and jellyfish (A. aurita & C. capillata) on gill pathology in Atlantic salmon (S. salar)

Raelyn L. McCurdy, Crystal V. Veltman, Shona K. Whyte, Sara L. Purcell, Mark D. Fast

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown PEI (McCurdy, Veltman, Whyte, Purcell, Fast)

Over the past decade, gill pathology and associated complex gill disease (CGD) in farmed Atlantic salmon (Salmo salar) has become one of the most detrimental causes of mortality and economic loss on the West Coast of Canada and around the world. CGD is estimated to account for the loss of 12,500 tonnes of salmon annually in British Columbia alone. The etiology of CGD is not well defined, and includes a broad range of pathogens, environmental conditions and handling practices. To better understand the progression of CGD, we are developing in vivo laboratory models using etiologies identified in past literature including exposures to different bath treatments (i.e. formalin and hydrogen peroxide) and jellyfish Aurelia( aurita and Cyanea capillata). We are also assessing an in vitro method of examining jellyfish impacts on Atlantic salmon. The aim of our present work is to determine the histological effects of these etiologies on the gills and how they contribute to the development of different stages of gill disease and potentially CGD.To accomplish this, fish were exposed to treatment baths of formalin for a period of 20 minutes, or baths of jellyfish slurry for a period of 1, 6 or 10 hours. At 6 hours, 24 hours and 7 days post exposure to a respective treatment, fish (n = 6-10) from each treatment tank are euthanized and tissue samples collected for analysis. The left, first gill filament was collected for molecular analysis and the second gill arch on the right and left side was collected for histological analysis. The results of this study will be an important contribution to our understanding of CGD in Atlantic salmon, and will serve as an excellent framework for the development of treatment and preventative measures.

Research Grant: Boehringer Ingelheim Veterinary Scholars Program Veterinary Student Research Award Program- Atlantic Veterinary College Student Support: None

2021 National Veterinary Scholars Symposium • 235 Cataloging retinal lesions in Asiatic black bears (U. thibetanus) and Malaysian sun bears (H. malaynus)

Kylie McDaniel, Felicia Nutter, Rachel Sanki, and Stephanie Pumphrey

Cummings School of Veterinary Medicine at Tufts University (McDaniel), Department of Clinical Sciences (Pumphrey), and Department of Infectious Disease and Global Health (Nutter); Animals Asia (Sanki)

Approximately 17,000 captive bears live on bile farms across Asia. Cardiovascular disease is the third leading cause of death in previously bile-farmed bears, and systemic hypertension is believed to be a common problem in bears at Animals Asia sanctuaries. Because blood pressure cannot be measured consistently in rehabilitant bears, evidence of ocular target organ damage (TOD) is one criterion currently used to diagnose systemic hypertension. Until now, no published literature has characterized common ocular findings in a large population of bears of any species, making consistent diagnosis of hypertension difficult. This retrospective study of 172 Asiatic black bearsUrsus ( thibetanus) and 12 Malaysian sun bears (Helarctos malayanus) housed at Animals Asia Vietnamese Bear Rescue Center (n = 184) establishes the prevalence of retinopathy in previously bile-farmed bears, identifies the most common fundic lesions, and explores the relationship be- tween hypertension and ocular disease. Using a smartphone camera and 40D lens, fundic images were obtained for bears during their biannual routine health examinations. Images were assessed visually, recording lesion appearance, size, and location. Lesions were grouped by class and compared across medical records to determine if the presence of a specific class of retinal lesion correlates statistically with clinical outcomes, history, and signalment. Retinal lesions frequently occur in captive bears previously used for bile farming, with the most common lesions including retinal detachment, ret- inal hemorrhage, vessel abnormalities, and hyporeflective tapetal patterns. Bears with evidence of ocularTOD should be closely monitored and possibly treated for systemic hypertension.

Research Grant: None Student Support: Cummings School of Veterinary Medicine at Tufts University

Assessing northern sea otter (Enhydra lutris kenyoni) immune function in Alaska

Malea R. McGimsey, Tuula E. Hollmen, Katrina L. Counihan

College of Veterinary Medicine, Colorado State University, Fort Collins, CO (McGimsey), College of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, AK (Hollmen), Alaska SeaLife Center, Seward, AK (Hollmen, Counihan)

Southern sea otters (Enhydra lutris nereis) as well as the southwest stock of northern sea otters (E. l. kenyoni) are listed as threatened under the Endangered Species Act. These animals are susceptible to a number of infectious diseases and harm- ful toxins which could have damaging effects on their populations, yet current knowledge is limited regarding the immune system that defends them from adverse effects. While some immune system analyses have been done in southern sea otters, to our knowledge no studies have yet been done investigating immune health and function in northern sea otters. The primary goal of this study is to assess lymphocyte function in northern sea otters from Prince William Sound, Alaska. Lymphocytes were isolated from blood collected from 60 free-ranging otters. Function will be evaluated through mitogen induced proliferation of T and B cells, using concanavalin A and lipopolysaccharide, respectively. Dependent on available field data, individual and group comparisons can be made such as age and gender differences in immune function. Prince William Sound was the site of the 1989 Exxon Valdez oil spill, which impacted the sea otter population and the ecosystem as a whole. Investigating sea otter lymphocyte function will give insight as to the health status of this population and the potential risk posed by pathogens and toxins.

Research Grant: Unknown Student Support: NIH T35 Training Grant 5T35OD015130-09

236 • 2021 National Veterinary Scholars Symposium Assessment of an experimental PCV2d vaccine in the PCV2d challenge model

Jared McGinley, Tanja Opriessnig, Patrick Halbur, Alex Bishop, Gaurav Rawal, Jianqiang Zhang

Department of Veterinary Pathology and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA

Porcine circovirus type 2 (PCV2) associated disease is well controlled by commercial vaccines. Five PCV2 genotypes have been recognized, of which PCV2a, PCV2b and PCV2d are prevalent worldwide. Most commercial vaccines are pres- ently based on PCV2a while the most prevalent strain circulating on US farms is PCV2d. The objective of this study was to assess a novel PCV2d vaccine based on ORF2 expression in the baculovirus system. Forty pigs were randomly split into groups of 10: A NEG control, a POS control, and two vaccine groups. The COM vaccine group received a leading commercial PCV2a vaccine (CircoFLEX ), while the EXP vaccine group received the novel PCV2d vaccine. Four weeks after initial vaccination, all groups except the NEG controls were challenged with PCV2d intranasally. Blood and nasal swabs were taken weekly to monitor viremia and shedding. Serum was tested for anti-PCV2 antibodies using a commer- cial kit (INgezim Circo IgG). At 21 days post challenge (dpc) all pigs were euthanized, necropsied, and lymphoid tissues were collected. At challenge, anti-PCV2 antibodies were more prevalent in the COM group than the EXP group. By 14 dpc, 9/10 pigs in the EXP vaccine group were antibody positive compared to 0/10 in the POS control group. By 20 dpc, anti-PCV2 antibodies in both vaccine groups were significantly higher (P < 0.05) compared to all other groups but not significantly different from each other. The COM vaccine group had lower PCV2 DNA prevalence at dpc 7, 14 and 21. Histopathology results are pending. The obtained results indicate that the EXP vaccine based on PCV2d had no advantage compared to a COM vaccine under the study conditions in subclinically infected pigs.

Research Grant: Unknown Student Support: Boehringer Ingelheim

Examining urine pH, specific gravity, and urine protein over time in healthy dogs

Andrew McGlynn, Ryan Mrofchak, Rushil Madan, Christopher Madden, Adam Rudinsky, Jessica Hokamp, Vanessa Hale

The Ohio State University College of Veterinary Medicine (McGlynn, Madan), Department of Veterinary Preventive Medicine (Mrofchak, Madden, Hale), Department of Veterinary Clinical Sciences (Rudinsky), Department of Veterinary Biosciences (Hokamp)

Urine properties such as pH, urine specific gravity (USG), and protein profiles are routinely measured during a urinaly- sis to assess canine health. Clinicians have well-established reference ranges to assess urine in patients with urinary tract diseases, such as urinary tract infections. Despite this, less is known about the natural fluctuations of urine properties in healthy dogs over time. In this study, mid-stream free catch urine was collected from 10 healthy dogs over 12 time points that were hours, days, and months apart. We hypothesized that urine pH and USG would vary significantly over time, while protein profiles would be stable due to consistent protein filtration by the kidneys. Urine pH was measured via pH meter; USG was measured via refractometer, and proteins were profiled via 4-12% Bis-Tris gel electrophoresis. Prelimi- nary data showed that USG varied significantly (P < 0.0001) between dogs but was consistent over time within dogs (p = 0.285) while urine pH did not vary significantly between dogs (p = 0.588) because pH was so highly variable within dogs over time. Urine proteins ranged from none to trace amounts. The most commonly detected proteins were at molecular weights consistent with Tamm Horsfall and albumin. Statistics on urine proteins are forthcoming. Our hypothesis for USG was unsupported as it did not vary significantly over time, indicating that a USG measurement at any time should be rep- resentative for that dog, while pH was so variable that it should be measured at multiple time points before altering clini- cal decisions. These findings help define normal variations in urine properties, which can inform clinical decision-making around urine sampling and urinalyses.

Research Grant: Ohio State University College of Veterinary Medicine Canine Funds Student Support: Muffin Sniadowski Student Research Endowment Fund

2021 National Veterinary Scholars Symposium • 237 Immunometabolic profiling of Tpl2-/- innate immune cells

Danielle M. McNabney, Denise L. Fahey, and Wendy T. Watford

Department of Infectious Diseases (McNabney, Fahey, Watford), College of Veterinary Medicine, University of Georgia, Athens, GA

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase known to promote inflammation, including the induction of both TNF and IL-1b, via the activation of multiple pathways. Due to its pro-inflammatory functions,Tpl2 inhibitors are being considered as immunotherapies for autoimmune disorders; however, precise mechanisms by which Tpl2 controls inflammatory phenotypes of cells is poorly understood. It has been previously demonstrated thatTpl2 activates the PI3K/ Akt/mTOR pathway, which has been implicated in the regulation of cellular metabolism. The scientific premise is that Tpl2 promotes inflammation by altering the metabolic state of immune cells.We hypothesize that Tpl2 induces glycolysis, which supports inflammatory functions including cellular activation, proliferation, and cytokine production. Gene expres- sion analysis of bone marrow-derived macrophages (BMDM) treated with or without lipopolysaccharide (LPS) showed higher expression of glycolysis-related genes including glucose transporter 1 (Glut1, Slc2a1), phosphofructokinase (PFK), pyruvate dehydrogenase kinase (PDK), and pyruvate kinase M (PKM) in wild type (WT) compared to Tpl2 knockout (Tpl2-/-) BMDMs over a time course. Tpl2-/- BMDMs also exhibited higher expression of the M1 (pro-inflammatory) phenotype marker Nos2, indicating a greater capacity to differentiate into M1 phenotype compared toWT BMDMs. On- going studies are assessing Tpl2-dependent metabolic changes, specifically glycolytic and metabolic respiration. Overall, the data suggest that Tpl2 promotes glycolytic functions of LPS-stimulated BMDMs and regulates downstream effectors involved in the differentiation towards an M1 phenotype.

Research Grant: Support for research was provided by NIH, National Institute of Allergy and Infectious Diseases, Grant Number R21AI147003-01 awarded to WTW. Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

The porcine gut microbiome: Understanding effects of age, sex, breed, facility location and birthing method

Beverly Meena, Tamara D. Ford, Lynn M. Martin, Aaron C. Ericsson

Departments of Veterinary Medicine and Surgery (Meena, Ford, Martin) and Veterinary Pathobiology (Ericsson), University of Missouri, College of Veterinary Medicine, Columbia, MO

A major focus within the swine industry is the efficient production of healthy pigs. The gastrointestinal tract of pigs con- tains a complex microbial ecosystem. The intestinal microbiota has been shown to play an essential role in many physio- logical processes such as nutrient absorption and metabolism. Several studies have demonstrated the profound effects that gut microbial communities have on overall health and growth rate of pigs. These microbial communities begin to establish shortly after birth and are influenced by both genetic and environmental factors. It is still unclear as to which environ- mental and genetic factors play the greatest role in the shaping of healthy gut microbial communities. The purpose of this study is to determine the effects of age, sex, breed, location, and method of delivery (vaginal versus caesarean section) on the pig microbiome. Fecal samples will be collected from 147 pigs from four different swine facilities. DNA will be ex- tracted and undergo 16S rRNA sequencing to determine the differences in microbial communities present in each sample. We expect to see differences in the microbiota of pigs of various ages, locations, and types of delivery while we do not expect to see significant differences between breed and sex. The results of this observational study can be used in future research and the development of managerial plans and nutritional diets that promote healthy gut microbial communities at various stages of swine production, leading to greater production efficiency.

Research Grant: University of Missouri Metagenomics Center Student Support: IDEXX-BioAnalytics

238 • 2021 National Veterinary Scholars Symposium The Fecal Metabolomic Profile of Dogs Transitioning from a Chicken Raw Meat Diet to a Plant-Based Diet

Tarun R Mehta, Janina Strobeck, Annika Linde, Tonatiuh Melgarejo

Translational Medicine & Antimicrobial Resistance Laboratory, College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA; & Zymo Research, Irvine, CA

Over the last decade feeding non-traditional diets, have quickly gained popularity amongst canine owners. Also, certain nutritional deficiencies have been allegedly linked to owners feeding non-traditional diets, such as unbalanced home- cooked diets, raw meat diets, and BEG pet foods. Scientific evidence is emerging strongly suggesting that dogs fed a meat-free plant-based diet (PBD) can not only maintain good health, but also perform well under extreme physical de- mands. While previous studies have focused on the lack of amino acids (e.g. taurine) and its relationship to dilated cardio- myopathy (DCM), no studies have evaluated the potential effect of raw-meat chicken diets vs Plant-Based Diets (PBD) on the fecal metabolome and microbiome of patients. The purpose of this study is to analyze the different populations of microorganisms (bacteria and fungi) and their metabolites between subjects eating a raw-meat chicken diet and PBD. The hypothesis of this study is that dogs eating a meat-based diet possess a population of fungi and bacteria (and their metabolites) that are proinflammatory and that transitioning to a PBD will modify the intestinal microbiome producing fewer proinflammatory metabolites. Using next generation sequencing and mass spectrometry we are currently analyzing the fecal microbiome and metabolome, respectively, in 3 subjects. As part of an ongoing project, the present study will describe the potential differences in the fecal metabolomics pattern of dogs fed originally a raw-meat chicken-based diet which were then transitioned over a week to a commercially available nutritionally balanced PBD, and fed this diet for a three-month period. Results will be ready by the time of the poster presentation.

Research Grant: Translational Medicine & Antimicrobial Resistance Laboratory, College of Veterinary Medicine, Western University of Health Sciences. Student Support: Western University of Health Sciences – Office for Research and Biotechnology CVM

Validation of a novel goat kid health scoring system

Tanzania M. Menn, Whitney A. Knauer, Emily A. Barrell

Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota

Goat kids are at an increased risk of morbidity and mortality during the pre-weaning period due to respiratory and diarrhe- al disease, yet a standardized scoring system to screen for and detect disease is lacking. Our objective was to validate the previously developed UMN-Goat Kid Health Scoring System (GKHSS) as a screening and diagnostic tool on a commer- cial goat dairy. On a single observation day, 560 goat kids ages 2-7wks were evaluated. Each kid was first evaluated by a small ruminant veterinarian, considered the gold standard test, and diagnosed as healthy or ill (respiratory, diarrheal, or other disease). Then, study technicians evaluated each kid’s mentation, body posture, respiratory secretions (RS), belly fill, fecal score (FS), and rectal temperature using GKHSS and conferred a score of 0-2 for each category. A kid was diagnosed with diarrheal disease if FS = 2, with respiratory disease if RS = 2, and as sick if total health score $4. Additionally, fecal samples were collected from kids diagnosed with FS = 2 and from matched healthy controls within the same pen with FS = 0. Samples from 244 kids were submitted to SDSU ADRDL and tested for common diarrheal pathogens such as coccid- ia, Cryptosporidium, and Rotavirus. Farm health treatment data was collected from farm computer records. Diagnostic test characteristics (sensitivity, specificity, accuracy) were calculated from 2x2 tables for the following: 1) ability of GKHSS to detect a sick kid (vs. veterinary diagnosis); 2) ability of GKHSS to detect diarrheal disease (vs. diagnostic testing); and 3) ability of GKHSS to detect a kid that received treatment for respiratory or diarrheal disease in the 63d surrounding the health score. Data collection and analysis is ongoing.

Research Grant: USDA NIFA Animal Health Project 1017915 Student Support: NIH T35 Training Grant T35OD011118

2021 National Veterinary Scholars Symposium • 239 Characterization of CFTR protein expression in gallbladder epithelium of dogs with mucocele formation

Bryanna K. Meredith, Stephen H. Stauffer, Jody L. Gookin

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Mucocele formation occurs when the gallbladder epithelium secretes excessive abnormal mucus often leading to obstruc- tion or rupture. It is among the most common and deadliest causes of gallbladder disease in dogs and its cause is currently unknown. Hydration of gallbladder mucus is mediated by the cystic fibrosis transmembrane regulatory channel (CFTR) via secretion of chloride and bicarbonate. Prior studies showed that CFTR knockout laboratory animals (piglets and fer- rets) develop gallbladders similar to canine mucoceles. Accordingly, the objective of our study was to examine the expres- sion of CFTR in gallbladder epithelium of dogs with mucocele formation. We hypothesized that mucocele formation is associated with decreased epithelial expression of CFTR. Protein was extracted from the gallbladder mucosa of dogs with and without mucocele formation (8 each) and used in Western blotting to detect CFTR and E-Cadherin (a tight junction protein known to be present in gallbladder epithelium). Canine kidney protein was used as a positive control. Preliminary results demonstrate expression of CFTR and E-Cadherin in both mucocele and normal gallbladder samples. Further study will use densitometry to compare CFTR expression between normal and mucocele gallbladders based on the quantity of epithelium (determined by E-Cadherin) present in each sample. These findings will contribute to the understanding of CF- TR’s role in gallbladder epithelial secretion in dogs with mucoceles and provide a possible treatment target for this deadly disease.

Research Grant: Morris Animal Foundation D17CA-068 Student Support: Veterinary Scholars Program at North Carolina State University's College of Veterinary Medicine

The effect of the COVID-19 pandemic on student performance at WesternU CVM

Danielle Merichko, Rhea Hanselmann, and Brian Oakley

College of Veterinary Medicine, Western University of Health Sciences, Pomona, California

In this study, we examined the effects of virtual instruction in the WesternU College of Veterinary Medicine curriculum on student performance and evaluated potential predictors of student performance. We compared student performance and student satisfaction in two third-year non-clinical courses (Population Health and Production, and Food and Feed Safety) between in-person (pre-COVID-19) and virtual (during-COVID-19) delivery. We also compared student performance data to overall academic performance in the veterinary program and to prior exposure to other courses of the third-year curric- ulum to examine whether these variables correlate with student success in the two courses and how virtual delivery has affected these relationships. We found significant increases in several outcome measures for virtual versus in-person -de livery for both courses. For all students, performance was generally higher in the virtual format than in-person. For Food and Feed Safety, the improved performance during virtual delivery was most dramatic for the bottom quartile of students who nearly all passed the threshold from a B to A letter grade. Interestingly, we found no decreases in student satisfaction during virtual versus in-person delivery. For both courses, we found few significant effects of prior exposure to other 3rd year courses on student performance for either in-person or virtual delivery. The most important predictors of student success were performance in the other course, cumulative GPA, and mode of delivery. Identifying predictors of student success such as these may allow targeted interventions to improve student learning and performance for both in-person and virtual curricula.

Research Grant: None Student Support: Western University of Health Sciences CVM and Office for Biotechnology and Research.

240 • 2021 National Veterinary Scholars Symposium Method optimization for studying effects of cannabinoids on canine PBMC immune responses

Madison Mertz, Todd Archer, and Barbara Kaplan

Department of Comparative Biomedical Sciences (Mertz, Kaplan) and Department of Clinical Sciences (Archer), College of Veterinary Medicine, Mississippi State University, Mississippi State, MS

In recent years, cannabinoid-containing products have grown increasingly popular for their putative anti-inflammatory properties but limited conclusive data have been gathered to demonstrate their efficacy or consistency. As veterinarians face a clientele which is gaining interest and access to these products, they require further data on the response of the canine patient. It is commonly assumed cannabinoids such as tetrahydrocannabinol (THC) and cannabidiol (CBD) are immune suppressive and prior studies from this lab have shown inhibition of interleukin-2 (IL-2) and interferon gamma (IFN-g) mRNA in canine peripheral blood mononuclear cells (PBMCs) by THC. The goals of this study were to optimize new methods to detect cannabinoid effects on canine PBMCs and to characterize the effect of cannabinoids on canine PBMC immune function. We hypothesized that CBD and THC would downregulate IL-2 and IFN-g protein production. Our initial results showed that while there was little cross reactivity of human antibodies with canine proteins, human anti-CD3 stimulated cells better than canine anti-CD3. Perhaps most importantly, our data from flow cytometry showed that both CBD and THC induced statistically significant increases or decreases in cytokine production across various conditions. Together these data suggest that cannabinoids must be considered immunomodulatory, rather than strictly im- munosuppressive, and that the immune response to cannabinoids may be patient- or condition-specific, but more detailed studies are required to further characterize the effects. With better understanding, clinicians can more confidently advise their clients on potential cannabinoid benefits (or lack thereof).

Research Grant: NIH T35OD010432 NIH P20GM103646 COBRE Core C Mississippi State University College of Veterinary Medicine Pharmacodynamic Laboratory (Archer) Student Support: NIH T35OD010432

Development of an online behaviour rating tool to enhance veterinary students’ knowledge of equine behaviour

Andrea Messina, Laurie McDuffee, William Montelpare

Department of Health Management (Messina, McDuffee), Department ofApplied Human Sciences (Montelpare), Univer- sity of Prince Edward Island, Charlottetown, Prince Edward Island, Canada

The majority of first year veterinary students have little to no prior experience interacting with horses which may nega- tively impact student safety and teaching horse welfare. The ability to correctly identify and interpret equine behaviour is a cornerstone to improving safety and welfare when working with horses. By recognizing an escalation in undesirable behaviours, students can modify their interaction with the horse before dangerous scenarios arise. Our goal is to create an online tool that will enhance knowledge of equine behaviour in first year veterinary students.The programming portion of the online tool was produced by computer science students at UPEI while the computational portion of the tool was com- pleted by one author (AM). Videos showing horses exhibiting various behaviours were obtained by recording teaching horses in common environments, including in pastures, paddocks, and teaching laboratories. A series of 22 short videos, each containing at least three distinct observable behaviours, were uploaded into the tool. A list of eight behaviours and four interpretations were composed for use in a multiple-choice list to accompany videos. In the validation phase of the study, volunteers with advanced equine knowledge were asked to identify the correct behaviours and interpretation for each video. The data was analyzed by calculating the consistency of the rater’s selection of the correct behaviours for each video using the Cramer’s V coefficient. The Cramer’s V value between raters overall was 0.68, considered moderate to good, with a range of 0.52 to 0.89. Future improvement of the tool will include analysis of individual videos and be- haviours to find common sources of inconsistency among the raters.

Research Grant: None Student Support: Atlantic Veterinary College Veterinary Summer Research Award (AVC VetSRA)

2021 National Veterinary Scholars Symposium • 241 Stance analysis and activity tracking in six dogs with osteosarcomas undergoing palliative radiation therapy

Walter Meyers, James K. Roush, Wei-Wen Hsu, Chieko Azuma

Departments of Clinical Sciences (Azuma, Meyers), Small Animal Surgery (Roush), and Department of Statistics (Hsu)

Osteosarcomas are a painful bone cancer affecting large dog breeds today. Palliative radiation therapy (RT) can be used to alleviate pain when surgical removal of the affected bone is not elected. Although RT has been proven to increase a dog’s survivability with pain control, a method to quantify and assess their pain or quality of life has yet to be established. Pain assessment by weight distribution analysis using a Stance Analysis platform (SA) and activity tracking by accelerometer (FitBark 2) data propose potential methods of effectively measuring patients’ pain level. Six dogs with osteosarcomas af- fecting a front limb were identified. Minute by minute activity data were collected by attaching FitBark monitors to dogs’ collars. The weight distribution data were collected prior to each radiation treatment and after RT was completed. Mea- surements were averaged and used to calculate a symmetric index. Changes in weight distribution and daily activity were graphed to visualize via RStudio. Among these six dogs immediate pain control by RT was assessed by SA which showed 3 positive pain control and 3 stable pain level. Patients tended to follow a trend of having a better quality of life with pain control post RT but then slowly decline over time. As a preliminary study, FitBark and SA provided complimentary data to assess patients’ pain and quality of life in response to RT.

Research Grant: Research Grant: Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars and Kurz Family Scholarship

Defining the Hbz/YBX1 interaction and its role in HTLV-1 pathogenesis

Amanda K. Midkiff, Susan M. Smith, Patrick L. Green, and Amanda R. Panfil

Department of Veterinary Biosciences, Center for Retrovirus Research (Smith, Green, Panfil), Comprehensive Cancer Center and Solove Research Institute (Green, Panfil), The Ohio State University, Columbus, OH

Human T-cell leukemia virus type 1 (HTLV-1) infects an estimated 5-10 million people worldwide and ~5-10% of infect- ed people develop clinical disease (T-cell leukemia or neurodegenerative disease) after a prolonged clinical latency period of several decades. However, progression of disease is not well understood, and patient prognosis is poor with limited treatment options available. The viral protein Hbz was initially identified as an antagonist to the viral oncoprotein and transcriptional activator, Tax. Hbz is believed to play a role in infected cell maintenance and oncogenesis. This viral gene is often the only viral transcript expressed in patient cells and its precise role in cell survival remains poorly understood. Our lab recently identified that Hbz interacts with the cellular proteinYBX1 via mass spectrometry. YBX1 is a transcrip- tion factor involved in growth-associated gene expression. Thus, we hypothesized that the Hbz/YBX1 interaction may play a key role in oncogenesis. Our studies have found that YBX1 activates viral transcription, and we find a synergistic positive effect on transcription with both Tax and YBX1. Hbz is able to repress Tax-mediated viral transcription, and exogenous YBX1 alleviates this repression. Using affinity pulldowns, we found YBX1 interacts with the central bromo- domain of Hbz. Interestingly, we also found that cellular distribution of YBX1 is both cytoplasmic and nuclear, suggesting possible roles for YBX1 outside of HTLV-1 transcriptional regulation. Our work will help to further define the mechanism of action of Hbz and the proteins it interacts with during HTLV-1 pathogenesis. These studies are critical for defining potential targets in future gene editing therapeutic approaches.

Research Grant: NCI P01CA100730 Student Support: NIH T35OD0977

242 • 2021 National Veterinary Scholars Symposium A Western diet promotes non-small cell lung cancer and affects PD-1-based immune checkpoint inhibition

Alison Mihailovich, Krista Pearman, and Jeffrey W. Norris

Department of Pharmacology, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Lung cancer is the leading cause of cancer-related death in the US, with 85% of cases histologically classified as non- small cell (NSCLC). The most common mutation found in NSCLC occurs in the KRAS gene and leads to constitutive activation of the encoded GTPase. After normalization for smoking, epidemiological studies provide evidence that NS- CLC risk is positively associated with consumption of a Western diet (WD). Using the C57Bl/6LA2 model of KRAS-driv- en NSCLC, our laboratory found a 2.5-fold increase in the number of tumors that form in mice fed a WD compared to those fed a standard chow diet (SCD). This increase is associated with decreased numbers of lymphocytes in tumors, increased expression of the immunosuppressive receptor programmed cell death protein 1 (PD-1) by CD4+ lymphocytes, and decreased expression of interferon-gamma. Therefore, we hypothesize that feeding a WD reduces responsiveness to immune checkpoint inhibition of PD-1. We are testing this hypothesis using C57Bl/6LA2 mice randomized at weaning into diet groups that include SCD, WD, and a synthetic diet that controls for macronutrient sources. Additionally, mice are divided into treatment groups, including 5 mg/g of murine anti-PD-1 blocking antibody or a nonspecific isotype control. Ten weeks after enrollment, mice are sacrificed and lung tumor multiplicity, volume, and burden are determined by exam- ination of all lung lobes at 10x magnification. In the future, additional mice will be enrolled for the purpose of collecting mRNA and determination of interferon-gamma levels in tumor and normal lung tissue from these mice. This research informs how dietary pattern may influence dosing regimens with PD-1-based immune checkpoint inhibitors.

Research Grant: Research Grant: Midwestern University College of Graduate Studies Intramural Funding Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program

Spatio-temporal analysis of raccoon rabies animal cases in eastern North America

Grace Miller, Kim Pepin, Matthew Michalska-Smith, Amy Davis, Amy Gilbert, Meggan Craft

Department of Veterinary Population Medicine (Miller, Michalska-Smith, Craft), College of Veterinary Medicine, University of Minnesota, St Paul, MN; USDA Animal and Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center; 4101 Laporte Ave, Fort Collins, CO (Pepin, Davis, Gilbert)

Over the course of the last century human-wildlife contact has changed dramatically. As a result of increasing human and livestock populations along with dramatic anthropogenic changes to our physical environment, human-wildlife interac- tions have increased substantially and with that, the potential for emerging infectious diseases and zoonotic transmission have also risen. Understanding the spatial and temporal distribution of disease outbreaks across landscapes can help in the prevention, preparation, and management for future outbreaks. Using raccoon rabies case data, we investigated two landscape factors that impact disease dynamics: 1) latitude and 2) urbanness. In the U.S., rabies virus (RABV), a zoonotic pathogen, is enzootic in raccoons along the entire Atlantic coast. Using more than 70,000 records of tests for RABV span- ning 2006-2017, we constructed a hurdle model to evaluate the importance of latitude and urbanness on RABV occur- rence and the number of positive rabies cases detected. We found that in northern latitudes, rabies occurs more frequently in rural areas relative to urban areas but, when it does occur in urban areas there are more cases. However, in southern latitudes, the occurrence of raccoon rabies is similar across urban and rural areas. But, in rural areas the occurrence and prevalence are similar across northern and southern latitudes. The current model does not have much predictive power, so future work would be to include a more explicit consideration of the observation process. Ultimately, by contributing to our understanding of rabies disease ecology, we hope to assist in the optimization of disease control techniques in the future.

Research Grant: none Student Support: University of Minnesota College of Veterinary Medicine Summer Scholars Program

2021 National Veterinary Scholars Symposium • 243 Co-Localization of Glucocorticoid Receptors and KDM1A in four placenta types

Seunga Min, Gaby Gonzalez, Agata Parsons, Garrit J. Bouma

College of Veterinary Medicine (Min, Gonzalez) and Department of Biomedical Sciences (Parsons, Bouma), Colorado State University, Fort Collins, CO.

In humans and animals, placental insufficiency caused by stress or malnutrition can lead to intrauterine growth restrictions (IUGR). IUGR is suboptimal growth and low weight of the baby during gestation. IUGR can result in serious postnatal health consequences, such as cardiovascular disease. Studies have shown altered expression of glucocorticoid receptors (GR) in placentas of women with IUGR. Glucocorticoids are stress hormones critical for fetal development. An appropri- ate concentration of cortisol, for example, is required for neural development and reaching the optimal birth weight of the fetus. Alternatively, in GR knockout mice, decreased embryo implantation and termination of pregnancy was observed, suggesting glucocorticoids are important for the proper development of the fetus. KDM1A is a co-regulator of steroid receptors and demethylates specific lysines on histone 3. KDM1A knockout mice had decreased trophoblast development and died within 8.5 days of gestation, which suggests KDM1A has essential roles in placental development. Additional studies demonstrated KDM1A co-localizes and bind to androgen receptors in the sheep placenta. Therefore, we wanted to determine if KDM1A binds to other steroid receptors like GR, in the placenta. Our goal was to co-localize KDM1A and GR in placental cells in the four mammalian placenta types using immunofluorescence microscopy: endotheliochorial (dogs), epitheliochorial (horses), synepitheliochorial (sheep), and hemomonochorial (guinea pigs). We hypothesize that KDM1A and GR co-localize in trophoblast cells in all four placenta types. This is the first step in understanding the func- tion of the GR-KDM1A complex in trophoblast cells and placental function.

Research Grant: CVMBS College Research Council Student Support: USDA Animal Health & Diseases

A survey of pet-owners to identify potential barriers to access veterinary care in the Cumberland Gap Region

Sarah Mithani, Karen Gruszynski, Dawn Spangler, and Charles Faulkner

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, Tennessee

This project attempted to identify factors influential pet caretakers’ decision to use veterinary services in the Cumberland Gap Region (CGR). A survey was created using Qualtrics software and pet owners provided information relative to their demographics, use of veterinary services, and perceived barriers to veterinary care. The survey was distributed as an anon- ymous link through local print media, and social media accounts of CGR animal shelters. Results indicated 92%of clients had a standing relationship with a veterinarian. Although 87% of pet owners affirmed that and tick preventative was important to their pet’s health, 79% of clients used flea and tick products. Similarly, 81% of dog owners affirmed that heart worm and internal parasite prevention is important to their pet’s health, 69% acknowledged use of heartworm and internal parasite prophylaxis. When asked if there was a time they were unable to provide care for their pet,19% of respondents answered yes. Word cloud analysis based on responses to open-ended questions of survey participants identified fixed, money, and vaccineas predominate descriptors associated with veterinary care. The CGR is historically characterized as a region with social and economic challenges affecting health care access for people and their pets. Data from this research will help identify perceived barriers and strategies to mitigate them.

Research Grant: PetSmart Charities Student Support: None

244 • 2021 National Veterinary Scholars Symposium Thermographic evaluation of the ventral abdomen and jugular vein in healthy horses

Ava Mohiuddin, Jarred Williams, Kira Epstein, Naomi Crabtree

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Surgical site infection and jugular vein thrombophlebitis are major postoperative complications in hospitalized hors- es. Current diagnostic methods do not allow for early detection of such complications. Thermography may be used as a diagnostic tool for early detection of inflammatory processes based on abnormal body surface temperature. Normal thermographic data must be established for the ventral abdomen and jugular vein prior to clinical evaluation of celioto- my incisions and jugular vein catheterization sites, respectively. This study was conducted in order to validate the FLIR C5 thermographic camera for use in horses, identify a normal thermographic profile of the equine ventral abdomen and jugular vein, and evaluate the effects of clipping, sedation, and time of day on thermal imaging. It was hypothesized that thermographic images would be reproducible for a given horse, and that thermographic pattern would be unaffected by clipping, sedation, or time of day. Images of the ventral abdomen and jugular vein were obtained before and after seda- tion with an alpha-2 agonist, as well as before and after clipping with a #40 clipper blade. Following this, images of the clipped sites were obtained in the morning, afternoon, and evening over three consecutive days. Images were obtained in triplicate, with standardized camera settings and distance from the region of interest. Images were analyzed with standard- ized parameters using the camera manufacturer’s software. Initial image acquisition was easy to perform. Subjectively, clipping did not change the thermographic pattern of the region of interest, but the overall temperature of the clipped area appeared higher. Full data set and results pending.

Research Grant: Steve Lee Summer Research Fund, UGA College of Veterinary Medicine Student Support: Steve Lee Summer Research Stipend, UGA College of Veterinary Medicine

Development of a novel mRNA vaccine platform for Bovine Respiratory Syncytial virus

Michele Moncrief, Allen Miller, Michael Kimber, David Verhoeven

College of Veterinary Medicine and Department of Veterinary Microbiology and Preventive Medicine (Moncrief), Department of Plant Pathology (Miller), Department of Biomedical Sciences (Kimber), Department of Veterinary Microbiology and Preventive Medicine (Verhoeven), Iowa State University, Ames, Iowa

Due to the 2019 SARS-coV-2 pandemic, mRNA vaccine technology has clearly demonstrated high efficacy over other vaccine approaches. Moreover, mRNA vaccine technology offers a highly potent, efficacious, and adaptive alternative to traditional vaccines in preventing and controlling infectious diseases. However, current mRNA technology has significant hurdles before adoption for use as production animal vaccines, prompting a need for refinement of the technology. Here, we investigated an alternative mRNA expression and delivery system, seeking to overcome the economic, thermostabil- ity, and delivery issues currently limiting this technology. Our expression cassettes, based on plant viruses, allow for use of non-modified nucleotides and 5’ methyl cap-less and poly(A) tail-less mRNA that resist host endonucleases, stabilize expression in cells for days, and significantly decrease the cost of production.The use of extracellular vesicle expression through traditional cell line transfection was investigated for synthesizing our mRNA as well as packaging the transcripts for delivery as a vaccine carrier and as a further stabilizing element. Immune responses to a reporter protein were eval- uated versus traditional protein vaccines as a first proof of principle. In tandem, development of expression cassettes for bovine RSV proteins were developed for future testing. In summary, our mRNA technology should allow for further development and use of these vaccines for production animals and possibly enhance the efficacy of vaccines of traditional approaches

Research Grant: None Student Support: NIH T35 Training Grant T335OD012199

2021 National Veterinary Scholars Symposium • 245 Surfactant protein D in serum and bronchoalveolar lavage fluid from asthmatic horses and age-matched controls

Saira M. Ortega Morales, Ashley Duncan, Joey Morgan, Paul Wood, Undine Christmann

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, Tennessee

Equine asthma is a common noninfectious lower respiratory tract disease in horses. Diagnosis is based on history and clinical signs combined with an inflammatory profile on bronchoalveolar lavage fluid (BALF) cytology. Surfactant protein D (SP-D) has been suggested as a potential serum biomarker for mild equine asthma (MEA) with neutrophilic airway inflammation (MEA-N). Its utility has yet to be assessed in severe equine asthma (SEA) or MEA with eosinophilic airway inflammation (MEA-E). The purpose of our study was to evaluate SP-D levels in serum and BALF from horses with dif- ferent severities and inflammatory profiles of asthma and those of age-matched control horses. Horses for the study were recruited according to specific inclusion criteria. A detailed history and physical examination were obtained. Blood and BALF samples were collected and analyzed to obtain CBCs and BALF cytologies. Serum and BALF SP-D were mea- sured using a commercially available ELISA test. Twelve horses met the inclusion criteria for SEA, 8 horses were diag- nosed with MEA-N, and 10 horses were classified as MEA-E. Serum SP-D levels were significantly higher (P = 0.0189) in horses with SEA (median = 99.7 ng/ml; range = 11.4-249.7) compared to controls (26.8 ng/ml; 7.6-192.5). No signif- icant difference was seen in BALF SP-D levels in SEA versus control horses. In horses with MEA-N or MEA-E, neither serum nor BALF SP-D levels were significantly different compared to age-matched control horses. In conclusion, serum SP-D may aid in the diagnosis of equine asthma. However, elevated serum SP-D levels are likely tied to the severity of neutrophilic airway inflammation rather than to a specific disease.Additional studies are needed to investigate this further.

Research Grant: Lincoln Memorial University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program

Canine perineal hernia and comorbidities: a retrospective study

Mike Morgan, F. A. (Tony) Mann

Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Canine perineal hernia is associated with weakness of pelvic diaphragm muscles resulting in herniation of various ab- dominal contents into the perineal region. Most dogs with perineal hernia are presented when the owner notices perineal swelling or difficulty to defecate or urinate. Dogs with this condition are middle-aged or older and can have concurrent medical conditions. We propose to determine what comorbidities are common in dogs with perineal hernia and how those comorbidities influence case management. We hypothesize that most dogs with perineal hernia presented to the Veterinary Health Center (VHC) at the University of Missouri will have at least one comorbidity and that comorbidities will not af- fect the decision to proceed with hernia surgery. Medical records of the VHC will be searched from January 2005 to June 2021 to locate all cases of canine perineal hernia. Data recorded will include types and number of comorbidities, whether surgery was pursued, and case outcome. There were 118 canine perineal hernia cases during this time span. Of the 13 cases studied to date, 12 had at least one comorbidity and 9 had surgery performed. Comorbidities common to most cases were not identified. Of the 4 that did not have surgery, 1 was transferred to another facility, 1 had no outcome reported, and 2 had euthanasia. Euthanasia was performed due to multiple comorbidities in both cases. So far, it appears that most dogs with comorbidities can have perineal hernia surgery, but comorbidities can influence an owner’s decision to pursue surgery. More cases without surgery will be required to determine if any particular comorbidities influence the decision against surgical intervention.

Research Grant: None Student Support: American Society of Laboratory Animal Practitioners Foundation and IDEXX-BioAnalytics

246 • 2021 National Veterinary Scholars Symposium Domestic Animal Injuries and their Impacts on Vulnerable Wildlife Species in central Ohio

Clarice Morris

College of Veterinary Medicine, The Ohio State University, Ohio Wildlife Center

Human-wildlife conflicts have become a daily occurrence in urban areas. Some of these conflicts are linked to high rate of mortality in vulnerable wildlife species. An analysis of the Ohio Wildlife Center’s (OWC) admittance data shows an over- whelming number of wildlife presenting to the rehabilitation center with injuries or as orphaned infants due directly to an interaction with a domestic animal, cats and dogs. I reviewed 30,029 representing seventy-six different species admitted to the hospital over a four year period (2016-2020). I identified that domestic animal interactions make up 18% of total wildlife deaths brought to OWC. This conclusion supports the need for further investigation of domestic animal interac- tions with wildlife to understand and address the need to protect vulnerable wildlife species from domestic animal injuries. This information will aid veterinarians in the effort to both directly protect domestic animals from zoonotic diseases and indirectly protect wildlife populations from increasing rates of mortality due to inappropriate predation.

Research Grant: None Student Support: Ohio State University College of Veterinary Medicine, Global Health Veterinary Summer Research

Effects of Veterinary Handling on Canine Behaviour and Physiology

Allison Morse, Dr. Karen Overall, Dr. Camille Squair

Department of Behavioural Medicine, Atlantic Veterinary College, Charlottetown, Prince Edward Island

Signs of distress in canine patients during routine veterinary care has been a topic too often normalized and neglected in this field. Many owners admit to delaying care for their pets because they are aware of the mental and emotional that takes place during veterinary visits. The goal of this project is to develop science based recommendations for veter- inarians to reduce fear and anxiety in their patients. Recent studies in Sweden have suggested that allowing dogs to have some type of control and participation during examination is critical in minimizing their distress (Hernander 2008). We wish to evaluate if repeated exposure to the veterinary exam setting makes the behaviour of canine patients better, worse, or if it has no effect. We then want to determine if this behaviour is correlated with the physiological profile of the dog’s response to the exam. We are taking blood samples to measure L:N ratios, serum cortisol, and creatine phosphokinase. We also will be measuring heart rate and respiratory rate. We are hypothesizing that standard veterinary handling will result in higher physiological indicators of stress compared to those handled using fear reduction techniques. Animals should be free to exhibit natural behaviours during veterinary examinations and freedom from unpleasant states. Mental and emo- tional suffering of canine patients is a major welfare concern and through this research we aim to provide veterinarians learning techniques to mitigate this suffering and create a new precedent for the gold standard of care.

Research Grant: Sir James Dunn Animal Welfare Centre Student Support: Sir James Dunn Animal Welfare Centre Veterinary Summer Research Award (SJDAWC VetSRA)

2021 National Veterinary Scholars Symposium • 247 Effects of 25(OH)D3 on the transcriptome and cytokine production of canine peripheral blood mononuclear cells

Benjamin Morse, Seth Peng, John Loftus

Department of Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY

The bioactive Vitamin D metabolite 25(OH)D3 modulates gene expression and cytokine production in murine and human lymphocytes. We previously demonstrated canines with immune-mediated disease (IMD) have low serum 25(OH)D3 concentrations. Given the potential of 25(OH)D3 to attenuate immune responses, we wondered if the low vitamin D levels contributed to the disease state. Here we report the effect ofin vitro incubation in 25(OH)D3 on the cytokine secretion and gene expression profiles of canine peripheral blood mononuclear cells (PBMCs).Whole blood was harvested from healthy Alaskan sled dogs (n = 3). PBMCs were isolated, incubated in calcitriol-supplemented medium, then RNA was extracted. RNA samples were sequenced using a next-generation Illumina sequencer, aligned with a standard canine genome, and quantified. Differential gene expression analysis identified differentially expressed transcripts across sample conditions. ELISpot assays on isolated PBMCs quantified cells secreting IFNg, IL-2, IL-4, IL-10, and IL-17 after stimulation with mitogens. A magnetic bead cytokine panel further characterized the general secretion profiles of the PBMC populations.

We hypothesize the immunomodulatory effects of 25(OH)D3 will cause reduced secretion of the pro-inflammatory cyto- kine IFNg and increased secretion of the anti-inflammatory cytokine IL-10.We also hypothesize the differential gene ex- pression analysis will reflect the changes in the genes for cytokines and downregulation of cell differentiation and prolifer- ation pathways. These results will probe the association between serum 25(OH)D3 deficiency and IMD and provide clues as to the viability of Vitamin D as a supplementary intervention for IMD.

Research Grant: Nestlé Purina Resident’s Research Grant Cornell University Faculty Start Up Grant Student Support: Cornell Canine Health Center

Evaluating treatments for bovine trichomonosis in a natural host model

Erica Moscoso, Katy A. Martin, Kris Kovach, Matt Brewer

Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes early embryonic death in cattle and results in severe economic loss. In the United States, there are no drugs currently approved for treatment of this parasite. While numerous compounds have been shown to kill T. foetus trophozoites in vitro, very few studies have attempted to treat infected cattle. In this study, we explored the effect of chlorhexidine gluconate (CG) onT. foetus. In vitro CG studies revealed EC50 values in the micromolar range. These EC50 values were significantly lower than the CG concentration used on mucosal surfaces in other applications. We investigated the utility of CG as a treatment for bovine trichomonosis in a heifer calf model. Infected calves were treated on 3 consecutive days by CG vaginal lavage. Histopathology, parasite cul- ture, and qPCR revealed decreased parasite loads in treated animals. Chlorhexidine gluconate lavage is a tractable option for treating bovine trichomonosis as it can be used in food animals. Future studies will address the use of preputial CG lavage for treatment of infected bulls.

Research Grant: This research was supported by funds from the Iowa Livestock Health Advisory Council and the Frank Ramsey Endowed Chair in Veterinary Medicine provided to Matt Brewer Student Support: The Iowa Livestock Health Advisory Council

248 • 2021 National Veterinary Scholars Symposium Plasma and tissue disposition of ceftazidime after multiple intravenous doses in healthy neonatal foals

Christina J. Mozeika, Clare A. Ryan, Londa J. Berghaus, Brenton C. Credille

Department of Large Animal Medicine (Mozeika, Ryan, Berghaus), Department of Population Health (Credille), College of Veterinary Medicine, University of Georgia, Athens, Georgia

Sepsis in neonatal foals has high mortality rates, ranging from 30-80%. Rapid recognition of illness and early, appropri- ate antimicrobial therapy are required for the successful management of septic foals. Nevertheless, certain antimicrobials (i.e.aminoglycosides) are contraindicated in foals with hypovolemia and azotemia. Ceftazidime, a third-generation ceph- alosporin, provides coverage against common pathogens, a good safety profile, and penetration into cerebrospinal fluid (CSF), making it ideal for use in septic foals with comorbidities such as meningitis. The plasma disposition of single intravenous (IV) and intramuscular (IM) doses of ceftazidime in neonatal foals has been recently described, but further evidence is needed to support the routine clinical use of ceftazidime in septic foals. This study examines the plasma dis- position of ceftazidime in neonatal foals after multiple doses (25 mg/kg IV, q 8 hr, 6 doses). Ceftazidime administration was well tolerated in all foals. The concentration of ceftazidime in CSF, pulmonary epithelial lining fluid (PELF), synovial fluid, and urine are being measured using an agar well diffusion microbiological assay. Multiple dose pharmacokinetics of ceftazidime in neonatal foals and the tissue concentrations achieved after multiple doses will be described. The informa- tion gained from this study will have immediate clinical impact for equine practitioners treating septic foals with azotemia or hypovolemia, or for foals with sepsis-related meningitis. This study provides critical information needed for future studies establishing susceptibility breakpoints of ceftazidime for veterinary isolates and comparing these breakpoints to tissue concentrations described in this study.

Research Grant: For the Love of the Horse – Equine Endowment Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

Weighted-incidence syndromic antibiograms for urine cultures in cats and dogs

Cassie Munroe, Kendall Leet-Otley, Kelly Chambers, Annie Wayne, Claire Fellman

Department of Clinical Sciences (Wayne, Fellman), Cummings School of Veterinary Medicine (Munroe, Leet-Otley, Chambers), Tufts University, North Grafton, Massachusetts

Antibiograms are a useful tool to guide empirical antimicrobial selection but traditional antibiograms are most helpful when the infecting organism is known. In this project, weighted-incidence syndromic antibiograms were created for urine cultures from cats and dogs from a veterinary tertiary care center. Syndromic antibiograms present the probability of sus- ceptibility for a specific infection location taking into account the relative frequency of pathogens causing the syndrome. Susceptibility results and antimicrobial minimum inhibitory concentrations were recorded over the course of two years for dogs and five years for cats. Syndromic antibiograms were created using a weighted average of susceptibility data for bacterial species with at least 30 cultured isolates. E. coli, mirabilis, sp., Staphylococcus pseudinterme- dius and Enterococcus faecalis were the most common bacteria cultured from dog urine. E. coli, Staphylococcus felis and Enterococcus species were most common in cat urine. Amoxicillin, cephalexin, amoxicillin-clavulanic acid, enrofloxacin and trimethoprim sulfa (TMS) are common antimicrobials prescribed to dogs and cats with positive urine cultures. Syn- dromically in dogs, amoxicillin was susceptible only 58% of the time whereas cephalexin was susceptible at 67%, amox- icillin-clavulanic acid at 80%, enrofloxacin at 84%, and TMS at 83%. In cats, amoxicillin was susceptible only 71% of the time whereas cephalexin was susceptible at 79%, amoxicillin-clavulanic acid at 84%, enrofloxacin at 87%, andTMS at 85%. Syndromic antibiograms are a promising tool for veterinary prescribers. Continued annual data collection will increase the accuracy and identification of syndromic susceptibility.

Research Grant: None Student Support: Blue Buffalo

2021 National Veterinary Scholars Symposium • 249 Cannabinoids modulate adipogenesis and lipogenesis in periparturient dairy cows

Madison N. Myers, Miguel Chirivi, Ursula Abou-Rjeileh, Maya Zachut, Joseph Tam, G. Andres Contreras

Dept. of Large Animal Clinical Sciences (Myers, Chirivi, Abou-Rjeileh, Contreras), College of Vet. Med., Michigan State U, East Lansing, MI, USA. Dept. of Ruminant Science (Zachut), Volcani Center, Rishon LeZion, Israel. Obesity and Metabolism Laboratory (Tam), Faculty of Medicine, Hebrew U of Jerusalem, Jerusalem, Israel

Increased adipose tissue (AT) lipolysis and suppressed adipogenesis and lipogenesis characterize the periparturient period of dairy cows. The intensity of these processes recedes with the progression of lactation; however, disease risk is exacer- bated when dysregulation occurs. Cannabinoids (CBs) activate CB receptor 1 (CB1) in monogastric AT and enhance the energy-conserving capacity of adipocytes, yet their effects in dairy cow AT remain unknown. We determined the effects of the synthetic CB arachidonyl-2’-chloroethylamide (ACEA)+/-the CB1 antagonist rimonabant (RIM) in cultured dairy cow adipocytes. AT was collected from multiparous, nonlactating, non-gestating Holstein cows (n = 8). Stromal vascular fraction was obtained via liberase digestion. Pre-adipocytes were isolated and induced to differentiate upon confluency for 14d. Adipocytes were exposed to ACEA at 10mM (A10)+/-RIM pre-treatment at 0.1mM for 2 (adipogenesis) or 4d (lipogenesis). Viability, adipogenesis, and lipogenesis were evaluated using commercially available plate-based assays and the IncuCyte S3. Adipogenic efficiency calculated as # of cells with 1+ lipid droplet/total # of cells/well. Statisti- cal analyses performed in JMP. Cell viability was unaffected by treatments. Adipogenesis was enhanced (P < 0.05) by A10 (54.564.3%) vs. CON (44.2364.3%). RIM reduced adipogenesis in ACEA-treated cells (46.2564.1%) vs. CON (53.5964.1%). Increased lipid accumulation (RFU/ng DNA6SEM) was observed in A10 (3115.436278) compared to CON (1849.45). Lipid content was reduced in A10+RIM (2681.19), but not RIM (1840.40). Collectively, these findings suggest that adipogenesis and lipogenesis may be enhanced through activation of CB1 by CBs in dairy cow AT.

Research Grant: US-Israel Binational Agricultural Research and Development Fund, USDA NIFA 2019-67015-29443, and the MSU CVM (East Lansing, MI) Student Support: NIH Grant 5T35OD016477-19 to Michigan State University

Evaluation of diet-induced obesity on mRNA and ion channel expression in murine hypoglossal motoneurons

Mikaela N. Myers, Ann L. Revill

College of Veterinary Medicine (Myers), Department of Physiology, College of Graduate Studies (Revill), Midwestern University, Glendale, AZ

Obstructive sleep apnea (OSA) is characterized by loss of airway patency during sleep, in part due to a lack of tongue muscle tone. Obesity is a strong risk factor for OSA; disease prevalence is approximately double that of normal weight patients. This trend also extends to animals, especially rats, pigs, and English bulldogs. Obesity is associated with hyper- leptinemia in peripheral circulation. Leptin is normally transported across the blood brain barrier (BBB) via transport pro- teins to act on leptin receptors in the central nervous system (CNS), including brainstem regions involved in the control of breathing. Upon leptin binding to its receptor, the activated signaling cascade leads to restructuring of actin skeletons to activate ion channels, including KATP and BK potassium channels. The leptin transporters in the CNS are saturable, so peripheral hyperleptinemia leads to receptor downregulation at the BBB in obese patients, and leptin deficiency in the CNS. CNS leptin deficiency may be partially responsible for OSA because, regardless of weight, OSA patients have plas- ma leptin levels 50% higher than controls, and the severity of OSA is proportional to the degree of hyperleptinemia. We hypothesize that CNS leptin deficiency due to diet-induced obesity will alter potassium channel trafficking and decrease excitability of hypoglossal motoneurons, which innervate the tongue. Using microarray analysis and immunofluorescence methods, we will investigate mRNA, ion channel, and receptor level changes in the hypoglossal motor nucleus, in re- sponse to diet-induced obesity and leptin deficiency in the CNS.The results of this study will seek to elucidate a mecha- nism between obesity and the onset of OSA.

Research Grant: Midwestern University Start-Up Funds Student Support: Boehringer Ingelheim Veterinary Scholars Program

250 • 2021 National Veterinary Scholars Symposium The role of the gut microbiome on neurogenesis and cortical development in germ free piglets

Julia Mykins, Paul Morton

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA

Neurodevelopmental disorders such as autism often have a pattern of comorbidities including a higher occurrence of gastrointestinal disease. The presence of gastrointestinal disorders in a large portion of patients with autism suggests there may be a physiologic basis to this neurodevelopmental disorder. A bidirectional communication between the central ner- vous system and gastrointestinal tract has previously been established and known as the gut-brain axis in which the two communicate through the vagus nerve, circulatory system, or the immune system. Preliminary data from our lab found a significant reduction in the number of neural stem cells/progenitor cells and proliferation in the subventricular zone of 16-day old piglets raised under germ-free conditions. The brain of 16-day old piglets is equivalent to that of a 6 month old human and is a critical time for cortical development. The aim of this study is to determine the effect of reduced neurogen- esis on gross anatomy of the cortical regions in 16-day old germ free piglets. Results of MRI analysis showed a trending but insignificant decrease in the cortical volume of germ-free piglets compared to the control.The gyrification index of the germ-free also trended downward but was ultimately statistically insignificant. Graphing the gyrification index rostral to caudal revealed differences in cortical folding among the two groups with hotspots in the somatosensory, insular, and visual cortices.

Research Grant: R15NS108183 awarded to PDM Student Support: T35OD011887D

ATP7A, ATP7B, and RETN genotypes in Labrador retrievers with and without copper associated hepatitis

Brendan Nagler, Rebecca Smedley, Daniel Langlois, Vilma Yuzbasiyan-Gurkan

Small Animal Clinical Sciences (Nagler, Langlois, Yuzbasiyan-Gurkan), Veterinary Diagnostic Laboratory (Smedley), and Department of Microbiology and Molecular Genetics (Yuzbasiyan-Gurkan), College of Veterinary Medicine, Michigan State University, East Lansing, MI

Copper associated hepatitis (CAH) is one of the most common causes of chronic liver disease in dogs. A genetic muta- tion leading to CAH has only been identified in the Bedlington terrier. However, the disease is prevalent in several breeds including the Labrador retriever. Coding variants in the copper transporting genes ATP7A and ATP7B have been associ- ated with decreased or increased risk of CAH, respectively, in a Dutch population of Labradors. Genetic testing for these variants is widely available, but the prevalence and significance of these variants inAmerican Labradors is unknown. Recently, a variant in the RETN gene has also been associated with higher copper levels in Dutch Labradors. The aim of this study is to evaluate ATP7A, ATP7B, and RETN genotypes in Labrador retrievers to determine if there is a significant difference in the prevalence of these variants in dogs with and without CAH in the United States.Archived formalin fixed paraffin embedded (FFPE) liver samples from Labrador retrievers (n = 95) collected between 2013-2021 were evaluated for CAH by assessing histopathologic features and copper accumulation. DNA is being extracted from these FFPE tissues and genotyped using TaqMan custom probes with real time PCR. We will evaluate allele frequencies at these loci in Labradors with and without CAH. Our findings will inform clinicians and breeders as they make treatment and breeding decisions.

Research Grant: Michigan State University College of Veterinary Medicine Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

2021 National Veterinary Scholars Symposium • 251 Heavy Metal Associated Resistance in Avian Pathogenic Escherichia coli (APEC)

Charlize Nakatsu, and Catherine M. Logue

Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Avian pathogenic Escherichia coli (APEC) is an extra-intestinal pathogen that causes respiratory and systemic disease in poultry production. Impacts of APEC include increased mortality in young chickens (up to 53.5%) losses due to poor quality, health, and carcass condemnation at slaughter. Here, we assessed APEC from cases of colibacillosis collected from international collaborators for metal resistance using broth microdilution for phenotype analysis and polymerase chain reaction (PCR) to detect metal associated genes. A survey of 80 isolates from regions such as Peru, Colombia, Bra- zil, Mexico, and the Dominican Republic were examined to determine resistance to copper, silver, zinc, lead, chromium, cadmium, arsenic, cobalt, manganese, nickel, and mercury. All isolates were assessed for minimum inhibitory concentra- tion (MIC) of metals using the broth microdilution assay at concentration ranges of 1600 mg/ml to 0.78 mg/ml. Isolates were also screened for metal resistance associated genes using PCR analysis. The results found that all APEC isolates displayed growth in arsenic, copper, lead, chromium, manganese, and nickel at high levels with MICs ranging between > 1600 mg/ml to 800 mg/ml. APEC exhibited lower resistance to the metals silver and mercury where the MICs ranged from 6.25 mg/ml to < 0.78 mg/ml. Using PCR, genes detected included pbrT (lead), chrA (chromium), and zntB (zinc) among others. In addition to providing valuable insight as to what metals could potentially be used therapeutically to help control the spread of APEC, it is beneficial to characterize APEC’s resistance to heavy metals as well as the genetic mechanisms used in order to gain this resistance as a key to better understanding APEC as a whole.

Research Grant: US Poultry and Egg Association Project #726 Student Support: Boehringer-Ingelheim Georgia Veterinary Scholars Program

Improving neonatal dairy calf mucosal immune response via micronutrient supplementation

Arpita Nayak, Ángel Abuelo

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Although there have been several advancements in calf management in recent years, it remains that there has been lit- tle to no decrease in the number of neonatal calf deaths. As a response to this, strategies such as intranasal vaccination have become points of interest in calf management. Intranasal vaccines stimulate mucosal immunity; therefore, maternal antibodies cannot interfere with the vaccine response. One issue with intranasal vaccines is that the immune response is short-lived. Another issue is that newborn calves also experience oxidative stress which limits their immune response. Though there are very few guidelines on micronutrient supplementation in calves, it was shown that antioxidant micronu- trient supplementation in cows lead to an improved immune response. For these reasons, this study aims to test the extent to which commercial antioxidant micronutrient supplements improve the mucosal immune response to intranasal vaccines in calves. 21 neonatal calves were randomly assigned at birth to placebo or one of two commercially available micronu- trient supplements. Nasal secretion samples were collected one time before administration of an intranasal vaccine, and once weekly for the next four weeks. These samples were used to quantify immunoglobulin A, an antibody used to assess mucosal immunity. It is anticipated that the calves that received one of the commercial micronutrient supplements will have an increased mucosal immune response to the intranasal vaccine compared to the calves that received the placebo. This result would be significant because it depicts how calf immunity can be improved with micronutrient supplementa- tion, which could lead to development and optimization of better calf health practices.

Research Grant: Michigan Animal Health Foundation, Michigan Alliance for Animal Agriculture Student Support: NIH T-35 Grant 5T35OD016477-19

252 • 2021 National Veterinary Scholars Symposium Real-time PCR compared to modified Knott’s for detecting heartworm microfilariae in routine heartworm testing

Veronica Negron, Meriam N. Saleh, Caroline Sobotyk, Guilherme G. Verocai

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M Universi- ty, College Station, TX

The canine heartworm, Dirofilaria immitis, is a mosquito-borne filarial nematode that affects dogs globally. Heartworm infection is highly prevalent in areas of the United States. If left untreated, heartworm disease can lead to severe mul- tisystemic effects or even be fatal. The American Heartworm Society recommends annual screening of every dog for heartworm infection using both a microfilariae detection test (MFDT) and antigen test. It is hypothesized that real-time PCR (qPCR) assays will have higher sensitivity in detecting microfilariae (mf) than traditional microscopy based MFDT, such as the modified Knott’s test (MK). We aimed to compare the performance of a qPCR assay to MK for detection of D. immitis in canine blood. We collected 237 matching blood and serum samples from shelter dogs in Texas. Blood was assessed for the presence of mf of filarial nematodes by MK andD. immitis DNA by qPCR. Serum samples were tested for the presence of heartworm antigen using a commercial ELISA pre- and post-immune complex dissociation (ICD) by heat treatment. Qualitatively, MK detected mf in 23.2% (55/237) of samples, 46 of which were morphologically identified as D. immitis (19.4%), and qPCR detected D. immitis DNA in 21.9% (52/237) of samples. Agreement between qPCR and MK for D. immitis was almost perfect (Kappa = 0.897). The combination of qPCR and antigen detection pre- and post- ICD detected 26.6% and 30.4% of dogs as infected, respectively; while MK combined with antigen detection pre- and post-ICD detected 27.0% and 30.8%, respectively. Our results suggest that qPCR is more sensitive than MK for the detec- tion of microfilaremia byD. immitis in dogs and is comparable to MK when both are combined with antigen testing.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

Early identification of canine benign prostatic hyperplasia using prostatic fluid metabolomic biomarkers

Alexis Nelson, Theresa Beachler, Jean-Sebastien Palerme, Margert Musser, Joseph DiBenedetto

Department of Veterinary Clinical Sciences (Nelson, Palerme, Musser, DiBenedetto), Department of Veterinary Diagnostic and Production Animal Medicine (Beachler), College of Veterinary Medicine, Iowa State University, Ames, Iowa

By five years of age, over 80% of intact male dogs will show signs of benign prostatic hyperplasia (BPH). In early dis- ease, BPH can be challenging to detect until there is enough prostatic enlargement that clinical signs such as difficulty urinating, painful ejaculation, or prostatitis occur. In breeding males, BPH can cause a reduction in fertility due to poor spermatozoa morphology and nuclear DNA fragmentation. The current challenge is knowing when medical treatment should begin prior to when clinical signs and fertility issues become apparent. This study’s objective is to use metabolom- ics in order to create a metabolite profile of canine prostatic fluid using mass spectroscopy in order to identify biomarkers indicative of BPH. Routine semen analysis consisting of concentration analysis using a fluorescence-based cell counter and motility and morphology assessment using light microscopy was performed following manual semen collection to collect prostatic fluid for metabolomic analysis. Additionally, prostatic fluid cytology was performed in addition to an ultrasonographic assessment of prostatic volume and architecture to identify males affected by prostatic disease. Prostatic metabolomic analysis via mass spectrometry remains pending. To date, six intact males have been enrolled from 1.5 to 7 years of age. There has been a range of breed sizes from toy to giant breeds, including Papillon, Poodle, Labrador Retriev- er, and Bernese Mountain Dog. Mean prostatic volume has ranged from 5.01 6 1.53 cm3 in toy breeds (n = 2), 6.97 6 3.67 cm3 in medium breeds (n = 2), and 19.54 6 7.74 cm3 in giant breeds (n = 2) with no evidence of prostatic abnormalities.

Research Grant: ISU VCS Incentive Grant Student Support: Iowa Livestock Health Advisory Council

2021 National Veterinary Scholars Symposium • 253 Utilization focused evaluation to develop a sustainable program of outcomes assessment

Caitlyn M. Nelson, Tamara S. Hancock

Department of Veterinary Pathobiology (Hancock) and Veterinary Research Scholars Program (Nelson), College of Veterinary Medicine, University of Missouri, Columbia, MO

The University of Missouri College of Veterinary Medicine’s Veterinary Research Scholars Program (VRSP) provides mentored research experiences to veterinary students. The objectives of the VRSP are to expose students to research ca- reer opportunities and develop a community of research scientists. Currently the VRSP uses outcomes assessment prac- tices to determine if they are meeting these objectives. However, it is uncertain if this program of assessment sufficiently captures whether or not the VRSP effectively meets their goals. Furthermore, these practices are self-described as limited and labor intensive. We are collaborating with VRSP leadership to develop and pilot an improved program of outcomes assessment to support their program evaluation and improvement needs. This utilization focused evaluation project uses various methods, including informal interviews and pre/post surveys, to develop suggestions for an improved program of assessment that will be sustainable for the VRSP. Evaluation projects can “contribute to dialogue and help shape under- standing of social programs” (Savin-Baden & Howell-Major, 2013, p. 275). Mentored research experiences, such as those in the VRSP, can impact students’ intentions to pursue careers in research, outcomes including scholarly productivity, and disciplinary persistence (Hu et al., 2007; Aikens et al., 2017). Generating a more accurate view of student outcomes from mentored research experiences in the VRSP will enable programmatic refinement to better capture goal achievement and document sustained impacts of their program.

Research Grant: None Student Support: University of Missouri College of Veterinary Medicine Office of Research

Effects of steroid implants and differing tall fescue endophyteAcremonium ( coenophialum) levels on cattle

Joel Nelson, Blaine Johnson, Jaymelynn Farney, Bob Larson

Department of Clinical Sciences (Nelson, Johnson, Larson), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas; Department of Animal Science (Farney), Kansas State University, Parsons, Kansas

The endophyte (Acremonium coenophialum) commonly infecting tall fescue costs the American cattle industry up to 2 bil- lion dollars annually. The endophyte is known to cause decreased weight gain, increased body temperature, and retained hair coat. The objective of this study was to determine if there is a difference in the magnitude of toxic effects in cattle treated with different types of growth-promoting implants and grazing differing levels and types of endophyte infected tall fescue. We hypothesized that cattle grazing endophyte-free and novel-endophyte tall fescue will have better growth performance and lower body temperature than cattle grazing low- or high-endophyte tall fescue, and that growth implants will modulate the endophyte effects. To execute this study, sixty-four growing steers were used in a multi-factorial study to investigate 4 levels of endophyte: K31 high endophyte (HIGH), K31 low endophyte (LOW), endophyte free (Free), novel endophyte (Novel) tall fescue and 4 implant treatments: no implant (NO), Ralgro (Ral), Revalor-G (RG), and Syn- ovex One Grass (SOG), and 2 legume treatments: no legumes and clover. When analyzing ADG to day-84, we found an interaction between endophyte type and implant (P < .01). We identified interactions among endophyte type and implant type that impacted ADG, and hair length, but not cortisol. We found that cattle grazing high endophyte fescue showed slower growth and longer hair length, whereas cattle grazing Free, LOW, and Novel endophyte showed fewer negative effects. In the first 84 days, this study did not identify a consistent effect of growth implant ADGon across types of tall fescue.

Research Grant: This work was supported by the USDA National Institute of Food and Agriculture, Hatch-Multistate project 1021429. IACUC number 4372 Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program

254 • 2021 National Veterinary Scholars Symposium Using DAPI-PI staining to determine Cryptosporidium parvum oocyst viability compared to qPCR quantification

Holly M. Newberne, Sierra R. Guynn, Jennifer A. Zambriski

Department of Population Health Sciences (Newberne, Zambriski) and Large Animal Clinical Sciences (Guynn), Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA

Cryptosporidium spp. are protozoal, zoonotic parasites causing significant diarrheal morbidity and mortality due to diar- rhea. Cryptosporidiosis is an important cause of illness in dairy calves and is the second leading cause of diarrheal death in children < 5 worldwide. The calf model of cryptosporidiosis is an important tool in studying the efficacy of novel che- motherapeutics targeting Cryptosporidium spp. for use in children, as the severity of clinical illness and intestinal pathol- ogy mirrors that of calves. Several novel chemotherapeutic agents have demonstrated efficacy in the calf model resulting in improved clinical health and diarrheal severity. Previous studies in the calf model have also demonstrated a significant reduction in fecal oocyst shedding via real-time polymerase chain reaction (qPCR). The limitation of qPCR is that it detects and enumerates the DNA of all oocysts in the sample, regardless of oocyst viability. Thus, parasite quantification is a reflection of parasite shedding from the GI tract and not parasitological cure. Novel chemotherapeutics targeting Cryp- tosporidium spp. may have a direct parasiticidal effect that is undetected by qPCR.The goal of this study was to quantify fecal oocyst shedding via qPCR in calves treated with novel chemotherapeutics known to be efficacious againstC. parvum and determine the effect of the chemotherapeutic on oocyst viability. Viability testing was conducted using 4’,6-diamidi- no-2-phenylindole (DAPI), propidium iodide (PI), and fluorescein. Oocysts stained with DAPI and PI signified a ruptured cell wall and non-viability. Viability was reported as a proportion of non-viable (DAPI+/PI+) to viable (fluorescein +) oocysts. Data analysis is ongoing.

Research Grant: PATH Student Support: Virginia-Maryland College of Veterinary support

Investigating feline endogenous retroviruses as immunotherapy targets for squamous cell carcinoma

Victoria L. Newberry, Jennifer C. Holmes, Paul R. Hess

Department of Clinical Sciences, NC State College of Veterinary Medicine, Raleigh, North Carolina

An important component of developing immunotherapies for cancers resistant to traditional treatments is to identify tumor-exclusive proteins (antigens) that serve as targets for killer T cells. A cutting-edge human study showed that intra- tumoral T cells are attracted to both patient-specific mutations and de-repressed endogenous retroviruses (ERVs), which would be shared by patients with the same tumor type and valuable for “off-the-shelf” therapy. ERVs constitute large chunks (~10%) of genomic DNA as relics of germ cell infections. Their expression is normally repressed by epigene- tic and intracellular immune mechanisms. Our hypothesis is that some ERVs that are prevalent in the feline genome are expressed in feline oral squamous cell carcinoma (FOSCC) but not in normal tissue and would be an important antigen target in this aggressive, incurable cancer. Since some feline ERVs are ancient and genetically fixed while others are still invading the genome at a variable prevalence, our first objective was to define the genomic prevalence of three ERVs, gamma4-X2, enFeLV, and DC-10, in cats from the US. Our second objective was to compare expression of any prevalent ERVs in FOSCC samples and normal peripheral blood mononuclear cells (PBMCs). We found that the prevalence of the gamma4-X2, FeLV and DC10 ERVs was 100%, 100%, and 18.75%, respectively, via PCR in cat gDNA. We probed for expression in normal PBMCs and FOSCC tumor samples via RT-PCR and discovered that none were transcribed in either tissue types, despite being gDNA-positive for the ERVs. Our sample size was small though (n = 1-3), and more research should be performed to determine if these ERVs could serve as a potential target for an anti-cancer vaccine in cats.

Research Grant: NC State Coat of Excellence Award Student Support: Student Support: VLN NC State Veterinary Scholars Program and the BIVSP

2021 National Veterinary Scholars Symposium • 255 The effects of chronic nicotine exposure on cardiac remodeling and arrhythmia susceptibility in a rabbit model

Lena Ngo, Amanda Guevara, Jessica L. Caldwell, Eric I-Ju Lee, and Crystal M. Ripplinger

School of Veterinary Medicine (Ngo), Department of Pharmacology (Guevara, Caldwell, Lee, Ripplinger), University of California, Davis, Davis, CA

Habitual tobacco use increases the risk of atrial and ventricular arrhythmias. While the cardiotoxic behavior of tobac- co smoke is well established, the individual factors contributing to pro-arrhythmic changes are unknown. The objective of this study is to determine the effects of nicotine on cardiac electrophysiology, structural remodeling, and molecular function. It is hypothesized that chronic nicotine exposure will result in sympathetic hyperactivity, diminished sympathetic responsiveness, structural remodeling of cardiomyocytes, and alterations in key ion channel activity and Ca2+ handling proteins. Optical imaging was performed on fully innervated hearts from New Zealand White rabbits exposed to nico- tine (NIC, 21mg/day, transdermal patch, 28 days, n = 7) and control (CT, n = 4) hearts. Preliminary results suggest that baseline heart rate (HR) was higher in NIC vs CT (214.5 vs 176.9 BPM, P = NS). Sympathetic nerve stimulation (SNS) thresholds were higher in NIC vs CT (6.25 vs 1.17 Hz, P = NS). Maximal HR increase with SNS trended lower in NIC vs CT (36.37 vs 64.91%, P = NS). Sympathetic nerve density was unchanged in NIC vs CT (7.86 4.0% vs 9.96 4.5%, P = NS) as determined by immunolabeling and quantification of tyrosine hydroxylase. Fibrotic density was decreased in NIC vs CT (14.26 1.8% vs 17.66 1.5%, P < 0.05) as determined by Masson’s Trichrome staining. Western blotting assays are currently underway to assess the expression and phosphorylation of key ion channels and Ca2+ handling proteins. Given the increasing popularity of alternative tobacco products among teenagers, this study’s investigation into the arrhythmo- genic properties of nicotine may provide novel insight into the deleterious effects of chronic nicotine use.

Research Grant: California Tobacco-Related Disease Research Program #T29IP0365C Student Support: University of California, Davis, School of Veterinary Medicine STAR Program

Assessing changes in myeloid derived suppressor cells in canine melanoma patients treated with immunotherapy

Symphony L. Nguy, Hansjorg Rindt, Jeffrey N. Bryan, and Carol R. Reinero

Comparative Internal Medicine Laboratory (Nguy, Rindt, Reinero), Comparative Oncology Radiobiology and Epigenetics Laboratory (Bryan), College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Myeloid derived suppressor cells (MDSC) cause immunosuppression in humans with cancer and promote tumor tolerance. While MDSC are found at low levels in the circulation of healthy individuals, increases can occur with cancer that result in suppression of T cell proliferation, synthesis of inhibitory soluble factors, and T regulatory cell proliferation. These effects on immunity may interfere with immunotherapy used to treat cancer. Canine melanoma is an excellent translation- al model of human melanoma for which there is an experimental adjunctive immunotherapy consisting of an autologous deglycosylated vaccine that reactivates tumor-specific lymphocytes. The study objective is to test whether the percentage of MDSC in cryopreserved peripheral blood mononuclear cells (PBMC) of dogs with melanoma treated with the canine melanoma vaccine will decrease and if there is a relationship between MDSC percentage (baseline and post-vaccine) and remission as a clinical outcome. We hypothesize that dogs with melanoma have a higher %MDSC before treatment than healthy dogs, and that post-vaccination %MDSC will decrease either by half compared to baseline values or to similar percentages of cryopreserved PBMC from healthy control dogs. Dogs with the most marked post-vaccinal reduction in %MDSC are also hypothesized to have a better outcome (i.e., dogs with complete and partial remissions). We will use lymphoid and myeloid surface markers to identify MDSC in banked cryopreserved PBMC via flow cytometry. We expect in dogs with minimal post-vaccinal decreases of MDSC failure to achieve clinical remission. A decrease in MDSC after treatment could support future studies evaluating MDSC as a potential therapeutic target and prognostic factor.

Research Grant: Siteman Investment Program Grant and Investigator Discretionary Funds Student Support: Boehringer Ingelheim

256 • 2021 National Veterinary Scholars Symposium Developing canine atopic dermatitis biomarkers

Jeffrey T. Nguyen, Chen Xie, Charli Dong, Gagandeep Kaur, Jijun Hao

Western University of Health Sciences, College of Veterinary Medicine, Pomona, California (Nguyen, Xie, Kaur, Hao); Animal Dermatology Clinic, Pasadena, California (Dong)

Canine atopic dermatitis (cAD) is a genetically predisposed, allergic skin disease characterized by inflammation, pruritus, and secondary microbial infection. Currently, the diagnosis of cAD is complex since predictive tests are not available. Diagnosis requires detailed patient history, clinical signs, and the elimination of other similarly presenting skin diseases. Our preliminary data suggests that phosphodiesterase 4D (PDE4D) gene expression in peripheral blood mononuclear cells (PBMCs) and miR-203 concentrations in plasma are significantly changed in atopic patients in contrast to the healthy ones. This current study seeks to investigate the sensitivity and specificity of these two biomarkers in atopic dogs (n = 27), dogs with cAD-like diseases (n = 27), and healthy controls (n = 27). We will examine if expression levels of PDE4D in PBMCs and miR-203 in plasma are specific and sensitive to patients with cAD. In addition, we seek to determine if expression levels of each biomarker correlate with severity of clinical signs observed. Currently, we have processed the samples of 2 cAD patients, 3 cAD-like patients, and 17 healthy controls.

Research Grant: Boehringer Ingelheim Veterinary Scholars Program Student Support: WesternU College of Veterinary Medicine - Veterinary Summer Research Program

Longitudinal assessment of structural brain alterations in a mouse model of Gulf War Illness

Sarah H Nguyen, Jessica M Carpenter, Kim M Mason, Nikolay M Filipov

Department of Physiology and Pharmacology (Nguyen, Carpenter, Filipov), College of Veterinary Medicine, University of Georgia, Athens, GA and Bio-Imaging Research Center (Mason), University of Georgia, Athens, GA

Gulf War Illness (GWI) is a chronic illness affecting 30% of GW veterans. Overexposures to chemicals such as the nerve agent prophylactic pyridostigmine bromide (PB) and pesticides such as permethrin (PM) are linked to GWI etiology. While laboratory GWI models have mirrored many of the behavioral deficits seen in GWI veterans, comparative struc- tural brain changes within these models remain unknown. This study aimed to assess the progression of structural brain changes in a GWI model over 9 months. Male C57BL6/J mice (8-9 weeks old) were co-administered PB and PM (0.7 and 200 mg/kg IP) or DMSO control for 10 days. Using a Varian 7T Bore MRI System, structural (coronal T1- and sagittal T2-weighted) scans were performed at 3 and 6 months post-chemical exposure. Regions of interests (ROIs), including the hippocampus, cerebellum, lateral and third ventricles, were demarcated in MATLAB (Aedes toolbox), followed by volu- metric analysis in ImageJ. Of the data analyzed to date, there were no treatment differences observed for ROIs compared at 3 or 6 months. However, there were significant longitudinal decreases in hippocampal volume for the PB/PM group and in cerebellar volumes for both treatment groups from 3 to 6 months; there were no significant changes for the ventricles’ volumes. These structural brain alterations align with the clinically observed decreases in hippocampal volume of veterans with GWI as well as behavioral deficits previously observed in this model. Cerebellar volume changes seen in the controls indicate this brain structure may succumb to the effects of age earlier than the rest of the brain. Overall, this improved characterization affirms the utility of this model for future therapeutic evaluations of GWI.

Research Grant: This research was supported by Franklin Foundation Imaging Award awarded to Jessica Carpenter and Department of Defense grant W81XWH-16-1-0586 to Nikolay Filipov Student Support: NIH Office of Research Infrastructure Programs, Grant Number 5T35OD010433-14

2021 National Veterinary Scholars Symposium • 257 BRAF mutations in feline tumors

Lucie G. Noall, Jeffrey N. Bryan, Gayle C. Johnson, Shirley Chu

Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO and Department of Veterinary Pathology, College of Veterinary Medicine, University of Missouri, Columbia, MO

BRAF is one of multiple RAF proteins responsible for activation of the MAPK cell signaling cascade involved in cell growth, differentiation, and survival. Oncogenic mutation of theBRAF gene leads to prolonged activation of the MAPK pathway. A hotspot non-synonymous point mutation, V600E, in exon 15 of BRAF is a driver in 100% of human hairy cell leukemias, 50-60% of human melanomas, 30-50% of human thyroid carcinomas, 10-20% of human colorectal carcino- mas, and 85% of canine bladder transitional cell carcinomas and prostatic adenocarcinomas. Many feline cancers share similar cellular signaling pathways with canine and human cancers, including BRAF, and BRAF is highly conserved across species, but a gain of function mutation has yet to be discovered in cats. Conventional PCR and gel electrophoresis were performed on FFPE samples of malignant feline tumors from the MU VMDL. Bidirectional Sanger sequencing of the full sequence of exon 15 (ENSFCAT00000037954.3) was conducted to determine whether the V597E BRAF muta- tion, orthologous to the human V600E and canine V5895E mutations, is present in feline cancers. The identification of BRAF mutations in feline cancers would provide the necessary rationale to evaluate BRAF or other kinase inhibitors as chemotherapy targets in cats. Additionally, BRAF mutations may serve as diagnostic markers, markers of treatment effica- cy, and provide rationale for the use of feline models of human cancers.

Research Grant: None Student Support: MU College of Veterinary Medicine Department of Veterinary Medicine & Surgery

Immunohistochemical evaluation of the modular vascularity in the inferior colliculus of the mouse

Josefina Nordenstahl, Nathiya Vaithiyalingam Chandra Sekaran, and Daniel A. Llano

College of Veterinary Medicine (Nordenstahl), Beckman Institute for Advanced Science and Technology (Vaithiyalingam Chandra Sekaran, Llano), Department of Molecular and Integrative Physiology (Llano), University of Illinois at Urba- na-Champaign, Urbana, IL

The inferior colliculus (IC), located within the midbrain, serves as a major auditory integration center in the brain. Our laboratory has contributed to the mounting evidence that suggests the IC plays a key role in multisensory processing. Thus, understanding IC microvascular structure is necessary to investigate multisensory integration disorders. In previous studies, we found a pattern of clearly defined modular regions that develop in the lateral cortex of the inferior colliculus (LCIC) during early development and remain distinguishable across the mouse lifespan. The goal of the current study is to further our physiological understanding of the LCIC modular regions by characterizing their microvascular density. We hypothesized that a higher microvascular density is present inside the modular regions in comparison to the surrounding matrix as they are concentrated hubs for neurochemical activity. Experiments were conducted on glutamic acid decarbox- ylase-green fluorescent protein (GAD67-GFP) knock-in mice in which LCIC modular regions were apparent due to their concentrated GAD67-GFP expression. Antibodies against endothelial markers were used to visualize blood vessels in brain sections. Overlapping images of GAD67-GFP and endothelial markers were captured microscopically and analyzed. Early results suggested that microvascular density was uniform across the LCIC. Greater understanding of brain micro- vasculature and IC structure will contribute to knowledge about multisensory processing, a critical process in both humans and animals.

Research Grant: R01 DC013073 Student Support: Office of the Director, NIH, T35 OD011145

258 • 2021 National Veterinary Scholars Symposium Immune responses elicited in calves administered the conditionally-licensed anaplasmosis vaccine

Shannon C. O’Day, Naemi P. Bickmeier, Ivy J. Schmid, Tippawan Anantatat, Kathryn E. Reif

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Bovine anaplasmosis is a tick-borne disease of cattle endemic in the United States that causes significant economic loss in the cattle industry through animal deaths and disease-associated management costs. Currently, the primary management strategy is use of antimicrobials to control active disease. As the need for improved antimicrobial stewardship increases, alternative management strategies such as vaccination are sought. Currently, an anaplasmosis vaccine sold under a condi- tional USDA license is available to U.S. cattle producers; however, no efficacy data is available. As numerous Anaplasma marginale strains exist in the U.S., whether or not this vaccine, developed using a single A. marginale strain, broadly protects against disease caused by diverse strains is also unknown. The objective of this study is to evaluate the immune response of calves to vaccination with the conditionally licensed anaplasmosis vaccine. The conditionally-licensed ana- plasmosis vaccine was administered to ten naive three-month-old Holstein steer calves while a matched group of eight unvaccinated calves served as controls. Serum was collected and tested weekly for antibodies to A. marginale using both a commercial cELISA and an in-lab developed crude total antigen ELISA . As of two-weeks post-immunization, all calves are seronegative for A. marginale. The procedure for total A. marginale antigen preparation for use in western blot and ELISA is in process to screen immunized calves. In the second phase of this study, calves will be challenged with one of two A. marginale strains 28 days post-booster immunization to evaluate vaccine efficacy in preventing clinical disease caused by diverse A. marginale strains.

Research Grant: USDA NIFA Award #: 2020-67015-31873 Student Support: We acknowledge Elanco Animal Health for veterinary scholar support

Evaluation of intestinal alkaline phosphatase as a novel therapeutic for equine intestinal epithelial injury

Kerry O’Donnell, Jamie Kopper, Liara Gonzalez, Amy Stieler Stewart

Department of Clinical Sciences (O’Donnell, Gonzalez, Stewart), College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA, Department of Veterinary Clinical Sciences (Kopper), College of Veterinary Medicine, Iowa State University, Ames, IA

Gastrointestinal (GI) disease remains a leading cause of morbidity and mortality in horses. Therapeutic options remain limited, and it is necessary to investigate novel or alternative treatments to improve patient outcome. In recent literature, intestinal alkaline phosphatase (iALP) improved restoration of barrier function in murine models of GI disease. We hy- pothesized that treatment of equine intestinal epithelial cells (IESCs) with iALP would improve epithelial repair following injury. Equine jejunum (n = 3) was collected following euthanasia. Intestinal epithelial stem cells were isolated, plated, and supplemented with growth factors. Enteroids were dissociated and replated for a 100% confluent monolayer. Mono- layers were scratched and treated with media (control) or iALP at 1 unit/mL (low) or 10 units/mL (high). Scratch margins were monitored every 6 hours for cell migration and wound closure. Closure-percentage was calculated and compared between groups (P < 0.05 considered significant). Wound closure occurred by 18-24 hours in all groups. Six hours post-in- jury, controls had increased closure-percentage (68.8%) compared to both low (57.4%, P < 0.0001) and high dose iALP (59.8%, P = 0.0003). At twelve hours, control monolayers closed 92.2% compared to low (90.1%) and high (86.7%) iALP treatment. There was no difference between low iALP and control, however high iALP closure was significantly decreased (P = 0.01) compared to control. Preliminary results suggest that in vitro treatment of equine IESCs with iALP does not improve epithelial wound closure following scratch injury. Additional studies are needed to investigate effects on epitheli- al barrier function, dose-response, and alternative models of injury.

Research Grant: American College of Veterinary Emergency & Critical Care Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 259 Intramedullary stents as a device to prevent post-radiation fractures in canine patients with osteosarcoma

Vivian L. Ojeda, Bernard Seguin, and Ben Gadomski

Department of Clinical Sciences (Seguin) and Orthopedic Bioengineering Research Laboratory (Gadomski), College of Veterinary Medicine and Biomedical Science, Colorado State University, Fort Collins, CO

Appendicular osteosarcoma, presenting in tens of thousands of primarily giant and large breed dogs in the United States, commonly results in limb amputation as the primary treatment. Amputation subsequently can lead to further complica- tions like orthopedic issues and/or behavioral changes. Stereotactic radiation therapy (SRT) has become an appealing approach for a subset of patients as an alternative to amputation. However, estimates from studies show that over 50% of dogs fracture their bone at 11 months post SRT due to a loss of structural integrity from the tumor and radiation therapy. These fractures unfortunately often result in amputation or euthanasia. It is expected that surgically implanting an intra- medullary stent along with cementoplasty will aid in preventing these post-SRT fractures. In this study, three groups will be tested with 6 pairs of left and right humeri in each. The first will be the control, the second will be to test the effect of cementoplasty alone, and the third will allow for testing of the stent with cementoplasty. Because torsional forces acting on bone are suspected to be a significant cause of these bone fractures, it is predicted that the placement of the stent with cementoplasty will provide the bone models with the greatest mechanical properties to protect them from fractures due to torsional forces as compared to the other two groups. We hypothesize that the stent will allow for a transfer of the forces from the weakened bone to the cement. The aim of achieving these results will be to provide a basis for implanting these intramedullary stents in live patients with osteosarcoma treated with SRT with a final goal of preventing the likelihood of fracture and decline in quality of life.

Research Grant: College Research Council, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO Student Support: NIH Fellowship Grant Trainee

Chemerin: What Can We Learn

Ebube H. Okpechukwu , Emma Flood, Stephanie W. Watts

BRUSH Summer Research Program (Okpechukwu), College of Veterinary Medicine, Pharmacology and Toxicology Department (Watts, Flood) Michigan State University, East Lansing. MI

Chemerin is a protein found in plasma, the levels at which correlate to obesity and high blood pressure in humans. It is also an adipokine, being made in adipose tissue. Obese humans (BMI > 30 kg/m2) have a greater risk for diseases of non-vascular smooth muscle, including asthma, inflammation, and bladder dysfunctions. Our hypothesis is that chemer- in-9 will contract non-vascular isolated smooth muscle (bladder body, stomach fundus, trachea and small intestine), thus contributing to diseases of non-vascular smooth muscle. I performed isometric contractility using the agonist chemerin-9 on organs which serve the above-described dysfunctions from male Sprague Dawley Rat. Organs were placed in a tis- sue bath system designed to simulate the organs biological environment. Once we validated the tissue was living with a challenge of KCL (80 mM), chemerin-9, an agonist for the Chemerin1 receptor, was added in a cumulative fashion and isometric contraction measured. The following maximum concentration of chemerin-9 (3 x 10 -6 M ) normalized to KCl (80 mM) were observed: stomach fundus a three percent cumulative contraction on average, bladder did not cause a con- traction with baseline remaining between fifteen and twenty, intestine there was no contraction observed remaining at zero baseline, and trachea no contractions were seen while having a baseline range of two to four. Explanations could include a lack of chemerin-1 receptor in tissues, or that chemerin causes relaxation instead of contraction.

Research Grant: NIH grant R25HL103156, BRUSH Summer Research Program, College of Veterinary Medicine, Mich- igan State University, East Lansing, MI Student Support: Unknown

260 • 2021 National Veterinary Scholars Symposium Esophageal T-cell infiltration in a pig model for Eosinophilic Esophagitis (EoE)

Jasmine Olivares, Lizette M. Cortes, David Brodsky, Madelyn Mirador, Cheryl Assante, Liara Gonzalez, Scott M. Laster, Tobias Kaser

Dep of Population Health and Pathobiology, NCSU, Raleigh, NC (Olivares, Cortes, Mirador, Assante, Kaser), Dep of Biological Sciences, NCSU (Brodsky, Laster), Dep of Clinical Sciences, NCSU (Gonzalez), Dep of Medicine, Comparative Medicine Institute, NCSU (Olivares, Cortes, Brodsky, Mirador, Assante, Gonzalez, Laster, Kaser)

Food allergies are a serious concern costing the US $25B annually in children alone. A chronic form of food allergies known as eosinophilic esophagitis (EoE) is growing in prevalence and can impact people of all ages. EoE can lead to a child’s failure to thrive or esophageal fibrosis in adults. On top, there are currently no prevention or FDA-approved treat- ments for EoE. To address this health problem, the Kaeser lab works towards the establishment of the pig as a relevant biomedical animal model for EoE: long-term goals are i) to promote the development of treatment and prevention strate- gies, and ii) to improve the understanding of the underlying immune mechanisms of this T-cell driven disease. This project shall answer the question: Do food allergic pigs have more esophageal T cells? To create these food allergic pigs, pigs were sensitized and challenged with the food allergen hen egg white protein. Post challenge, control and food allergic pigs were sacrificed to collect their esophagi for fluorescent immunohistochemistry: Frozen tissue sections were stained for i) CLDN4 to visualize the different esophageal tissue layers, ii) DAPI to show cell nuclei and iii) theT-cell marker CD3. Stained sections were imaged using fluorescent microscopy. Currently ongoing are the quantification of the area and the infiltrated T cells of the esophageal lamina propria and epithelium. Preliminary results indicate that food allergic pigs have more esophageal T cells. If the full quantification confirms these preliminary results, it would indicate that T cells may play an active role in the local immune response in EoE. Future studies will then investigate if and how these esophageal T cells drive EoE in pigs.

Research Grant: NIH R21 (grant # 1R21AI149098-01) Student Support: NIH Interdisciplinary Biomedical Research Training Program T35-T35OD011070

Searching for chronic wasting disease (CWD) in the southeastern United States

Zoe Olmsted, Nathaniel Denkers, Mark Ruder, Candace Mathiason, Edward Hoover

Prion Research Center, Department of Microbiology, Immunology, and Pathology, Colorado State University (Olmsted, Denkers, Mathiason, Hoover) and Southeastern Cooperative Wildlife Disease Study, University of Georgia (Ruder)

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) occurring in wild and captive cer- vids, caused by exposure to an abnormally folded isoform of a prion protein, PrPRES. CWD was first recognized in Col- orado and is now found throughout North America, Korea, and all 3 Scandinavian countries. The extent to which CWD is spreading remains uncertain due to limitations in surveillance and sensitive detection methods. The current diagnostic gold standard for CWD is immunohistochemistry (IHC). More recently, a rapid amplification assay, real-time quaking induced conversion (RT-QuIC) showed a higher level of sensitivity than IHC, making it useful for early antemortem detection of the disease. Here we assess the emerging presence of CWD in the southeastern United States using both IHC and RT-QuIC as part of a surveillance study with the Southeastern Cooperative Wildlife Disease Study (SCWDS). Forty one (41) randomly-selected free-range white-tailed deer were collected from Georgia (negative controls), Arkansas, Ten- nessee, and West Virginia. Retropharyngeal lymph node (RPLN) and the obex region of the brain were analyzed for the presence or absence of: (a) CWD prion seeding activity by RT-QuIC, and (b) abnormal prion protein (PrPRES) deposition by IHC. Results demonstrated 20/37 (54%) deer were positive in either RPLN (19/37) and/or obex (20/37) by RT-QuIC, whereas 17/37 (46%) deer tested positive in either RPLN (16/37) and/or obex (10/37) via IHC. Further analysis is re- quired on 12 samples to validate results. All controls (n = 4 Georgia deer) remained negative in both assays. In conclusion, CWD appears to be established within the study areas and the sensitivity of RT-QuIC is higher than that of IHC.

Research Grant: MGR: SCWDS (US Department of Interior F20AP00172), CKM: R01-NS-076894 (National Institutes of Health), EAH: RO1-NS-061902, PO1-AI-077774, T32-OD-010437 (National Institutes of Health) Student Support: CVMBS Veterinary Summer Scholars Program and NIH T35 USDA Grant NI20AHDRXXXXG005

2021 National Veterinary Scholars Symposium • 261 Non-surgical Approach to Sterilizing Avian Species Using a Domestic Chicken Model

Brianna O’Malley, Jennifer Graham, Sandra Ayres

Department of Clinical Medicine (Graham), Department of Comparative Pathobiology (Ayres), The Cummings School of Veterinary Medicine, Tufts University, Grafton, MA

The goal of this study is to develop a non-surgical solution to overpopulation and reproductive diseases in avian spe- cies using domestic chickens as a research model. This study will investigate the effects of a high-dose single injection of lipid-nanocomplex (LPN) in a small group of female chickens, as well as provide understanding on the lethality of LPN to determine effective non-lethal dosing in future studies. To reduce egg production saporin, a cytotoxin, is carried by LPN and guided via anti-Mullerian hormone II receptor (AMHIIR) antibodies in hope of causing apoptosis in thecal and granulosa cells in gonadal tissue. Granulosa cells and theca cells are support cells that produce hormones needed to produce viable eggs. Membrane bound AMHIIR are almost exclusively found on the theca and granulosa cells. Test group egg production will be monitored and compared to control group for 4 weeks. Gonadal tissue from both groups will be harvested and observed histologically for any apoptotic morphology and follicular activity. This pilot study will evaluate three hypothesized effects of the nanocomplex injection. This will be done by using a Fisher’s exact test, mixed model ANOVA, and descriptive statistics. Results from this study can have implications beyond just poultry, including wildlife and companion avian species, providing a foundation for further research on non-surgical methods of permanent sex steroid suppression in domestic chickens. Developing a single-injection sterilization technique could potentially address overpopulation issues in various avian species, as well as serve as an alternative to other forms of reproductive control for hormonally-related diseases in companion birds.

Research Grant: USDA Formula Funds Student Support: USDA Formula Funds

Temporal comparison of plasma total mercury in Steller sea lions (Eumetopias jubatus) of the Aleutian Islands

Julia Orluk, J. Margaret Castellini, Stephanie Crawford, Lorrie Rea

College of Veterinary Medicine and Biomedical Sciences (Orluk), Colorado State University, Fort Collins, CO and Marine Ecotoxicology and Trophic Assessment Laboratory (Castellini, Crawford, Rea), Water and Environmental Research Cen- ter, Institute of Northern Engineering, University of Alaska Fairbanks, Fairbanks, AK

Populations of Steller sea lions (Eumetopias jubatus) in Alaska experienced a sharp decline in the late 1970’s to early 2000’s. Hair samples in pups from the Western Aleutian Island populations (WAI) have shown increased total mercury ([THg]) from 2011-2019, which may relate to the poor reproductive success of these areas. No hair samples were avail- able prior to 2011 to study temporal changes, however the capability of new instruments to detect lower limits of mercury have made it possible to use archived plasma samples to assess Steller sea lions sampled prior to 2011. This study incor- porates new data from 1991-2007, expanding the temporal analysis of [THg] to further assess the increasing trend in the WAI populations. A Nippon MA3000 analyzer was used to measure [THg] in archived plasma samples. Agattu Island (WAI) has had increasing [THg] in hair samples since 2011, however no significant difference among individual years of plasma samples [THg] was found (Kruskal-Wallis ANOVA H = 10.58, P = 0.10). When Agattu samples were binned by time periods of 1997-2007, 2011-2013, and 2015-2017, the 1997-2007 time period was found to be significantly lower than the other bins (H = 11.16, P = 0.0038). Similar increases in plasma [THg] were not found at Ugamak or Bogoslof Islands (Eastern Aleutian Islands) during these same time periods (H = 2.77, P = 0.25 and H = 1.71, P = 0.43 respectively). These results suggest changes occurring sometime between 2007 and 2011 have resulted in an increase in the bioaccumu- lation of mercury in the WAI Steller sea lions, prompting further inquiry as to the nature of such changes and the need for continued monitoring in this region.

Research Grant: Ocean Peace Research Partnership Student Support: NIH T35 Training Grant 5T35OD015130-09

262 • 2021 National Veterinary Scholars Symposium Herpesvirus prevalence in neotropical bat species across disrupted landscapes in northwestern Ecuador

William Orrico, Hannah K. Frank, Carlos A. Carrion, Juliana Gilson, Phoebe Reuben, Thalia Williamson

Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA (Orrico); Department of Ecology & Evolution, Tulane University, New Orleans, LA (Frank, Gilson, Reuben, Williamson); Department of Biology, The University of New Mexico, Albuquerque, NM (Carrion)

Anthropogenic habitat disturbance results in changes to regional biodiversity and novel interactions between and within local species. These interactions can alter host-pathogen relationships and affect population level infection dynamics. Globally, bats account for 20% of all mammal species and are both taxonomically and ecologically diverse, occupying many important niches. For this reason bat diversity is highly sensitive to anthropogenic disruption. Bats are known reservoirs for at least nine viral families, many of which have known zoonotic potential. However, little work has been done to elucidate how infection patterns are altered in the context of anthropogenic landscape disruption, particularly in the neotropics, where bat diversity is greatest. The goal of this study is to use herpesvirus prevalence as an indicator of general pathogen transmission in neotropical bat species in relation to anthropogenic disruption. To accomplish this, we performed mist-net sampling for a 7-week period within different environmental categories in the FCAT Reserve, an area of the Mache-Chindul Ecological Reserve in northwestern Ecuador. Oral-pharyngeal swabs, blood samples, and fecal samples were collected from captured bats. Approximately 600 individual bats have been sampled spanning 39 species. Samples will be analyzed in early Fall 2021 for the presence of herpesvirus using PCR primers for pol and glycoprotein B genes. Additional phylogenetic analysis will occur to compare relatedness of herpesvirus between species. Screening for other additional viral and bacterial pathogens is occuring concurrently. This study also serves as a baseline for a longitudi- nal biodiversity analysis of regional reforestation efforts.

Research Grant: None Student Support: Cummings School of Veterinary Medicine at Tufts University

Surveillance for coronaviruses in North American bats

Gabrielle Orsini, Nicole Castaneda, Anna Fagre, Katrina Morris, Diana Hews, Joy O’Keefe, and Rebekah Kading

College of Veterinary Medicine (Orsini, Fagre, Kading), Colorado State University, Fort Collins, CO; Indiana State University (Castaneda, Hews), Terra Haute, IN; Georgia Department of Natural Resources (Morris); University of Illinois (O’Keefe), Urbana, IL

Bats are reservoirs for numerous viruses of concern, necessitating increased surveillance of North American populations to comprehend potential for zoonotic spillover. Presently, coronaviruses, such as SARS-CoV-2, represent a significant threat to public health worldwide and have demonstrated the ability to spillover from bats to humans. However, there is a paucity of data investigating circulation of coronaviruses within North American bats. We hypothesize that bat corona- viruses are endemic to populations in North America. We chose bat feces as our primary sampling source, as it is con- venient, non-invasive, and can be a strong indicator of viral presence and shedding. Fecal pellets, representing 50 bats, were collected from roosts in Indiana, Missouri, Georgia, and Colorado. Additionally, the three main bat species found within these roosts were the evening bat (Nycticeius humeralis), big brown bat (Eptesicus fuscus), and the Indiana bat (Myotis ). RNA was extracted from all samples and subjected to a pan-coronavirus PCR assay. One sample from a big brown bat located in Indiana yielded a positive amplicon at 328bp, suggesting the potential presence of a coronavirus. Bat species will be confirmed via cytochrome c oxidase I PCR and sequencing. Coronavirus strain will also be supported with a more specific pan-coronavirus PCR assay and sequence confirmation. Currently, big brown bats have not been a successful host for SARS-CoV-2, though, a spillover event could occur from a native bat coronavirus. Recognition and genomic characterization of coronaviruses within their bat reservoir hosts can provide an understanding of host ranges and susceptibility to pathogens.

Research Grant: NIH Fellowship Grant Student Support: CVMBS Veterinary Summer Scholars Program

2021 National Veterinary Scholars Symposium • 263 Visual Performance of Dogs in Low Light Levels

Maria P. Ortiz Fullana, Claire E. Ludwig, Jacklyn H. Salmon, Brian C. Gilger, Annie Oh

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Dogs exhibit superior vision in low light settings due to several factors, including the presence of a tapetum lucidum. The extent of superior vision in low light intensities is unknown. The purpose of this study was to objectively assess visual performance of dogs in low light levels. We hypothesize that dogs can see low light levels up to 0.005 cd/m2. Normal bea- gle and hound dogs (n = 15; age range: 1.00 to 12.9 years old) completed trials in a four-choice vision testing device. The four-choice vision testing device consisted of a junction box with four tunnels. Dogs were placed in the box and given one vision-based choice for exit. The time to exit (seconds) and first-choice tunnel (correct choice fraction) were recorded and analyzed. Seven trials were completed for each light intensity: 0.005, 0.01, 0.1, 1, and 10 cd/m2. The control trial consisted of the dogs wearing bilateral opaque contact lenses, when tolerated, or completed runs at 0 cd/m2 to mimic blindness. Data was analyzed via one- and two-way ANOVA with statistical significance set at P < 0.05. Results revealed that there was no significant difference in time to exit and first choice tunnel between light levels 0.005, 0.01, 0.1, 1, and 10 cd/m2. There was a significant difference between time to exit and first choice tunnel when comparing the control trial to all other light levels (P < 0.001). Factors such as age, age-related lens changes, and fearful behavior did not affect measured parameters. In conclusion, beagle and hound dogs can see low light levels up to 0.005 cd/m2. For future studies, visual performance at lower light intensities such as 0.003, 0.001 cd/m2, and ultraviolet light will be assessed.

Research Grant: Army Research Office (ARO) 2021-2420 Student Support: NIH T35 Training Grant T335OD012199

Comparative distribution of VTA glutamate- and dopamine-releasing terminals in the medial prefrontal cortex

Nashelys M. Ortiz Negron, Olalekan Ogundele

Department of Biomedical Sciences, College of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

The VTA is a nucleus in the midbrain and a part of the mesocorticolimbic pathway. Dopamine neurons make up ~65% of the total VTA neuron population and Glutamate (2-3%). There is evidence that VTA neurons project axons to brain centers involved in reward/aversion learning (Nucleus Accumbens), and cognition (prefrontal cortex and hippocampus). The distribution of VTA projections in these target brain areas may be indicative of their primary functions within the broad cognition circuitry. As such, dopamine-releasing VTA terminals predominantly innervate the nuclei in the meso- corticolimbic pathway for which dopamine release is pertinent. Likewise, VTA glutamate terminals are dominant in the hippocampus and mPFC where glutamate-mediated long-term plasticity is required for cognitive processes. The proposed research sought to examine the distribution of VTA glutamate and dopamine presynaptic terminals in the prefrontal cortex, a primary hub for executive memory function. We hypothesize that the percentage distribution of VTA glutamate and do- pamine presynaptic terminals that innervate the pyramidal cells in the input layer (Layer IV), will vary in comparison with the output layers (Layers Va/Vb, VI). The goal of this research is to define the anatomical map for VTA dopamine and glutamate presynaptic terminals in the input and output layers of the prefrontal cortex. To achieve this goal, I will combine conventional and genetic neuroanatomical tracing methods, with 3D-fluorescence imaging. From the results of this study, we will create a model to describe the neural mechanism involving VTA regulation of cognition.

Research Grant: unknown Student Support: Kenneth F Burns Clinical Clerkship

264 • 2021 National Veterinary Scholars Symposium Comparative analysis of osteocytes-canalicular networks among vertebrates and their role in bone adaptation

Milton J. Ortiz-Rivera, Brennan T. Flannery, Russell P. Main

Department of Basic Medical Science, College of Veterinary Medicine (Ortiz-Rivera, Main), Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907 (Flannery, Main)

Osteocytes are the most abundant cells in the bone, comprising more than 90% of the cells within the mineralized bone matrix. Far from being a passive cell, osteocytes are indispensable for bone homeostasis and normal skeletal function. Osteocytes are hypothesized to be sensitive to mechanical loading and produce signals that alter bone formation by osteo- blasts, but the mechanisms are poorly understood. The goal of this study is to characterize the osteocyte lacunar-canalic- ular networks between different species of vertebrates and how these networks may affect bone adaptation to mechanical loading in these different taxa. Tibiae (or tibiotarsi, in the case of birds) were harvested from different vertebrate species including birds (guinea fowl, chukar, emu, ostrich), mammals (rat, mouse, opossum), and reptiles (monitor lizard, iguana). A total of 15 tibiae were studied. Harvested bones were fixed in 10% neutral buffered formalin and embedded in epoxy for structural support. Transverse sections (~700mm) originating near the midshaft of the bones were collected using a dia- mond blade saw and hand-ground to a thickness of approximately 100mm. Finally, samples were stained using Alexa 488 for imaging by confocal microscopy. A Matlab program developed in our laboratory was used to characterize the lacunae (geometry, orientation) and the dendritic canalicular processes from each osteocyte in the posterior region of the bone. We expect that this comparative research approach will provide novel insights into the role of lacunar-canalicular networks of osteocytes in bone remodeling.

Research Grant: None Student Support: Boehringer Ingelheim, Purdue University College of Veterinary Medicine

Comparative microbiome analysis of Eptesicus fuscus and Myotis grisescens

Alexandra Osterland, Sybill Amelon

College of Veterinary Medicine, University of Missouri, Columbia, Missouri (Osterland), USDA, Forest Service, Northern Research Station, Columbia, Missouri (Amelon)

Bats are an important part of our ecosystems and the agriculture sector, as they provide essential ecosystem services in the form of pest control. The health of bats is indispensable, yet many bat species are threatened by the spread of WNS. Bat fecal samples can be used to profile the gut microbiome to gain a better understanding of the differences and similarities of their microbial composition. Learning more about the composition of microbiomes between different bat species may give us insight into why species vary in susceptibility to certain diseases. There is not much available information about bat gut microbiomes particularly among Eptesicus fuscus and Myotis grisescens. The objective of this study is to compare the microbiomes of wild E. fuscus to wild M. grisescens and to compare the microbiomes of captive and wild E. fuscus. Additionally, the objective is to determine how the microbiome changes with age by comparing captive juvenile and adult samples in E. fuscus. We hypothesize that there will be significant differences between theE. fuscus and M. grisescens, between wild and captive E. fuscus, and between E. fuscus of different ages. We will extract the DNA from fecal sam- ples using the Zymo Quick-DNA Kit, and perform 16S rRNA sequencing using standard amplicon primer sets through the Genomics Technology Core (GTC) facility. Microbiome composition data received from GTC will be processed using QIIME2 software. We expect that the gut microbiome will vary in composition between our different observation- al groups. Such results could reveal areas of further research to determine how differences in the composition of the gut microbiome translate to functional differences in diseased versus healthy states.

Research Grant: Funded by Grant Award: 0056825 to University of Missouri from Missouri Department of Conservation. Student Support: IDEXX-BioAnalytics

2021 National Veterinary Scholars Symposium • 265 Effects of early life stress on the aged myocardium: potential sex differences in hypertrophy and fibrosis

Nicolette Paci, Carley Dearing, Rachel Morano, Lawson Wulsin, Brent Myers

Department of Biomedical Sciences (Paci, Dearing, Myers), Colorado State University, Fort Collins, CO and Department of Psychiatry and Behavioral Neuroscience (Morano, Wulsin), University of Cincinnati, Cincinnati, OH

Chronic stress is a risk factor for the development of cardiovascular disease, the leading cause of death worldwide. Expo- sure to chronic stress during adolescence promotes metabolic dysregulation that may affect cardiac structure.While our group has previously reported sex differences in rodent endocrine and metabolic responses to chronic stress, it is unclear how this may affect sex-specific susceptibility to cardiac pathology. The aim of the current study is to determine how bi- ological sex impacts cardiac health in rats after experiencing chronic stress in adolescence. We hypothesize that impaired female metabolic function after chronic stress increases risk for cardiac hypertrophy and fibrosis in adulthood.To test this hypothesis, we evaluated cardiac structure in male and female rats that were either chronically-stressed in adolescence or remained as unstressed controls. Histological staining and quantitative analysis of left ventricular wall thickness and cell surface area were used to assess hypertrophy, while collagen deposition was used to measure cardiac fibrosis. These results will improve our understanding of how adolescent chronic stress differentially impacts cardiac physiology in males and females, which is vital to improving the prevention and treatment of cardiovascular disease.

Research Grant: R00 HL122454 and R01 HL150559 Student Support: NIH T35 Training Grant 5T35OD015130

An insight to Ornithobacterium rhinotracheale through a time space investigation in turkeys

Miranda Painter, Brittany Skaggs, Amro Hashish, Mohamed El-Gazzar, and Yuko Sato

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA

Ornithobacterium rhinotracheale (ORT) is a bacterium implicated in a respiratory disease affecting poultry, particularly turkeys. However, the role of ORT in disease, its source of infection and its transmission patterns are not well understood. The objective of this study is to investigate if this bacterium is a primary or secondary pathogen. Additionally, we aim to investigate the potential source of infection and transmission patterns of ORT. Samples were collected to investigate the prevalence of ORT in birds and the environment of apparently healthy flocks as well as investigating ORT source of infection and how it spreads among the flocks. Sequential longitudinal respiratory (tracheas and lungs) and environmental (feed, water, litter and varmints) samples were collected from commercial turkey flocks with a known history of ORT in- fections (case, n = 2) and without a history of ORT infections (control, n = 1). Samples were collected at eight time points from hatch to processing in the summer of 2020. Additionally, cross-sectional respiratory samples (tracheas and lungs) were collected from 40 - 50 different age flocks in one week in February of 2020 and 2021. Collected samples were tested for ORT using quantitative real-time PCR (qPCR). Results showed that ORT was present in 23.73% of the longitudinal samples with detection occurring as soon as three weeks of age and the highest prevalence occurring 5 to 12 weeks of age. Moreover, 17 out of 42 flocks (29.35%) of the cross-sectional samples revealed positive results. Obtained results from this study showed that ORT can be detected within a flock regardless of their clinical status. This study will expand the under- standing of ORT, improving disease prevention and control.

Research Grant: None Student Support: Boehringer Ingelheim

266 • 2021 National Veterinary Scholars Symposium Using digital enrichment to enhance parrot wellness

Dillin Parsons, Constance Woodman, and Donald J Brightsmith

College of Veterinary Medicine and Biomedical Sciences (Parsons), Schubot Avian Health Center, Department of Veterinary Pathobiology (Woodman, Brightsmith), Texas A&M University, College Station, TX

Captive animals tend to have sedentary lifestyles, and this has negative health impacts. Digital enrichment is an emerging field to combat this issue without the need for constant human interaction.This study worked with fifteen birds housed in seven cages: twelve blue-throated conures (Pyrrhura cruentata) among five cages, one pair of African grey parrots (Psittacus erithacus), and one cape parrot (Poicephalus robustus). They were all given access to a digital enrichment consisting of a movement tracking electronic tablet connected to a food dispenser to evaluate the efficacy of this technolo- gy to increase activity levels. Birds went through a preliminary three day trial where the enrichment system automatically dispensed rewards from 7:00-9:00am and 7:00-8:00pm. This was to acclimate the birds to the device and associate the device with the rewards. Following these three days, the ten-day trial began with nine 10-minute control sessions sepa- rated by eight, 5-minute game times in the morning block and five control sessions and four game sessions in the evening block. Two cages of conures and one cage of African greys moved significantly more during game sessions than game off times and won the game more times per day as the trial progressed (Least squares regression, P < 0.0001 for all). How- ever, the lone cape parrot, moved significantly less during the experimental sessions and won less as the trial progressed (LS regression, P < 0.0001 for both). Three other cages of conures showed no significant differences in movement or wins over time (LS regression, P > 0.05 for all). Digital enrichment equipment and software, with proper training can provide healthy alternative stimulus to captive animals.

Research Grant: National Science Foundation PFI:AIR-TT award # 1701026, Quaker Parakeet Society. Student Support: Boehringer Ingelheim VSP, Texas A&M College of Veterinary Medicine & Biomedical Sciences.

ACTH stability in frozen equine plasma

Meera Patel, Ann Chapman, Nicole Hazard, Jon Fletcher, Heidi Banse

Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana (Patel, Chapman, Fletcher, Banse), Veterinary Endocrinology Laboratory, Louisiana Animal Disease Diagnostic Laboratory (Fletcher, Hazard)

Pituitary pars intermedia dysfunction (PPID) is one of the most common endocrinological diseases of older horses. Deter- mination of adrenocorticotropin (ACTH) concentration or ACTH concentration following thyrotropin releasing hormone (TRH) stimulation are diagnostic tests used to evaluate for PPID in horses. In research settings, samples may be frozen long term (months to years) prior to analysis. We hypothesized that ACTH stability in frozen samples would be influenced by TRH stimulation and PPID status. In this study, blood samples were collected from 51 horses before and after TRH stimulation. Prior to and at 6 months, 12 months, 18 months, and 24 months after freezing at -80 C, samples were ana- lyzed for ACTH concentration. Horses were categorized as above reference interval (PPID) or within reference interval (healthy). Data were analyzed using a Friedman’s test; a Dunn’s post-hoc test was applied when differences were found. Change in concentration over time for baseline samples ranged from -4% to 16% and for post-TRH samples ranged from -19% to 42%. There were changes over time in all horses (healthy and PPID) for baseline ACTH (P < 0.0001) and post- TRH ACTH (P < 0.0001) concentration. These findings indicate that ACTH concentration is not stable over time in frozen samples, which may have implications for diagnosis of PPID from stored samples.

Research Grant: Unknown Student Support: Boehringer Ingelheim LSU Summer Scholars Program

2021 National Veterinary Scholars Symposium • 267 Evaluating the basis for severe inflammation caused by pathogenic human coronaviruses

Titus Patton, Muneeswaran Selvaraj, Rudragouda Channappanavar

College of Veterinary Medicine (Patton) and Department of Pathobiology (Selvaraj, Channappanavar), College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma

Coronaviruses have been a cause of great public health concern for the last two decades with the emergence of pathogenic human coronaviruses (hCoV) such as MERS-CoV, SARS-CoV, and SARS-CoV-2. Excessive inflammation upon hCoV infections is a key trigger for acute lung injury and fatal pneumonia. However, the basis for this hCoV-induced inflam- mation has not been well defined. GU-rich viral single-stranded RNA (ssRNA) causes robust inflammatory cytokine/ chemokine production. Consequently, we hypothesized that toll-like receptor 7 (TLR-7) stimulation by GU-rich hCoV ssRNA plays a significant role in exuberant inflammatory mediator production. To test our hypothesis, we stimulated mouse monocytes with viral nucleic acid mimics and found that monocytes stimulated with a GU-rich ssRNA mimic (R837) showed robust expression of inflammatory mediators (TNFa and MCP-1) than those stimulated with viral dsRNA (Poly I:C) and DNA (CpG) mimics. We then examined key molecules in TLR7 signaling by treating stimulated cells with inhibitors of key inflammatory signaling molecules. TRAF6, MEK, and IKK were identified as the most effective targets for reducing inflammatory cytokine/chemokine output. Developing pharmaceuticals to target these key components of the TLR7/TRAF6 pathway would help reduce the harmful effects of excessive inflammation in hCoV patients, thereby reducing the mortality rate in severe infections, while facilitating the protective antiviral TLR7/TRAF3 pathway-mediated interferon response. Collectively, we show that hCoV ssRNA induces robust inflammatory mediators and identify TLR7/ TRAF6 signaling as the key target to moderate hCoV-induced inflammation.

Research Grant: Oklahoma State University Research Start-up Grant Student Support: Oklahoma State University, College of Veterinary Medicine, Office of the Dean

Investigating recombinant mammalian orthoreovirus expressing HER2 peptides as a HER2+ breast cancer vaccine

Alexandrea S. Paulson, Nicole A. Jandick, Cathy L. Miller

Department of Veterinary Microbiology and Preventive Medicine (Jandick, Miller), College of Veterinary Medicine (Paul- son), Iowa State University, Ames, Iowa.

Mammalian orthoreovirus (MRV) is a clinically benign virus that infects and preferentially kills cancer cells. Currently, MRV is progressing in trials to treat various subtypes of breast cancer. One aggressive subtype, human epidermal growth factor (HER)2+, is characterized by excessive growth and increased likelihood to metastasize. To augment MRV’s ther- apeutic potential against HER2+ breast cancer, we have replaced one of the two known binding domains of the MRV attachment protein with three HER2 peptides, creating a recombinant virus termed S1/HER2nhd. Each HER2 peptide included in S1/HER2nhd has been shown individually to induce a specific immune response resulting in targeted killing of HER2 overexpressing cells in human trials. By adding these peptides to MRV, we have created a potential vaccine candidate for HER2+ breast cancer. Pre-clinical studies assessing viral replication, cell death induction, and HER2-specif- ic immune activation are necessary prior to moving S1/HER2nhd into in vivo studies. To determine the extent by which alteration of the binding protein in S1/HER2nhd affects MRV replication, we infected four cell lines expressing varying HER2 levels with either S1/HER2nhd or wildtype MRV. We found that S1/HER2nhd maintains the capacity to infect each of the breast cancer cells though replication is delayed compared to wildtype MRV. It is unclear how this replication delay may impact the efficacy of S1/HER2nhd, however previous studies examining replication deficientV MR showed significant viral replication is not required for tumor regression or antitumor immunity. Future studies will investigate the immune response elicited by S1/HER2nhd.

Research Grant: Margaret Barry Cancer Research Award Student Support: National Institutes of Health T35 Training Grant

268 • 2021 National Veterinary Scholars Symposium The novel innate immune-antagonistic effects of the multifunctional ectromelia virus C15 protein

Elise Peauroi, Katherine S Forsyth, Laurence C Eisenlohr

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA (Peauroi, Forsyth, Eisenlohr) and Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA (Eisenlohr)

The success of poxviruses as pathogens depends upon their extensive antagonism of host immune responses by a large arsenal of immunomodulatory proteins. The C15 protein of ectromelia virus (ECTV, the agent of mousepox) is the larg- est of the ECTV immunomodulatory proteins and is a member of a well-conserved poxviral family previously studied as inhibitors of T cell activation. We have recently determined that C15 also facilitates viral spread in vivo by 3 days post in- fection, suggesting a second non-adaptive function of C15. Accordingly, we sought to further investigate this new function and identify the cellular target. We found this replication-promoting effect persists in the absence of T cells but is lessened in NK cell-deficient animals, implying the targeting of NK cells. Further investigation of NK cell function both ex vivo and in vitro shows that C15 selectively antagonizes degranulation of NK cells but not production of antiviral cytokines. Preliminary data suggests that the full impact of C15 in vivo is also reliant upon CD8 T cells, even at this early time point. These results prompt further investigation into the mechanism used by C15 to inhibit these cell types and demonstrate the discovery of a novel second function of the protein, which can selectively antagonize both the innate and adaptive murine immune responses.

Research Grant: NIH R0I3210871219 & R21AI160063 Student Support: T32AI070077 & F30AI149864

In vitro modeling of canine myotendinous junction using induced pluripotent stem cells

Savannah Pees, Masatoshi Suzuki

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin

Traumatic muscle injuries occurring at the myotendinous junction (MTJ) are a common case of musculoskeletal ab- normality in dogs. The novel creation of in vitro MTJ models is a crucial step toward improving understanding of MTJ development and muscle-tendon injury treatment. Stem cell technology is the ideal path to developing in vitro models of the MTJ. Particularly, induced pluripotent stem cells (iPSCs), which can be generated by cell reprogramming from adult somatic cells derived from patients, represent an attractive tool for in vitro modeling. Thus far, the derivation of skeletal muscle and tendon cells from iPSCs has not been achieved in any species, including canines. The specific aims of this study are to newly derive skeletal muscle progenitor cells (SMPCs) from canine iPSCs and to explore the ability of these cells for in vitro MTJ modeling. We tested a culture protocol using free-floating spherical aggregates, which has been developed for SMPC derivation from human pluripotent stem cells. Spherical cultures termed EZ spheres were success- fully prepared from canine iPSC colonies. EZ spheres were maintained in progenitor expansion medium supplemented with high concentrations of growth factors (FGF-2 and EGF) for six weeks. The spheres were dissociated into single cells, plated down on coverslips, and maintained in terminal differentiation medium for an additional two weeks. Immunocyto- chemistry and RT-qPCR were performed to identify the presence of myogenic progenitors (Pax7, MyoD, and Myogenin) and skeletal myocytes (myosin heavy chain). This work demonstrates that canine SMPCs and mature myocytes can be differentiated from canine iPSCs, which is the initial step in developing a canine model of the MTJ.

Research Grant: School of Veterinary Medicine, University of Wisconsin Madison National Institute of Health, R01 AR077191 Good Food Institute Student Support: NIH T35 OD011078

2021 National Veterinary Scholars Symposium • 269 Understanding alpha-synuclein aggregation propensity in exotic, wildlife, and zoo animals

Hailey B. Penticoff, Ashley A. Hetak, Jessica S. Fortin

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI

Alpha-synuclein (a-syn) is an intrinsically disordered presynaptic protein of the central nervous system, and its aggrega- tion has been described to play a central role in the pathogenicity of Parkinson’s Disease (PD). Although the mechanism is not fully understood, certain mutations in the a-syn protein can increase its susceptibility to oligomerization, fibrilla- tion, and cell permeabilization that creates the cytotoxic effects defined in PD and other forms of neural degeneration. The a-syn amino acid variability could further our understanding of the mechanisms involved in the aggregation process. This study aims to characterize the aggregation propensity of a-syn via a library of fragment peptides designed based on substitutions (or variability) found in various animal species. We used biophysical assays such as Thioflavin T (ThT) and electron microscopy (EM) to identify critical amino acid residues for misfolding and aggregation. ThT and EM analysis concluded that a-syn sequences in non-mammalian species do not misfold in the same frequency as in mammals. These sequences included the 1-25 and 26-50 regions in the wild turkey, two-lined caecilian, mainland tiger snake, Tanaka’s snailfish, Greenland sleeper shark, and tiger pufferfish. Region 88-113 was the most prone to aggregation, with all spe- cies being positive on EM and ThT. Region 1-25 was least prone to aggregation, with most species being negative. We are awaiting further confirmation of fibril formation with CD. The understanding of these identified protein motifs with increased risk for amyloidosis may serve as new potential therapeutic targets for PD and neurodegeneration.

Research Grant: Boehringer Ingelheim, The Graduate School at Michigan State University Student Support: Boehringer Ingelheim, The Graduate School at Michigan State University

Evaluating B7-H3 as a tumor antigen target in canine osteosarcoma for CAR T cell therapy

Luisanny Perez, Jennifer W. Cao, Jessica Lake, Lyndah Chow, Jade Kurihara, Renata Impastato, Dylan Ammons, Ashley Yingst, Michael Verneris, Steven Dow

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO; Department of Hematology/Oncology/BMT, University of Colorado Anschutz Medical Campus, Aurora, CO (Verneris, Lake, Yingst); Department of Clinical Sciences, Colorado State University, Fort Collins, CO

Chimeric antigen receptor (CAR) T cells target surface antigens of tumor cells to mediate an adaptive immune response. CAR T cell therapy has shown remarkable clinical success targeting CD19 to treat B cell malignancies. However, human clinical trials of CAR T cell therapy with solid tumors has not shown the same efficacy as seen in preclinical mouse mod- els. One of the challenges in applying CAR T cell therapy to solid tumors is the immune suppressive tumor microenviron- ment (TME). A large animal spontaneously occurring immune competent model would recapitulate the TME and account for individual tumor antigen expression variability, allowing for a more accurate evaluation of CAR T cell efficacy. Canine osteosarcoma (OS) has been established as a translational model for various other therapies and would serve has a robust model for CAR T cell therapy in pediatric OS due to the similar pathogenesis and disease progression. The costimulatory marker B7H3 is a promising target for CAR T Cell therapy and has been showed to be overexpressed in OS and correlated with increased metastasis, poor prognosis, and decreased tumor immune infiltration. Our goal is to develop a protocol to evaluate the prevalence of B7H3 within formalin fixed paraffin embedded (FFPE) canine OS and glioma tissue by immu- nohistochemistry. We established cross reactivity of a human B7H3 antibody to canine B7H3 by western blot. Antigen preservation of B7H3 was evaluated by varying fixation of cell lines that were then stained by IHC.An IHC protocol would allow for a reliable high through put screen for B7H3 on clinical biopsies of canine OS and gliomas, allowing for proper evaluation of the viability of B7H3 as a tumor antigen target for CAR T cell therapy.

Research Grant: Unknown Student Support: Unknown

270 • 2021 National Veterinary Scholars Symposium Delivery routes of antimicrobials against Mycobacterium tuberculosis

Martin Perez-Jaimes, Hadley Gary, Fabiola Silva-Angulo, Connor King, Brendan Podell

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO

Mycobacterium tuberculosis leads the world as the single deadliest infectious agent killing approximately 1.5 million people and infecting another 10 million globally each year. Although antimicrobial treatment against Mycobacterium tu- berculosis is available, current treatment requires a combination of antimicrobials over several months, which can lead to low patient compliance resulting in relapse of the disease and development of drug resistance. It is imperative to develop a more efficient treatment regimen to shorten treatment length while still providing sterility to prevent relapse.This study investigates the pharmacodynamic activity of bedaquiline and capreomycin administered by oral, intramuscular, and intrapulmonary delivery routes using guinea pigs infected with Mycobacterium tuberculosis HN878 to determine their ef- ficacy. We hypothesize that through intrapulmonary delivery of the antimicrobials, bacterial load and lesion burden can be reduced more effectively than standard routes. Additionally, through the combined use of bedaquiline and capreomycin we expect bacterial load and lesion burden to be less than single drug regimens. This study uses a low-dose aerosol infection of Mycobacterium tuberculosis HN878 on 48 guinea pigs for 10-weeks with a 7-week antimicrobial regimen. Bacterial load will be determined by CFU counts and lung lesion burden will be determined by H&E staining and analyzed through digital imaging software. As new antimicrobials against tuberculosis emerge, understanding the most effective delivery routes and drug combinations will not only aid in reducing treatment time and prevent infection relapse, but could also represent a novel approach to treating tuberculosis infections.

Research Grant: Seattle Children’s Research Institute subcontract Student Support: Boehringer Ingelheim

Effects of methadone on leukocyte cytokine production in dogs

Heather Perkins, Imani M. Carswell, Jared Jaffey, and Anderson da Cunha

Department of Internal Medicine (Jaffey), Department of Anesthesiology (da Cunha), College of Veterinary Medicine (Perkins, Carswell), Midwestern University, Glendale, AZ

Intravenous opioids are rapid acting and effective at managing pain in dogs.Although these drugs are typically regarded as safe when administered at clinically relevant doses, previous studies conducted in other species show that and other pure mu opioids have immunomodulatory properties. The effects these drugs have on the canine immune system remain poorly understood. Previous studies in dogs have yielded conflicting results and have been limited to the following drugs: buprenorphine, morphine, fentanyl, and tramadol. The objective of this pilot study was to determine if intravenous methadone, a schedule II synthetic opioid, alters leukocyte cytokine production in dogs. Between June 1, 2021, and July 12, 2021, ten dogs were enrolled to participate in this prospective cross-over study. Subjects were randomly selected to receive a 0.3 mg/kg dose of methadone intravenously, or the volume equivalent of 0.9% NaCl, once every six hours for four total doses. Blood was collected at baseline before intervention administration, and at ten minutes, six hours, and twenty-four hours after intervention administration. Dogs returned seven days later to receive the opposite intervention, and the same time points were used for administration and collection. Whole blood cultures were performed from blood obtained at each time point in the presence of PBS (negative control), LPS, or LTA for 24 hours. Plates were centrifuged and supernatant was stored frozen at -808C. Cytokines (GM-CSF, IFN-g, IL-10, IL-2, IL-6 and TNF-a) will be quantified from thawed supernatant in batch analysis using a canine multiplex bead-based assay.

Research Grant: Midwestern University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 271 Cerebrospinal fluid proteomic profiling for biomarker discovery in canine non-infectious meningoencephalitis

Lynsey Petersen, Luke Wittenburg, and Christine Toedebusch

UC Davis School of Veterinary Medicine, Department of Surgical & Radiological Sciences, Davis, CA

Non-infectious meningoencephalitis (NIME), representing three subtypes of immune-mediated brain disease, affects 25% of neurological dogs. Despite a high disease prevalence, a definitive ante-mortem diagnosis via brain biopsy is rarely made, which precludes standardized treatment and therapeutic monitoring protocols and has mitigated improvements in patient care. Consequently, there is a critical need for sensitive and specific diagnostic and therapeutic biomarkers to im- prove patient management. We hypothesized that cerebrospinal fluid (CSF) from dogs with NIME would have a unique, readily detectable protein signature via unbiased mass spectrometry that will aid ante-mortem disease diagnosis and ther- apeutic monitoring. Using tandem mass tag (TMT) mass spectrometry, we profiled the proteome of archived CSF from neurologically normal dogs (control; n = 6), dogs with granulomatous meningoencephalitis (GME; n = 5) and dogs with necrotizing meningoencephalitis (NME; n = 6). Quantitative proteomic analysis identified 748 proteins across groups. Using a 2-fold change threshold, comparisons between normal vs. GME samples yielded 141 differentially expressed proteins (DEPs), normal vs. NME yielded 203 DEPs, and GME vs. NME yielded 25 DEPs. Compared to controls, acid sphingomyelinase-like phosphodiesterase and chitinase-3-like protein were top DEPs in both GME (8.0-fold; P = 2.00e-8 and 3.8-fold; P = 4.06e-7) and NME (6.5-fold; P = 4.60e-7 and 2.0-fold; P = 0.01). Ferritin light chain was 3.0-fold higher in NME vs. GME samples, which may allow for discrimination between subtypes. These findings provide the rationale to examine these DEPs with a larger, more comprehensive cohort of samples to determine their diagnostic utility for NIME.

Research Grant: Center for Companion Animal Health (2020-6-G) NIH 5-K01 OD026526-03 Student Support: UC Davis School of Veterinary Medicine, STAR NIH T35 Training Grant 5T35OD010956-22

Epidemiological study of avian pathogenic Escherichia coli serogroups in Georgia poultry

Breck N. Peterson, Meaghan M. Young, Anne C. Devorak, Hilary Hsieh, Jerry Tsai, Catherine M. Logue, Nicolle L. Barbieri

Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Colibacillosis, one of the most common diseases in production poultry worldwide, is caused by localized or systemic infection with avian pathogenic Escherichia coli (APEC). Characterizing the E. coli present in the poultry population allows for epidemiological analysis to identify new or emerging strains and to define traits that are associated with infec- tion within a population and their potential risk factors. Because Georgia is the top poultry producer in the USA, with 1.3 billion broilers in 2020 valued at nearly $3 billion, improved disease control aimed at relevant targets is key to minimizing losses at the flock and producer level. This project aims to identify virulence factors and serogroups of 420 isolates ob- tained from chickens diagnosed with colibacillosis in Georgia. Samples were identified by the age of the bird, production type, lesion location, and date collected. Each was screened for virulence associated genes and O-serogroup using multi- plex PCR and gel electrophoresis. The results showed 38 unique fingerprint patterns of the 9 virulence associated genes. Out of the 9 virulence factors screened for, 65% (n = 273) of isolates examined harbored 3 or more genes. The two most prevalent genes detected were ompT and hlyF (65.7%). O serogroups were identified for 204 isolates, with 90% belong- ing to one of 7 common serogroups including O8 (n = 46), O78 (45), O2 (32), O25 (23), O18 (17), O15 (12), and O161 (8). In addition to disease control and prevention through risk identification and development of improved biologics and vaccines, characterization of APEC in Georgia provides valuable insights to its potential as a reservoir for virulence genes that may pose a threat to other species, including humans.

Research Grant: None Student Support: Boehringer-Ingelheim Georgia Veterinary Scholars Program

272 • 2021 National Veterinary Scholars Symposium Validation of standard Doppler cuff sizes in dogs

Jessica Peterson, Anderson da Cunha, Tokiko Kushiro, Teela Jones, Renata Costa, and Mark Acierno

Department of Specialty Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Blood pressure measurement is a critical component of anesthetic monitoring. The gold standard for blood pressure mea- surement is via invasive arterial catheterization. In clinical settings, this method is not practical; therefore, non-invasive methods such as Doppler ultrasound and oscillometry are used. The current ACVIM consensus statement recommends that when Doppler is used to measure blood pressure, a cuff whose width is 30-40% of the circumference of the limb over which it is placed should be selected; however, this recommendation is poorly studied and has not been recently validated. The aim of this research is to challenge the widely held belief that cuff size makes a difference in Doppler ultrasonography blood pressure measurement. If measurements are affected by cuff size, an additional objective is to determine which size provides readings that are in best agreement with invasive blood pressure measurements. Dogs presenting to the Midwest- ern University Companion Animal Clinic for routine anesthetic procedures had an arterial catheter placed in the median sacral artery of the tail. A single hose, inflatable cuff whose width was 30-40% of forelimb circumference was selected, as were two additional cuffs, one a size smaller and one a size larger. Each cuff, in random turn, was utilized, and simulta- neous Doppler and invasive blood pressure measurements were recorded. Doppler and invasive blood pressure measure- ments were then compared using Bland-Altman agreement analysis to determine which cuff, if any, was in best agreement with arterial pressures.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Pharmacodynamic evaluation of subcutaneous methadone in African pygmy hedgehogs (Atelerix albiventris)

Macy L. Peterson, Grayson A. Doss, Christoph Mans

Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin

African pygmy hedgehogs (Atelerix albiventris) are common exotic companion animals. Despite their popularity, there is minimal information about analgesia in this species. The purpose of this study was to evaluate the efficacy and safety of subcutaneous (SC) methadone, a potent mu-opioid, in African pygmy hedgehogs. We hypothesized that 1) SC metha- done at published doses for other species of 0.1 - 4.0 mg/kg would result in thermal antinociception and 2) SC methadone would not produce significant central nervous system effects when administered at antinociceptive dosages in hedge- hogs. Analgesic efficacy was assessed by measuring the change in hindlimb withdrawal latency following application of a thermal noxious stimulus using the Hargreaves method. Dose-finding trials identified that methadone at 0.6-5 mg/ kg produced antinociception. A high frequency of side-effects, including excessive vocalization, hyperactivity, ataxia and vomiting were noted with doses of 2-5 mg/kg. Therefore, the analgesic efficacy and safety of 0.5 (M0.5) and 1.0 mg/ kg (M1) methadone were evaluated in a randomized, blinded, placebo-controlled crossover study in 9 adult hedgehogs. Following baseline measurement, withdrawal latencies were measured again at 0.5, 1, 2, 4, 8, and 12 hours after injection. M0.5 failed to produce a statistically significant increase in withdrawal times. M1 demonstrated a statistically significant increase in withdrawal times at the 1-hour time point. Transient side-effects were noted in 3/9 and 1/9 animals in the M1 and M0.5 groups, respectively. Results suggest that M1 produces short-acting antinociception in hedgehogs lasting < 2 hours and M0.5 is not recommended for clinical analgesia in this species.

Research Grant: 2021 University of Wisconsin-Madison School of Veterinary Medicine Companion Animal Fund Student Support: 2021 University of Wisconsin-Madison School of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 273 Evaluation of hematological and biochemical profile trends in patients with thyroid disorders

Erin Pettibon, Athema Etzioni

Department of Pathobiology, College of Veterinary Medicine Tuskegee University, Tuskegee Institute, Alabama

The present study was conducted on 57 animals (14 cats and 43 dogs) presenting at varying ages and health conditions from October 2011 to August 2020 at Tuskegee University Small Animal Teaching Hospital. The aim of this investigation was to interpret hematology, serum chemistry and thyroid levels of each patient; and to discover trends or correlations within the data that were linked to either hypothyroidism or hyperthyroidism. From the study population, 21% (9) ca- nines displayed abnormal thyroid levels suggestive of hypothyroidism, while 29 % (4) felines thyroid levels demonstrated higher levels characteristic of hyperthyroidism. It was concluded that animals with abnormal T4 values shared an inverse relationship with platelet and plateletcrit levels. Both canine and felines followed this pattern. Canines with hypothyroid- ism are more likely to have an elevated cholesterol concentration. No correlation was seen in felines. A mild nonregenera- tive anemia was seen in patients with hypothyroidism.

Research Grant: Tuskegee University College of Veterinary Medicine Student Support: Boehringer Ingelheim

The effects of castration and early weaning on long-term immune esponsesr

Lauren K. Phillip, Mahsa Fardisi, Kyan M. Thelen, Adam J. Moeser

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI

A sex-biased mortality exists in swine with significantly higher mortality rates in castrated males, compared with intact males and females. The reason for increased mortality in castrates remains unknown. In this study, we are investigating how castration and early weaning (EW), a common early life production practice, interact to impact their long-term im- mune system development which may underly increased disease risk. We hypothesize that EW and castration negatively impacts immune system development and response to later life immunological challenges. Male pigs were either castrated (MC) at 8-10d of age or left intact (MI) and then randomly assigned to either an EW group, where pigs were separated from the sow at 15 days, or a late weaning group (LW) where pigs were separated at 28 days. Pigs were vaccinated with a Circovirus vaccine at 49 days of age. Serum was collected for 5 weeks and PCV2-specific-IgG titers were measured as an index of immune function. At 77 days of age, pigs will be injected with Lipopolysaccharide (LPS, 25 mg/kg; intramuscu- lar) or saline to evaluate inflammatory response. Rectal temperatures, clinical scores, and blood samples will be collected at basal (0h), 2h, and 4h after LPS challenge. To characterize the immune responses, plasma cytokines and endocrine hormones will be measured by using enzyme-linked immunosorbent assay (ELISA). Based on our preliminary results, we predict that EW and castration will have a deleterious impact on immune system development characterized by suppressed vaccine and LPS-induced immune responses. Findings from this study have the potential to provide insight into the en- hanced susceptibility to disease in castrates thus having significant economic implications.

Research Grant: Unknown Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University.

274 • 2021 National Veterinary Scholars Symposium Pilot Study on the Healthy Canine Blood Microbiome

Jill Piorkowski, Janina A. Krumbeck, Zachary Morris, Annika Linde

College of Veterinary Medicine, Western University of Health Sciences, Pomona, California (Piorkowski, Morris, Linde); MiDOG LLC, Irvine, California (Krumbeck)

Blood has historically been considered sterile in terms of composition, and the presence of microbes in circulation has been regarded as an indicator of disease. However, our regard for microbes as simply hematological intruders is evolving. Recent evidence depicts the presence of a core microbiome in the blood of clinically healthy individuals, while chang- es in the composition and abundance of these blood microbial species have been demonstrated in diseased individuals. Research has emphasized the human blood microbiome; however, veterinary studies are beginning to demonstrate similar findings. One very recent publication focused on the bacterial microbiome in blood of dogs, however, additional studies are needed to characterize the blood microbiome of this species more comprehensively and the implications of dysbiosis in different disease processes. The aim of this pilot study was to further explore the canine blood microbiome in clinically healthy dogs to define the baseline signature of both bacterial and fungal species during homeostatic conditions. Periph- eral blood samples from client-owned dogs have been collected and are currently being analyzed via next generation sequencing to generate detailed records of the bacterial and fungal blood microbiome as well as antimicrobial resistances in individual dogs. These microbial profiles will be evaluated in light of medical and environmental histories obtained through client questionnaires. Data from this pilot study will generate novel insight on the healthy canine blood microbi- ome and is expected to furthermore pave the way for future studies focusing on the interplay between nutrition, immunity and different diseases, viewed through the lens of evolutionary medicine.

Research Grant: College of Veterinary Medicine, Western University of Health Sciences Student Support: Boehringer Ingelheim

Assessment of zoonotic parasites and bacteria in fecal samples of shelter dogs

Milan Piva, Amie Burling, Katelyn Smith, and Craig Franklin

Shelter Medicine (Burling) and Department of Veterinary Pathology (Franklin), College of Veterinary Medicine, University of Missouri, Columbia, MO

The American Veterinary Medical Association states that the average American household owns more than one dog. Many of these animals are adopted from rescue and animal shelter settings with unknown health histories or backgrounds. As dogs become an increasingly integral part of households, they may pose an increased risk to human health due to the possibility of transmission of zoonotic diseases. A zoonotic diseaseis an infectious disease that is transmitted from animal to humans (or from humans to animals). Zoonotic diseases also present an occupational health risk to those who routinely handle dogs such as shelter workers, veterinary staff, kennel attendants, and more.This study aims to identify and assess the prevalence of zoonotic parasites and bacteria present in fecal material of shelter dogs. To this end, fecal flotation is performed to identify parasites present in the stool samples. DNA is then extracted from samples and subjected to next generation sequencing to fully characterize the bacterial fecal microbiome which may include zoonotic or opportunistic pathogens. Concurrently, surveys are completed by staff and volunteers with frequent interaction with dogs to assess their knowledge about zoonotic diseases. In coupling these findings, we will be able to evaluate the level of zoonotic risk these individuals face. Results from this study will be utilized to make recommendations for decreasing zoonotic disease trans- mission within animal shelters.

Research Grant: Franklin Research Incentive Funds Student Support: IDEXX-BioAnalytics

2021 National Veterinary Scholars Symposium • 275 Metabolic analysis of canine and murine osteosarcoma cells in comparison with mesenchymal stromal cells

Andrew C. Poon, Anita K. Luu, Anthony J. Mutsaers

Department of Biomedical Sciences (Poon, Luu, Mutsaers) and Department of Clinical Studies (Mutsaers), Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Metabolic adaptations fuel the outgrowth of tumor cells, yet less is known about the metabolic changes during cancer ini- tiation. Osteosarcoma (OSA) is an aggressive bone tumor of mesenchymal origin in dogs. We hypothesized that metabolic flexibility post sarcomagenesis would reveal unique, targetable signatures in the OSA landscape. Using a cross-species approach, we investigated the metabolic requirements of ten primary and metastatic canine and murine OSA cell lines, in comparison with two control mesenchymal stromal cell (MSC) lines. Measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were obtained using Seahorse respirometry. Cell viability was evalu- ated after addition of glucose, glutamine, pyruvate, or lactate. Cells were then grown on biomimetic hydrogel cultures re- capitulative of lung and bone stiffness, and mass spectrometry of metabolites was performed. Results showed that canine and murine MSCs were highly dependent on glycolysis (high ECAR/low OCR), while canine and murine OSA cells were highly energetic (high ECAR/high OCR). OSA cell lines preferred glutamine over glucose or pyruvate but also consumed lactate. Carbon intermediates glucose-6-phosphate, lactate and malate were upregulated in metastatic OSA lines compared to control, whereas succinate was decreased, especially in metastatic murine OSA lines. Collectively, our results suggest that glutamine and lactate fulfill a metabolic gap in respiration to confer aerobic cancer survival, and that changes to the TCA cycle may accommodate lactate consumption. These findings represent an investigation into the metabolic flexibility of OSA cells, and may hold future clinical implications for studying cancer metabolism.

Research Grant: Natural Sciences and Engineering Research Council of Canada (NSERC) PGSD3-535680-2019 [or] Ontario Veterinary College Pet Trust. Student Support: Art Rouse Memorial Scholarship in Veterinary and Comparative Cancer Studies.

Detection of Brugia malayi antigen in experimentally infected dogs using a commercial heartworm test

Catherine E. Pope, Katelin E. Greenway, Michael T. Dzimianski and Andrew R. Moorhead

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia

Lymphatic filariasis affects 120 million people worldwide.Brugia malayi, one of its causative agents, develops in dogs and cats, making them models for research and potential zoonotic reservoirs. In B. malayi-infected cats, we have previ- ously shown that heat-treatment of serum allows antigen detection on a commercial heartworm test presumably through immune complex dissociation. However, cross-reactivity in dogs remains uninvestigated. This study aims to determine if parasite antigen can be detected in heat-treated serum from B. malayi-infected dogs using the DiroCHEK Canine Heart- worm Antigen Test Kit. Twenty dogs were infected with 500 B. malayi third-stage larvae subcutaneously in either the dorsal foot or inguinal region, with ten dogs in each group. Half of the dogs received a single injection; the other half re- ceived four weekly injections of 125 larvae each time. Serum was collected every two weeks and stored at -808C. Samples were thawed and tested in triplicate pre- and post-heat-treatment using the DiroCHEK kit according to the manufacturer’s instructions. For heat-treatment, samples were heated to 1038C for 10 min and then centrifuged at 18,900 X g for 15 min. Samples were analyzed for color change, both visually and on a spectrophotometer at 490 nm. While no antigen was de- tected in pre-heat-treated samples, antigen was detected post-heat-treatment as early as 56 days post-infection. Nine dogs remained antigen-positive for the first year of the study. Overall, these findings demonstrate that commercial canine heart- worm tests can be utilized with heat-treated serum as a diagnostic tool in dogs experimentally infected with B. malayi.

Research Grant: None Student Support: Moorhead laboratory

276 • 2021 National Veterinary Scholars Symposium The impacts of COVID-19 on high-quality, high-volume, spay-neuter clinics

Tenley R. Porcher, Simone D. Guerios, Gina Clemmer, Elizabeth Fitzpatrick, and Julie K. Levy

Department of Small Animal Clinical Sciences (Porcher, Geurios, Levy) College of Veterinary Medicine, University of Florida, Gainesville, FL; Clinic HQ (Clemmer), Portland, OR; Alachua County Animal Services Department (Fitzpatrick), Gainesville, FL

High-volume spay-neuter services have proven beneficial in mitigating pet overpopulation in the US. In March of 2020, the country faced the novel COVID-19 pandemic and nonurgent surgical procedures ceased, including spay-neuters. The aim of this study was to determine the impact of the COVID-19 pandemic on the number of spay-neuter surgeries per- formed. This study included data from 212 clinics, obtained from Clinic HQ, a nationally recognized spay-neuter soft- ware, that had complete data throughout the study period. Species, sex, age category, source, and geographical locations were analyzed. Total number of spay-neuter surgeries in 2020 were 1,050,683, down 174,581 from a total of 1,225,264 in 2019. An abrupt decrease in surgeries was observed in March-April 2020, dropping 81%, which coincided with the initial lockdown and restriction to essential services. Surgery numbers began to recover in May-June, however remained lower than the previous year for most months. Surgeries were close to normal numbers by the end of 2020 and beginning of 2021. However, spay-neuters remained 2% lower in Jan-May 2021 compared to the same months in 2019, demonstrat- ing that spay/neuter capacity has not recovered to pre-COVID conditions. Total surgeries for 2020 were down 11% for cats and 20% for dogs. In an 18-month period, it was estimated 1,906,776 births. Assuming the studied shelters represent approximately 10% of total spay/neuter capacity in the US, a deficit of more than 1 million surgeries were performed in 2020. This may have a detrimental effect on efforts to control unwanted reproduction of pets, economic uncertainty, hous- ing insecurity, increased shelter admissions, and decreased adoptions threaten the welfare of homeless pets.

Research Grant: University of Florida College of Veterinary Medicine Department of Small Animal Clinical Sciences Student Support: Boehringer Ingelheim Veterinary Scholars Program

Survey of dental conditions and management in zoological animals

Vanessa R. Powers, June E. Olds, and Brenda L. Mulherin

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Dentistry for animals within zoological institutions is a steadily growing field, but there is a lack of research on dental practices conducted in zoos. Recommendations have been made regarding incorporating dental prophylaxis and treatment as part of routine veterinary care of zoo animals. The objective of this study was to survey zoological institutions regard- ing dental diagnostics, prophylactic care and procedures performed in zoos, dentistry training of veterinary staff, and use of specialists for dental procedures. Results were compared to a previous study to evaluate changes over time. Eighty-four responses were collected. Ninety-eight percent of responses to the survey were from accredited institutions. Similar to the previous survey, carnivores and primates were shown to have the highest incidence of dental issues and procedures. How- ever, our survey also showed tubulidentata and artiodactylids with a higher incidence of dental disease and/or trauma. This survey also reports an increased use of veterinary dental specialists to perform dental procedures at zoological institutions. In contrast to the previous survey, our survey identified several challenges zoological institutions face when attempting to perform dental procedures for zoo animals. These challenges included lack of animal training, lack of dental specialist and equipment, as well as concerns for procedural risks. The results of this study may provide possible opportunities to im- prove the veterinary dental care provided by institutions, and therefore improve the health and welfare of animals within zoo collections.

Research Grant: None Student Support: Boehringer Ingelheim Animal Health

2021 National Veterinary Scholars Symposium • 277 Microplastics and per- and polyfluoroalkyl substances (PFAS) in bottlenose dolphins along Mississippi’s coast

Hannah R. Pray, Chanaka M. Navarathna, Natalie R. Hampton, Todd E. Mlsna, Debra P. Moore, Stephen R. Reichley

Department of Clinical Sciences (Pray, Moore), Comparative Biomedical Sciences (Reichley), College of Veterinary Medicine, Mississippi State University, Mississippi State, MS; Department of Chemistry (Navarathna, Mlsna), Mississippi State University; Department of Biological Sciences (Hampton), Tougaloo College, Tougaloo, MS

With global plastic usage increasing annually, plastic pollutants, namely microplastics (#5 mm), are a growing concern. Microplastics have been shown to harbor and release harmful chemicals, such as per- and polyfluoroalkyl substances (PFAS), acquired from the environment. Microplastics have also been shown to accumulate in top marine predators, such as cetaceans. The aim of this study is to analyze the stomach and intestinal contents of bottlenose dolphins stranded along the Mississippi coast for microplastics and PFOS, a common PFAS. Gastrointestinal contents were digested with 10% KOH in 50% MeOH then analyzed for microplastics using Nile red microscopy, pyrolysis gas chromatography-mass spectrometry (Pyro-GC-MS), and Raman spectroscopy. Filtrate from digested samples were pre-concentrated using sol- id-phase extraction (SPE) and analyzed for PFOS with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The PFOS extraction and analysis was validated with pike perch fish certified reference material, which had a recovery of 98.6%. The Pyro-GC-MS results for two samples showed the presence of acetamide, which is used as a plasticizer. Raman spectroscopy results also showed characteristic plastic peaks corresponding to those of polystyrene in one sample. Addi- tionally, PFOS was detected in three samples ranging from 95.55 to 1,934.56 mg/kg. The results show that three samples potentially contain microplastics and three samples contain detectable levels of PFOS, while the majority of samples were negative for both microplastics and PFOS. This project is part of a long-term study that will continue in order to better understand microplastics and PFAS within the environment and their impact on bottlenose dolphins.

Research Grant: Mississippi Department of Marine Resources Student Support: National Institutes of Health

Surveillance for Salmonella and resistome profiling among hospitalized wildlife patients

Alexandra Presher, Kevin J. Cummings, Sara Childs-Sanford, Julie D. Siler, Niki Mitchell, and Laura B. Goodman

Baker Institute for Animal Health (Presher, Goodman), Department of Population Medicine and Diagnostic Sciences (Cummings, Siler), and Swanson Wildlife Hospital (Childs-Sanford, Mitchell), College of Veterinary Medicine, Cornell University, Ithaca, NY.

Antimicrobial resistance (AMR) is a growing public health concern, yet wildlife and the environment represent critical gaps in surveillance. The impact of multidrug resistance on wildlife and rehabilitation personnel is also largely unknown. The goals of this study are to examine how long-term antimicrobial treatment of hospitalized wildlife changes their micro- bial composition. We hypothesize that over time, treatment will select for resistance to drugs that patients may have been previously exposed to in the environment. Fecal samples were collected from patients shortly after admission to the Swan- son Wildlife Hospital. Individuals who were treated with antimicrobial therapy had further fecal samples collected on a weekly basis. DNA was extracted from the fecal samples and analyzed for AMR genes using a targeted deep amplicon sequencing approach. Samples were also cultured for Salmonella; confirmed isolates were characterized using whole-ge- nome sequencing. Upon analysis, 21 of 513 samples (13 of 451 individuals) were culture-positive for Salmonella, with five serovars being represented. Eight individuals (six species) had the same strain ofSalmonella . An important compo- nent of this study was the optimization of the DNA extraction protocol and sequencing workflow in order to maximize quality and quantity of DNA from fecal samples. Study samples came from a wide variety of hosts, each of which had different diets and biological properties, resulting in different compositions of feces. Many samples had a low mass, mak- ing it critical to optimize the protocol in order to yield viable results. Establishing this protocol will enable future studies examining AMR in wildlife to be conducted, helping to address the lack of data.

Research Grant: John T. and Jane A. Wiederhold Foundation Student Support: NIH T35 OD010941, Cornell University College of Veterinary Medicine

278 • 2021 National Veterinary Scholars Symposium Heavy metal fish: a band of potential health threats for local Philadelphian fish consumers

Rowan Prown, Lisa A. Murphy, Marilyn Howarth, Julie Carol Ellis

Department of Pathobiology, School of Veterinary Medicine (Murphy, Ellis) and Center of Excellence in Environmental Toxicology (Howarth), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

John Heinz National Wildlife Refuge (JHN), in southeastern Pennsylvania, is a popular site for wildlife and human activ- ity in an urban area. Unfortunately, it is threatened by pollution from neighboring landfills and industries. There is local concern regarding heavy metal pollution in fish and the potential effects on fish consumers. Our study is a dual toxicology and epidemiological survey. Locally-consumed fish will be collected, and edible tissues will be tested for arsenic, cad- mium, lead, mercury, selenium, and thallium using inductively-coupled mass spectrometry (ICP-MS). The toxicology data will allow us to estimate the potential risk of consuming fish from these waters by comparing the results to available national standards for heavy metal consumption. Additional data will be collected from people fishing in JHN. The first part of this survey will investigate consumption of fish in JHN waters, including species, location, portion of fish con- sumed, method of preparation, frequency of consumption, and whether consumption by pregnant women or children is occurring. The other part of the survey investigates attitudes towards the health risks or benefits of consuming fish from JHN and individuals’ understanding of local fish advisories. The results of this project will be shared with the Eastwick Lower Darby Creek area community advisory group, who will use the information to educate fishermen on potential risks of fish consumption and how to utilize and interpret current fish advisories.The researchers will also work with JHN staff to engage visitors in health and environmental outreach. Although the focus of this study is human health, all outreaches will include the risks that heavy metals pose to animal health.

Research Grant: Department of Pathobiology Student Support: NIH T35 Training Grant

Transduction efficacy of viral vector serotypes in canine joint cells for osteoarthritis gene therapy

Christian Quiles-Torres, Laurie Goodrich, Ah Young Kim, Jennifer Phillips

(Quiles-Torres) School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA; Department of Clinical Sciences (Goodrich, Kim), Orthopaedic Research Center (Phillips), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Osteoarthritis (OA) is an incurable and progressive disease that cripples the quality of life of millions of veterinary and human patients alike each year. Current treatments for OA fail to provide long-term therapeutic effects and mostly focus on pain management, emphasizing a need for a cost effective, minimally invasive, and sustainable treatment that promotes tissue regeneration. Gene therapy could meet those criteria, and the first step in developing a treatment for canine OA is to identify the self-complementary adeno-associated virus (scAAV) serotype that most efficiently transduces canine chon- drocytes and synoviocytes. Despite species differences in surface receptors of equine, human, and canine joint cells, we suspect that scAAV2 will continue to execute the highest efficiencies among all serotypes. We will test nine different- se rotypes at three different dose levels on joint tissue explants and monolayers from two individual specimens.The scAAV vectors contain the gene for green fluorescent protein, and the cells will be analyzed post-transduction with fluorescent microscopy and flow cytometry. The serotypes will then be ranked by their transduction rates in chondrocytes and synov- iocytes, respectively. Since the canine joint model is very similar to equines and humans, finding commonalities in vector serotype efficacy could facilitate translating advancements from one species to another. The knowledge gained from this study will drive the fields of gene therapy and orthopedics closer to an optimal treatment for canine OA patients.

Research Grant: Morris Animal Foundation Student Support: NIH T35 Training Grant 5T35OD015130-09

2021 National Veterinary Scholars Symposium • 279 Developing a rapid plasmid DNA extraction method

Sonia Rafique, Azam Ali Sher, Lixin Zhang, Linda Mansfield

Department of Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Plasmid DNA are small extrachromosomal circular DNA strands, mainly found in bacteria, that can be transferred amongst each other during a process known as conjugation. This exchange of genetic material via plasmid transfer has al- lowed bacteria to acquire and share antibiotic resistance genes (ARGs), posing severe threats to both humans and animals. The isolation and characterization of plasmid ARGs in bacterial populations has always been challenging. We found that commercial plasmid extraction kits were not successful in extracting larger sized plasmids, and additional literature re- vealed that these kits were ineffective for complex samples such as soil or cecal samples. In our study, we hope to develop a rapid and cost-effective method for DNA plasmid extraction. This method will facilitate the isolation of plasmids from simple bacterial cultures to complex microbial samples such as sewage, soil, and feces. We plan to use this method to screen human isolates for the presence of conjugative plasmids carrying the Extended-Spectrum Beta-Lactamase (ESBL) enzyme, which is responsible for resistance to many types of antibiotics including penicillin and cephalosporins. We are currently testing both commercial plasmid DNA extraction kits and in-house methods on overnight bacterial cultures to isolate plasmids of known sizes with various resistance genes. The preliminary results show that tested methods have different abilities to extract plasmids from pure cultures of bacteria.The findings from this study will enable us to advance the screening process for multi-drug-resistant bacteria carrying ARGs on conjugative plasmids.

Research Grant: Unknown Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

Determining the predictive value of radiographic change and ALP levels for metastases in an osteosarcoma model

Leah Ramsaran, Shun Ishiyama, Xin Guo, Aaron James, Kathy Gabrielson

College of Veterinary Medicine, Cornell University, Ithaca, NY (Ramsaran), Department of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, MD (Ishiyama, Guo, James, Gabrielson), Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD (Ishiyama, Gabrielson)

Osteosarcoma (OS) is the most common primary bone tumor in both canine and pediatric patients. As such, research to develop new treatment options is critical for this aggressive tumor. Variability in success in tumor implantation models and poor prognosis post-development of metastases leading to ineffective analysis of treatment efficacy has led to a need for an early predictive marker. This pilot study aims to assess radiographic changes and serum alkaline phosphatase (ALP) levels to determine their predictive value for lung metastasis. We used an immunocompetent, outbred, Sprague-Dawley, syngeneic rat cancer model and implanted an orthotopic UMR106 OS cell line into the tibias of three-week-old male and female rats to model pediatric OS. Radiographs were taken two times a week starting seven days post injection until eu- thanasia. Serum samples were collected at time of euthanasia. Elevated serum ALP level at euthanasia was found to have a 100% positive predictive value and specificity for gross OS lung metastases at necropsy. We qualified radiographic op- timal densities using Image J focusing on cortex and medulla osteoblastic and osteolytic bony changes at primary implan- tation site. We found a cortical and medullary optimal density deviation from normal limb values that was predictive of gross and histologic lung metastases at necropsy with 100% agreement. Future studies include assessing changes in serial ALP levels to determine the predictive value of percent change over time and determining the predictive value of density changes in bone via machine learning.

Research Grant: NIH funding through National Cancer Institute, grant # CA228582 Student Support: 5T35OD024982-03 Veterinary Research Scholars Program, Johns Hopkins University

280 • 2021 National Veterinary Scholars Symposium Ocular Pulse Amplitude in normal beagles and beagles with ADAMTS10-Open-Angle-Glaucoma (ADAMTS10-OAG)

Vanessa A. Raphtis, Dhruv Sharma, Amanda L. Anderson, Christine D. Harman, Andras M. Komaromy

Department of Small Animal Clinical Sciences (Raphtis, Anderson, Harman, Komaromy), College of Veterinary Medicine, Center for Statistical Training and Consulting (Sharma), Michigan State University, East Lansing, MI

The objective of this study was to evaluate the effect of age and G661R missense mutation in theADAMTS10 gene on ocular pulse amplitude (OPA) in normal and glaucomatous dogs with ADAMTS10-open-angle-glaucoma (ADAMTS10- OAG). Animals studied included 30 glaucomatous beagle dogs and 4 unaffected control dogs between the ages of 6 months and 12 years. Dogs were sedated with butorphanol/midazolam. For every dog, left or right eye was randomly selected to begin measurements for OPA. The Reichert Model 30 Pneumotonometer was used to measure OPA as well as provide measurements of intraocular pressure (IOP), a deviation index, and pulsation rate. Measurements were re- corded by placing the pneumotonometer probe directly on the central cornea and applying gentle pressure until a reading was produced. Each dog went through this process of measurement twice; once to become acclimated to the probe and noise of the machine, and a second time for a more accurate reading. Lastly, corneal thickness measurements were taken via PachPen Handheld Pachymeter. A descriptive analysis of the data will be used for interpretation as well as ANOVA, regression, and mixed effects models. Preliminary findings show that mutant beagles have a smaller AOP of 3.96 +/- 1.81 mmHg (mean +/- SD) compared to 5.13 +/- 2.02 mmHg of normal beagles. As expected, mutant beagles display a greater mean IOP of 28.36 +/- 4.24 mmHg compared to 20.00 +/- 6.01 mmHg of normal beagles. Although, these findings show no significant association, accurate inference will require a larger sample size of dogs. We anticipate determining an age effect on OPA as well as gathering a more reliable analysis on the mutation effect as we complete data collection within the upcoming weeks.

Research Grant: NIH Grant R01EY025752 Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

Editing the chicken genome by microinjecting CRISPR reagents into unfertilized oocytes

Ashley M. Rasys, Charles K. Meeks, Kristen J. Navara, Brian G. Condie, Jonathan Eggenschwiler, James D. Lauderdale, Andrew P. Benson, and Douglas B. Menke

Department of Cellular Biology (Rasys, Lauderdale), College of Veterinary Medicine (Rasys), Department of Poultry Sci- ence (Meeks, Navara, Benson), Department of Genetics (Menke, Condie, Eggenschwiler), Neuroscience Division of the Biomedical and Translational Sciences Institute (Lauderdale), University of Georgia, Athens, GA

Improving gene-editing technology in chickens is vital for industry growth, research purposes, and for developing strate- gies to mitigate pathogen spread and disease among birds. Current methods in chickens rely on isolating embryonic germ cells, culturing them in a dish, modifying a target gene with CRISPR, and then transferring the germ cells back into a host. This process is very expensive and time-consuming, taking anywhere between 1-1.5 years to generate stable mutant lines. Here, we adapt a surgical approach, originally developed in Anolis lizards, in an attempt to modify the chicken genome. This process involves anesthetizing adult female chickens, accessing their ovaries through a left flank surgical incision, and microinjecting their unfertilized oocytes with CRISPR reagents. The day before surgery, chickens are artificially inseminated once and then again at 2- and 8-days post-surgery. Eggs are collected daily for 3 weeks and incubated until embryonic day 10. Following incubation, embryos are isolated and DNA from these embryos is screened via PCR PAGE and Sanger sequencing to identify embryos that carry mutations in the targeted locus. In this pilot study we targeted the tyrosinase gene. This gene was chosen because inactivating mutations in tyrosinase result in albinism - a phenotype that can be easily scored visually. We report here our methodology and our preliminary findings.

Research Grant: University of Georgia TIR Pre-Seed Program. Student Support: Foundation for Food and Agriculture Research Vet Fellowship, UGA College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 281 A survey of parasites affecting wild fish populations in lakes across Uganda

Madison Rawdon, T. Graham Rosser, Stephen Reichley, David Kahwa, Margaret Khaitsa, and Robinah Nakabuye

Comparative Biomedical Sciences (Rawdon, Rosser), Pathobiology and Population Medicine (Reichley, Khaitsa), College of Veterinary Medicine, Mississippi State University, Starkville, MS; College of Veterinary Medicine, Animal Resources and Biosecurity (Kahwa, Nakabuye), Makerere University, Kampala, Uganda

Parasites affect wild and farmed fish populations on a global scale. Infection can be characterized by reduction in growth, reproduction, and quality of fish products. Many organs including gills, fins, gastrointestinal tract, muscles, and nervous system may be sites of development. Parasitic infections are particularly devastating in cultured fish populations where stressful conditions associated with water quality and crowding exacerbate disease. Our overall goal is to establish base- line prevalence data for parasites infecting wild fish populations in Uganda.Wild caught fish were collected from fish landing sites at Kisenyi, Lake Edward; Rubare, Lake Mburo; and Ggaba, Lake Victoria. Gill parasites were observed in 54.5% (18/33), 0.0% (0/15), and 50.0% (14/28) of fish at lakes Edward, Mburo, andVictoria respectively. Internal parasites were observed in 24.0% (6/24), 13.3% (3/15), and 10.7% (3/28) of fish in lakes Edward, Mburo, andVictoria. Parasites present in the gills included metacercariae and adult trematodes, myxozoan plasmodia, copepods, and motile and sessile ciliates. Parasites present in the gastrointestinal tracts included trematodes, nematodes, and myxozoan plasmodia. Currently, morphological and molecular characterizations are being made of the parasites collected. A Henneguya sp. found in the pyloric cecum of a Lates niloticus has been characterized morphologically and sequenced at the 18S rRNA subunit, and is suspected to be a novel species. The molecular and morphological data collected from these parasites can be used in future studies to complete parasitic life cycles and develop rapid diagnostic PCR assays for parasites found to be serious pathogens in Uganda’s growing aquaculture system.

Research Grant: Mississippi State University Global Center for Aquatic Food Security Student Support: Boehringer Ingelheim Veterinary Scholars Program and Mississippi State University CVM

Optimization of expansion techniques for adoptive NK cell transfer in dogs with cancer

Aryana M. Razmara, Sean J. Judge, Cordelia Dunia, Robert B. Rebhun, Michael S. Kent, Robert J. Canter

Department of Surgery (Razmara, Judge, Dunia, Canter), School of Medicine, UC Davis, Sacramento, California. Department of Surgical and Radiological Sciences (Rebhun, Kent), School of Veterinary Medicine, UC Davis, Davis, California

Natural killer (NK) cells can recognize heterogeneous cancer cell targets without prior sensitization, making them promis- ing prospects for use in immunotherapy. We have completed first-in-dog feasibility clinical trials in dogs with cancer using both autologous and allogeneic NK cells expanded from peripheral blood mononuclear cells (PBMCs). Previously, CD5 depletion of PBMCs has been used to enrich for a CD5dim expressing subset prior to NK co-culture with an irradiated feeder line, but this can limit the yield of the final NK product.The purpose of this study was to compare ex vivo culture conditions using standard CD5 depletion versus unmanipulated PBMCs plus feeder line co-culture in matched healthy donors, hypothesizing that PBMCs plus feeder cells would generate an equivalent or superior NK product to CD5 deple- tion. Cell count, fold change, and viability data were collected for both techniques in 12 dogs across five time points up to day 14. A mixed-effects model analysis showed no statistical difference in calculated cell counts, overall fold change, and viability (P > 0.05 all) between PBMCs with feeders and CD5 depleted cells with feeders. PBMCs had a higher mean than CD5 depleted cells at day 14 in all three categories, reaching a peak mean of 677 million cells from 5 million PBMCs at day 0. Killing assays against melanoma and osteosarcoma targets and immunophenotyping also demonstrated comparable results among PBMCs plus feeders versus CD5 depleted NK cells (P > 0.05). Overall, these findings support the use of unmanipulated PBMCs plus feeder line co-culture as an equivalent method to CD5 depletion in the expansion of canine NK cells for adoptive immunotherapy.

Research Grant: National Institutes of Health/National Cancer Institute grant U01 CA224166-01 (RJC) Student Support: SVM Center for Companion Animal Health Endowment funds

282 • 2021 National Veterinary Scholars Symposium Parasites in Captive Felids at the Uganda Wildlife Education Center and Their Zoonotic Implications

Kiera Reardon, Margaret Khaitsa, Celsus Sente, Delilah Namara, Rebecca Nakato, Rachael Mbabazi, James Watuwa, Victor Musiime, Jackson Bwambale Kananga, David Musingo

College of Veterinary Medicine, Mississippi State University, Starkville, Mississippi (Khaitsa, Reardon); College of V eterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala (Sente, Namara, Nakato), Uganda Wildlife Conservation Education Centre, Entebbe (Mbabazi, Watuwa, Musiime, Kananga, Musingo)

As endangered animal species continue to decline, zoos are serving an important role by creating reservoir populations of these animals. Keeping animals in captivity causes challenges such as zoonoses and drug resistance in pathogenic organ- isms. This study investigated occurrence of parasites found in captive felids and their implications on public health. The Uganda Wildlife Education Center (UWEC-Zoo) was the study site, and their animals are acquired through rescue. The hypothesis was that parasite management at UWEC-Zoo is effective thereby resulting in a low prevalence of parasites in their captive felids. Two data sources were utilized: cross-sectional data (June 2021) and retrospective data (2005-2020). For the cross-sectional study, fresh fecal samples were taken from 13 felids (8 lions, 2 tigers, 2 cheetahs, and a leopard). The sampling was repeated twice a week for three weeks. For each fecal sample, wet mounts and sedimentation assays were performed. Retrospective data from 2005-2020 were obtained from UWEC-Zoo and utilized in the study. Felids or feliform species sampled included: serval cats, lions, leopards, hyenas, cheetahs, caracals, and black backed jackals. Toxo- cara sp. and Cystoisospora sp. were recovered from the cross-sectional study while retrospective data reported Ascarid sp., Strongyle sp., Ancylostoma sp., Altenanz sp., Toxocara sp., Amoeba spp., Isospora spp., Taenia spp., Tapeworm sp., Oesophagostomum sp., and Coccidia spp. The presence of parasites in captive felids at UWEC-Zoo implied that parasite management was not as effective as previously thought. Also, presence ofToxocara spp. with zoonotic potential was a significant finding that warrants further study.

Research Grant: Boehringer Ingelheim Student Support: None

A novel xenotransplantation mouse model to study mammary gland regulation and pathogenesis

Alexandra C. Reddy, James L. Miller, Gerlinde R. Van de Walle

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York

The mammary gland is a hallmark of all mammals that is characterized by its postnatal development and hormone-reg- ulated cycles of expansion and regression. Although highly conserved in structure and function, lactation strategies and disease incidence (e.g. mammary cancer) greatly vary across mammals, with many mechanisms responsible for these variations not clearly understood. In vivo models for mammary gland studies beyond the traditional rodent models are scarce, in part due to logistical limitations such as size and cost for large mammals and availability for wild or rare mam- mals. To address this challenge, we aim to establish a xenotransplantation mouse model to study the mammary gland of non-traditional mammalian species in vivo under well-controlled conditions. To this end, the developing pubertal mam- mary gland of immunocompromised female mice is removed and replaced by mammary fragments, containing mammary stem/progenitors and stromal cells, from non-rodent mammals. Thus far, we successfully engrafted equine and canine mammary fragments and found that they recapitulated the mammary epithelial morphology and structure as observed in vivo. After mating the female mice with transplants, deposits of eosinophilic material/proteinaceous fluid were observed in the mammary xenografts indicative of hormonal stimulation caused by circulated hormones during pregnancy. Once fully characterized, this model will serve as a useful in vivo tool for better understanding important processes, both in health and disease, of the mammary gland from virtually any mammal.

Research Grant: The Schwartz Research Fund for Women in Life Sciences Student Support: NIH T35 AI007227, Cornell University Leadership Program for Veterinary Students

2021 National Veterinary Scholars Symposium • 283 Evaluating accuracy and precision of Next-Generation Sequencing using organisms causing canine otitis externa

Miriam E. Reed, Jason B. Pieper, Darren J. Berger

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Next-Generation Sequencing (NGS) technology is increasing in popularity for both microbiome research and clinical applications. However, the efficacy, accuracy, and repeatability of this test is yet to be confirmed by any independent -pub lished study. Standardized strains of the three of the most common infectious organisms causing canine otitis externa were used. Standards of Pseudomonas aeruginosa, Staphylococcus pseudintermedius, and Malassezia pachydermatis were made and sampled in triplicate for NGS testing. The standards were separated into triplicate with a 10-fold serial dilution performed with plate counts to identify a mean cell count for each standard to compare to NGS testing. Pseudomonas aeruginosa was identified by bacterial culture with a mean of 6.9 x 107 colony forming units (CFUs) compared to NGS with a mean of 3.2 x 106 cells per sample, which was statistically significant (P = 0.005). There was a higher variability with NGS with a coefficient of variation (CV) of 49.4% compared to bacterial culture with a CV of 12.9%. Staphylococ- cus pseudintermedius was identified by bacterial culture with a mean of 2.2 x 107 CFUs compared to NGS with a mean of 2.7 x 106 cells per sample, which was statistically significant (P = 0.001). There was a higher variability of NGS with a CV of 45.5% compared to bacterial culture with a CV of 2.7%. Malassezia pachydermatis was detected with NGS with a mean of 6.6 x 104 cells per sample, but no growth occurred on culture. NGS correctly identified all of the organisms from the samples, but there were a lower number of organisms identified. Future studies with a larger sample size are needed to evaluate the precision of NGS and its usefulness in clinical microbial testing.

Research Grant: Boehringer Ingelheim Student Support: Iowa State University College of Veterinary Medicine Summer Scholars Research Program

Characterizing the association of proteinuria with canine and feline periodontal disease

Heather N. Reid, Brenley Fuller, Sydney A. Hastain, Jack Guinan, and Kathryn L. Wycislo

Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

Periodontal disease is a common finding in veterinary companion animals and is frequently considered as a differential for proteinuria. The correlation between periodontal disease and proteinuria has been well established in human medicine, due to a persistent low grade inflammatory response that leads to vascular endothelial cell damage. However, in veterinary medicine, information on this correlation is scarce due to a lack of research in this area. This study aims to retrospectively determine the prevalence of proteinuria in dogs and cats evaluated for periodontal disease at the Midwestern University Companion Animal Clinic. Prospective evaluation of urine protein electrophoresis patterns in a group of patients sched- uled for a Comprehensive Oral Health Assessment and Treatment (COHAT) is also being performed to further charac- terize potential disease associations. Data from this study will help determine if periodontal disease and proteinuria are correlated in dogs and cats and give insight into whether proteinuria in these cases is due to periodontitis-associated inflammation or underlying kidney disease.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

284 • 2021 National Veterinary Scholars Symposium What happens to Paenibacillus larvae spores after cessation of antibiotic use in honey bee hives?

Lara Reitsma, M. Zabrodski, M. Castano Ospina, J. DeBruyne, I. Moshynskyy, S. Wood, I. Kozii, C. Klein, J. Thebeau, F. Masood, S. Biganski, A. Cloet, B. Brown, T. Epp, A. Ruzzini, G. Wilson, E. Simko

Vet. Pathology (Reitsma, Zabrodski, Castano Ospina, DeBruyne, Moshynskyy, Wood, Kozii, Klein, Thebeau, Biganski, Cloet, Brown, Simko), Vet. Microbiology (Masood, Ruzzini), Large Animal Clin. Sci. (Epp), WCVM, University of Saskatchewan, Saskatoon, SK; Ministry of Agriculture, Gov. of Saskatchewan (Wilson), Prince Albert, SK

American foulbrood (AFB) is a bacterial disease of honey bees caused by Paenibacillus larvae. Infection kills larvae and causes economic losses for beekeepers and farmers who rely on pollination services. Since all hives are considered con- taminated with spores of P. larvae, North American beekeepers use metaphylactic antibiotic treatment to prevent clinical signs of AFB. Treatment affects vegetative bacteria, but spores persist and spread disease, causing antimicrobial depen- dence with negative consequences including the development of resistant strains of P. larvae. In this study we 1) assessed changes in spore concentrations in hives after cessation of routine antibiotic use; and 2) screened P. larvae isolates from across the province for evidence of antimicrobial resistance. Brood chamber honey, bees, honey super honey, and bottom board debris were collected from commonly sourced hives with moderate spore contamination at sites with and without antibiotic use over a three-year period. During sampling, hives were inspected for evidence of AFB disease. Samples were cultured on semi-selective media, and spores enumerated from P. larvae colony numbers. There was no incidence of AFB in treated or untreated hives. Spore concentrations remained low across all hives throughout the study without any signifi- cant differences between groups. Antimicrobial resistant strains ofP. larvae were identified in two commercial beekeeping operations. The findings from this study will improve evidence-based decision-making for AFB prevention, including more judicious antimicrobial use. Metaphylactic treatments may not be necessary for all producers if combined with ap- propriate risk assessment and integrative disease management techniques.

Research Grant: Saskatchewan Agriculture Development Fund, Mitacs, Saskatchewan Beekeepers Development Commission, WCVM Interprovincial Graduate Student Fund, Project Apis m. Christi Heintz Memorial Scholarship Student Support: Boehringer Ingelheim Veterinary Scholars Program

In vitro effects of oxycodone and nitric oxide synthase inhibition on fetal rat striatal neuronal morphology

Laura Richards, Christopher Schonhoff

Department of Comparative Pathobiology, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts

Axonal extension and development of the dendritic arbor are critical components of neuronal cytoarchitecture and contrib- ute to connections among neurons during optimal brain development. Previous studies from our laboratory have investi- gated sex-dependent effects of opioids on striatal neuronal morphologyin vitro. These studies demonstrated that oxyco- done negatively impacts development of female, but not male, rat striatal neurons in vitro by inhibiting development of the dendritic arbor. The current studies focus on the potential role of nitric oxide (NO) in mediating striatal neuronal develop- ment. Since NO is involved in neuronal differentiation and is produced in pre- and post-natal striatum, we hypothesized that nitric oxide synthase (NOS) inhibition may be involved in striatal neuronal development. The present experiments aim to both extend previous findings regarding the effects of oxycodone on neuronal cytoarchitecture, and to investigate sex-dependent roles for NO in axonal extension and dendritic morphology. We exposed rat striatal neurons to oxycodone and the NOS inhibitor N(G)-Nitro-L-arginine methyl ester (L-NAME), followed by immunohistochemistry and fluores- cent photomicrographs to analyze the effects of these agents on neuronal morphology. Preliminary results indicate that NOS inhibition prevents axonal extension and decreases dendritic complexity in female, but not male, rat striatal neurons. We also find that both female and male axonal extension is reduced by oxycodone exposure. Taken together, these results indicate that female rat striatal neurons are particularly susceptible to developmental dysregulation by the influence of NOS inhibition and oxycodone exposure.

Research Grant: None Student Support: Cummings School of Veterinary Medicine at Tufts University

2021 National Veterinary Scholars Symposium • 285 Efficacy of a liposomal bupivacaine suspension (Nocita ) for disbudding in dairy calves

Sydney K Richardson, Patrick J. Gorden, Kelsey Meyer, Rebecca L. Parsons, Joe S. Smith, Bonnie Kraus, Suzanne T. Millman

Veterinary Diagnostic and Production Animal Medicine (Richardson, Gorden, Meyer, Parsons, Smith, Millman), Veterinary Clinical Services (Kraus), Biomedical Sciences (Smith, Millman), College of Veterinary Medicine, Iowa State University, Ames, IA

The goal of this study was to determine if a liposomal bupivacaine suspension product (LB) would provide extended pain mitigation when used as a local anesthetic for calf disbudding relative to bupivacaine (B) and lidocaine (L). Based on research in other species, we hypothesized that LB would mitigate pain responses for days versus hours as observed in L and B. Thirty Holstein calves were randomly assigned to one of three treatment groups (LB, B, L). Anesthesia was confirmed by pinprick test, followed by cautery disbudding if the block was successful. Cortisol, mechanical nociceptive thresholds (MNT) and infrared thermography were collected over a 72-hour time period at 0, 0.5 (cortisol only), 1, 3, 6, 9, 12, 24, 36, 48, 60, and 72 hours. Pain related behaviors were quantified from video recordings of the home pen 30 minutes prior to injection and 1, 3.5, 6.5, 12.5, 24.5, 36.5, 48.5 and 60.5 hours relative to disbudding. Preliminary results indicate of the 20 horn buds (HB; two per calf) enrolled in each treatment, 65%, 75% and 85% were successfully blocked within 60 minutes for LB, B and L, respectively. Of the 10 calves enrolled in each treatment, two calves had one HB block within L and LB, three calves had one HB block within B, two calves had neither HB block within L and B, and three calves had neither HB block within LB. There were no observed differences between treatments in the probability of the HB block- ing (1.260.6 P = 0.39). There were no observed differences between latency to block between treatments (14.862.9 min [LSMeans 6 SE]; P = 0.33). Analyses of additional pain related outcomes are pending.

Research Grant: Iowa Veterinary Medical Association and Iowa Livestock Health Advisory Council Student Support: College of Veterinary Medicine, Veterinary Summer Scholar Program

Colonization and effect of Nissle 1917 E Coli probiotic on the healthy feline microbiome

Jordan E Riha, Jenessa A Winston, James P Howard, Valerie J Parker, Adam J Rudinsky

Department of Veterinary Clinical Sciences, The Ohio State CVM and Comparative Hepatobiliary and Intestinal Research Program (CHIRP)

Probiotics are commonly used therapies in veterinary medicine. E. coli Nissle 1917 (EcN) is a commercially available, non-pathogenic probiotic strain that can produce bacteriocins that exhibit antimicrobial activity against closely related bacteria. Previous work has shown health benefits following administration of this oral probiotic in humans and dogs, however it has not previously been used in cats. The objectives of this study were to detect short term colonization of EcN in the feline microbiome, to observe strain level diversity of E. coli following EcN ingestion, and to look at the impact of EcN on the overall healthy cat microbiome. Five healthy research cats received EcN (108 viable EcN bacteria/ml; 5 ml/ dose) for 3 days, followed by a 25-day observation phase. Fecal samples were collected at baseline (days -2, -1, 0), during probiotic administration (days 1, 2, and 3) and then post-administration (days 4, 5, 6, 7, 14, 21, and 28). Each fecal sample will be analyzed for presence of the EcN probiotic using EcN specific PCR primers, total microbiome structure, and culture for strain specific shifts in E. colisubpopulations based on 20 individual isolates from each fecal sample collected. This project is ongoing. The animal portion of the study has been completed and all samples have been collected. DNA extraction of study samples is currently underway. The next steps in the project include identifying EcN in the fecal sam- ples, typing individual E coli isolates using the EcN specific PCR primers, and submission of fecal DNA for microbiome sequencing.

Research Grant: Dr. Adam Rudinsky research laboratory Student Support: Ohio State Veterinary Student Summer Research Program

286 • 2021 National Veterinary Scholars Symposium Evaluating canine aryl hydrocarbon receptor’s role in osteosarcoma progression, metastasis and chemoresistance

Justin Ringhofer, Dan York, Rob Rebhun

Center for Companion Animal Health, College of Veterinary Medicine, University of California: Davis, Davis, California

Aryl hydrocarbon receptor (AHR) is a cytosolic transcription factor that upon activation binds to DNA sequences and alters the activity of nearby genes. These DNA sequences are dispersed in many species’ genomes and may regulate thou- sands of genes. AHR can be activated by many ligands such as omeprazole and the toxin TCDD. Previous studies have found increased AHR expression in several canine cancers. The aim of this study was to identify the role AHR may have in canine osteosarcoma (OSA) progression, metastasis, and chemoresistance. The hypothesis was that activation of AHR inhibits OSA growth/migration and contributes to the cytotoxic effects of chemo. 3 OSA cell lines were used for majority of this study: Abrams, Gracie, HMPOS. As expected, qPCR showed that AHR inhibition by siRNA decreased AHR RNA expression by 3-fold. Treatment with TCDD (100nM) for 2 hr, increased CYP1A1 expression, which could be abolished with 24-hr pretreatment of AHR siRNA. Results from proliferation assays showed that both doxorubicin (DOX) and carboplatin (CAR) significantly decreased proliferation in OSA cell lines. In Abrams, the combination of CAR and AHR inhibition resulted in significantly less proliferation, as compared to CAR alone, but had no affect when combined with DOX. Interestingly, AHR siRNA alone also decreased proliferation in Abrams. However, AHR siRNA had no effect on proliferation for Gracie or HMPOS cell lines, either alone, or in combination with CAR or DOX. Preliminary data from scratch assays showed that both omeprazole and indol-3-carbinol (I3C) significantly inhibited wound healing in all 3 OSA cell lines. The results gathered so far suggests that AHR may play a role in the proliferation and migration of canine OSA.

Research Grant: Center for Companion Animal Health, UC Davis College of Veterinary Medicine Student Support: NIH T35 Training Grant T335OD012199

Repurposing b-blockers as anti-cancer immunotherapeutics to target myeloid derived suppressor cells

Cody Rinker, Dylan Ammons, Steven Dow

Flint Animal Cancer Center, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature myeloid cells which arise in re- sponse to chronic inflammation associated with cancer and other inflammatory disorders.There are two distinct subsets of MDSCs, polymorphonuclear (PMN) and monocytic (M), both of which correlate with poor outcomes in cancer patients. In human and murine models, b-adrenergic stress has been shown to promote the differentiation and immunosuppressive functions of MDSCs through interactions between catecholamines and b-adrenergic receptors. This study aims to evaluate the potential of b-adrenergic signaling antagonists (b-blockers) to decrease the MDSC burden and enhance anti-cancer immune responses. We hypothesize that the quantity and function of circulating MDSCs in canine cancer patients will positively correlate with degree of b-adrenergic stress and that treatment with b-blockers will decrease their immunosup- pressive capabilities and abundance. To this end, we quantified the MDSC burden in the blood of healthy dogs and cancer patients then correlated urine concentrations of metanephrine and normetanephrine with MDSC number. We confirmed that canine PBMCs express b1 and b2 receptors via flow-cytometry and western blotting, then investigated ifb -adren- ergic activation enhances MDSC immunosuppressive functions and differentiation. Finally, we assessed whether non-se- lective or b1-specific blockers could reverse the effects of non-specificb -adrenergic activation. These studies provide insights into the relationship between b-adrenergic stress, MDSC burden, and cancer immune suppression. Ultimately, the findings from these studies can inform how theb -adrenergic signaling axis could be targeted in the clinic.

Research Grant: Shipley Foundation and NIH U01 CA224182 Student Support: Unknown

2021 National Veterinary Scholars Symposium • 287 Nonhuman Primate pregnancies at advanced maternal age show markers of poor metabolic and cardiovascular health

Alexis A. Roach, Masha Block, Kennita Johnson, Rachel White, Kylie Kavanagh, Sarah Cilvik

College of Veterinary Medicine (Roach), North Carolina State University, Raleigh, NC; Department of Pathology (Kavanagh, Block), Department of Pediatrics (Cilvik), Wake Forest School of Medicine, Winston-Salem, NC; University of Chapel Hill (Johnson, White), Chapel Hill, NC

Advanced maternal age (AMA) is becoming increasingly common in developed countries and is associated with ad- verse pregnancy outcomes for both mother and offspring. Previous studies using rodent models of aged pregnancies have demonstrated evidence of cardiovascular (CV) risk in offspring, and fetal programming that is seen in the unfavorable intrauterine environment. Rodent models, however, are less ideal for studies of reproductive physiology, and there is a need for nonhuman primate (NHP) reproductive models to better recapitulate human pregnancy. The aim of this study is to determine the effects of AMA and explore placental features that may affect in utero development, and thus the risk for chronic disease, with a focus on CV biomarkers in the vervet (Chlorocebus aethiops sabaeus) NHP. We evaluated pla- cental perfusion using contrast-enhanced ultrasound (CEUS) and fetal sizing (young n = 3, AMA n = 2). Moreover, we assessed measures of metabolic syndrome (abdominal circumference, lipid profile, HgbA1c) and CV risk (blood pres- sure, plasma NT-proBNP concentration) in adult NHPs (n = 87-153) with archived plasma and health information. Adult offspring from AMA pregnancies have 2% large abdominal circumference and 14% lower HDL-C (P < 0.05 for both) compared to offspring from pregnancies at a young age. Plasma NT-proBNP concentration, a biomarker often associated with cardiac dysfunction, showed an inverse correlation with adiposity but was unrelated to maternal age at birth. In con- clusion, our study demonstrates that AMA is associated with signs of poor metabolic health in offspring. This necessitates ongoing research to determine in utero mechanisms that program chronic disease in offspring of older mothers.

Research Grant: NIH P40 D010965, UL1 TR001420, R01 HL142930 Student Support: T35 OD010946

Effect ofTTC37 gene variants on intestinal epithelial barrier function in THES patients

Daana M. Roach, Brooke E. Boyer, Gloria E. Soto, Tatiana Karakesheva, Noor Dawany, Judith R. Kelsen, Kathryn E. Hamilton

School of Veterinary Medicine (Roach) and Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia (Boyer, Soto, Karakesheva, Dawany, Kelsen, Hamilton), University of Pennsylvania, Philadelphia, PA

Trico-hepatic-enteric syndrome (THES) is a rare form of very early onset inflammatory bowel disease (VEO-IBD) with heterogeneous clinical presentation, including intractable diarrhea, liver abnormalities, immune deficiencies, cardiac de- fects, and facial dysmorphia. It is a monogenic disorder with mutations in either TTC37 or SKIV2L genes for which there is no cure and limited therapeutic options. The contribution of impaired gene function to THES pathogenesis, and particu- larly to epithelial barrier dysfunction, is poorly understood. Given preliminary data demonstrating phenotypic differences between THES and control colonoids, we hypothesize that epithelial-intrinsic defects underlie epithelial barrier dysfunc- tion leading to the gastrointestinal symptoms associated with THES. Using colonoids derived from patients with THES and age-matched control patients, this project assesses epithelial barrier function through evaluation of colonoid growth, ZO-1 staining, and transepithelial electrical resistance (TEER). So far, we find that colonoids from patients with THES grow at a reduced frequency compared to healthy control colonoids. Results from colonoid studies in patients with THES, and more broadly in patients with VEO-IBD and IBD, represent a valuable form of personalized medicine to mimic in vitro disease conditions and serve as a foundation to develop novel treatment plans that can be used in vivo.

Research Grant: Unknown Student Support: Boehringer-Ingelheim

288 • 2021 National Veterinary Scholars Symposium Effect of mosquito salivary proteins in animal endothelial physiology

Nicole D. Robben, Jessica D. Burger, Andres F. Holguin-Rocha, Pablo D. Becerra and Berlin Londono-Renteria

Vector Biology Laboratory, Department of Entomology, Kansas State University, Manhattan, KS

Mosquito saliva has garnered significant interest due to its potent immunomodulatory effects, yet most of its effects on- an imal cells remain unknown. Previous RNA sequencing of Human Umbilical Vein Endothelial Cells (HUVEC) incubated with mosquito salivary gland extracts (SGE) revealed altered expression of genes involved in the regulation of angiogen- esis, cell survival, and inflammation. This study aimed to investigate further how these changes impact the host in a way that may lead to increased susceptibility to diseases like cancer or arthropod-borne pathogens. Rt-qPCR was performed using RNA from HUVEC cells incubated with SGE from Anopheles, Aedes, and Culex mosquitoes, or the salivary peptide Apyrase 1 (Apy1). We also evaluated the effect of salivary proteins on angiogenesis and cell proliferation (MTT and LDH levels). Interestingly, when incubated with SGE and Apy1, HUVEC cells displayed differences in transcriptome expres- sion, with SGE upregulating pro-angiogenic genes while Apy1 upregulated inhibitory Neurofibromin and SP100. This difference is also seen in the angiogenesis assay in which Apy1 displayed a significant increase in individual cells. MTT and LDH Cytotoxicity assay results showed no significant differences.These results suggest the action of Apy1 is distinct from SGE.

Research Grant: This research was funded by the USDA National Institute of Food and Agriculture, Hatch-Multistate, project 1021430 and the COBRE–NIH 5P20GM103638-08 Student Support: This work is sponsored in part by an NIH T35 training grant T35OD029981

Hypothalamic-Pituitary-Adrenal Axis Assessment in Healthy Foals Utilizing Vasopressin Stimulation Test

Hannah P. Robertson, Erin Elder, Jenna Schirmer, Katarzyna Dembek

Department of Clinical Sciences (Elder, Dembek, Schirmer), College of Veterinary Medicine, North Carolina State University, Raleigh, NC.

Sepsis is the life-threatening organ dysfunction caused by the body’s dysregulated response to infection, and it is the leading cause of mortality in neonatal foals. A major complication of sepsis correlating to mortality is relative adrenal insufficiency (RAI), caused by dysregulation of the Hypothalamic-Pituitary-Adrenal axis (HPAA) and cortisol deficien- cy. There are strategies to diagnose RAI, though sensitivities vary. Little is known of the effect vasopressin (AVP) has on cortisol response in foals, though it plays a large role in the HPAA. This crossover study utilized AVP stimulations at 3 doses of AVP (2.5, 5, and 7.5 IU), given 12 hours apart. The stimulations quantified AVP’s effects on cortisol response in 13 healthy foals. Baseline blood samples were collected at 24h of age, then at 15, 30, 60, and 90 minutes after AVP admin- istration. Vital parameters were taken at 0, 5, 10, 20, 30, 60, and 90 minutes. Cortisol was quantified with a radioimmuno- assay. Vital parameters confirmed a large safety profile with mild, transient differences in heart rate, mucous membranes, and gut sounds at all doses. The cortisol levels were significantly increased at all doses. Concentrations peaked 30 minutes after AVP administration. Cortisol increased up to 45 times the baseline values reaching 20-40mg/dL for all doses. All 3 doses are safe and effective for testing HPAA function in healthy foals. Other HPAA hormone levels in the samples will be evaluated to determine AVP’s full effect. In the future, AVP tests will be performed in critically ill foals. This procedure could be a more sensitive test for RAI in septic foals, better informing veterinarians on the foal’s current state and the best plan for sepsis and RAI treatment.

Research Grant: Intramural Seed Grant Student Support: NC State CVM VSP and BIVSP

2021 National Veterinary Scholars Symposium • 289 Adaptions in the masticatory-associated musculature in the hypercarnivorous African wild dog (Lycaon pictus)

Felicia A. Rocco, Mia A. Felix, and Heather F. Smith

Department of Anatomy, College of Veterinary Medicine, Midwestern University, Glendale, AZ

African wild dogs (Lycaon pictus) are hypercarnivorous canids that consume meat almost exclusively and engage in long distance, coordinated hunts. The goal of our study is to determine whether the largely undescribed muscles of mastication and related muscles of L. pictus show morphological adaptations to hypercarnivory. Gross anatomic dissections of the masticatory musculature of an adult male African wild dog in comparison to a juvenile coyote (Canis latrans) and domes- tic dog (Canis familiaris) revealed size differences and morphological variations. Notably, the muscles of mastication, and other associated muscles, were appreciably more robust and contained more muscle fibers inL. pictus than either compar- ative canid taxon. C. latrans also showed stark differences fromC. familiaris. A distinct lack of deep fascial covering on m. pterygoideus medialis and inferior placement of m. pterygoideus lateralis illustrate potential adaptations. In C. latrans, the more transverse orientation of m. pterygoideus lateralis suggests it functions to protrude the mandible, rather than elevate it as in C. familiaris. In C. familiaris, m. digastricus traveled rostrally along the ventral portion of the mandible and inserted at M1. Conversely, m. digastricus of C. latrans had both a belly terminating in this fashion and a second belly continuing rostrally on the medial aspect of the mandible to insert at C1. This suggests a more powerful contribution to mouth opening. Continued dissections will reveal further comparative results pertaining to size, attachments, and function of the canid masticatory musculature. The results of our study will elucidate the evolution of cranial musculature of vari- ous canid species, from domestic to wild.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Role of polyamine inhibitors in pheochromocytoma/paraganglioma cancers with SDHB mutation

Mateo Rodriguez, Hans K Ghayee, Sergei G Tevosian, Heather M Hatch

Department of Endocrinology (Ghayee), College of Medicine, University of Florida, Gainesville, FL and Department of Physiological Science (Sergei, Hatch), College Veterinary Medicine, University of Florida, Gainesville, FL

Adrenal pheochromocytoma (PCCS) and parganglioma (PGLs) tumors carry high disease burden due to excessive hor- mone secretion by the tumor cells and high probability of metastasis in human and canine patients who carry a mutation of succinate dehydrogenase subunit B (SDHB) gene. The overall aim of the study is to put forward the first safe and effec- tive pharmacological treatment against metastatic tumors with a loss of the SDHB function and impacting the outcome of many patients. One of the essential steps is understanding, why polyamine inhibitors are effective against these tumor types, specifically, tumors withSDHB mutation. Preliminary experiments demonstrated the regression of PCCs with a mutation of SDHB gene when treated with N1,N11-diethylnorspermine (DENSPM) both in vivo and in vitro. Whereas, histone deacetylase inhibitors (HDACis) though known to differentiate cells and currently used in clinical tests to treat cancers, have proven ineffective on hPheo1 tumor cell lines. Our study focused on three cell lines, hPheo1,SDHB nega- tive hPheop1, and SDHB rescued hPheo1. Our hypothesis that we could increase the efficacy of DENSPM using HDACis was tested by challenging hPheo1 cells with and without an SDHB mutation with sodium butyrate, or CI-994, and using Quantitative Real Time PCR to test for differentiation protein. Our results showed HDACis had no significant effect on the cells in reference to differentiation. Western Blot demonstrated the successful rescue of theSDHB gene. Additional experiments of the effects of DENSPM on the rescued cells compared to the mutated cells is underway and genes will be tested using RNAseq approach.

Research Grant: College of Veterinary Medicine, University of Florida Student Support: Florida Veterinary Scholars Program

290 • 2021 National Veterinary Scholars Symposium Efficacy and safety of the Rho-kinase inhibitor ripasudil (Glanatec) in canineADAMTS10 -open-angle glaucoma

Olivia E. Rodts-Palenik, Christine D. Harman, Ava Cabble, Juan P. Steibel, Kumiko Kato, Andras M Komaromy

College of Veterinary Medicine (Rodts-Palenik, Harman, Cabble, Komaromy), Departments of Animal Science & Fisheries and Wildlife (Steibel), Michigan State University, East Lansing, MI, USA; Kumi Animal Hospital, Misato-City, Japan

Glaucoma is a common painful optic neuropathy, culminating in irreparable blindness. While current medications fail to effectively alleviate glaucoma, few innovative glaucoma medications have entered the market since the 1990s.This study utilized eleven beagles with ADAMTS10-open-angle glaucoma to assess efficacy and safety of ripasudil, a novel topical Rho-kinase inhibitor recently approved for human use in Japan. The four-week study included baseline (week 1), q24h administration (week 2), q12h administration (week 3), and washout measurements (week 4). Left and right eyes were randomly assigned to receive either BSS (balanced salt solution) sham treatment or ripasudil. Efficacy assessment includ- ed diurnal intraocular pressure (IOP) and pupil diameter, and safety measures included diurnal conjunctival hyperemia, routine eye examinations, central corneal thickness, and gonioscopy. The linear Gaussian model was used to evaluate the differences in least square means of all quantitative measurements between eyes that received BSS and ripasudil. There were no significant differences in mean diurnal IOP (baseline- treatment: 27.886 1.50 mmHg vs. sham: 26.976 1.48mmHg; q24h-treatment: 28.82 6 1.70 mmHg vs. sham 27.70 6 1.67 mmHg; q12h-treatment: 30.776 2.82 mmHg vs. sham: 29.716 2.78 mmHg; washout-treatment: 34.686 2.68 mmHg vs. sham: 32.946 2.64 mmHg) and mean diurnal pupil diameter or central corneal thickness. Although ripasudil appears to cause mild to moderate conjunctival hyperemia when administered q12h (baseline average score: 0.24; q12h 11am score: 1.32; q12h 3pm score: 1.6), no further adverse effects were detected. Together, these results do not support future use of ripasudil as treatment for canine glaucoma.

Research Grant: NIH R01-EY025752 Student Support: NIH grant 5T35OD016477-19

Exploring the role of Rhipicephalus sanguineus as a vector for Rocky Mountain spotted fever in Arizona

Nicolette Roe, Jeffrey Mutterperl, Alec Oliva, Mary Griffin Logue, Kayla Allwardt, Charles Schaefer, Jose Hernandez, Rachael Kreisler, Jung Keun Lee, Michael Quinlan, and John VandenBrooks

College of Veterinary Medicine (Roe, Logue, Allwardt, Kreisler, Lee), College of Graduate Studies (Schaefer, Hernandez, Lee, Quinlan, VandenBrooks), Arizona College of Osteopathic Medicine (Mutterperl, Oliva), Midwestern University, Glendale, AZ

Recently, there has been an outbreak of Rocky Mountain spotted fever (RMSF) in Arizona, which is a tickborne disease caused by the bacteria Rickettsia rickettsii. This zoonotic disease is of importance to public health officials because it is carried by domestic dogs. The brown dog tick (Rhipicephalus sanguineus) has been demonstrated to be the vector of RMSF in Arizona and the surrounding region, as the typical RMSF vectors are not present in these areas. Previous re- search in our lab shows there are three distinct populations of R. sanguineus in Arizona, with rickettsial bacterial infection rates varying between each. What is still unknown is how the rickettsial strains vary across these tick populations and regions, whether there is a unique strain present, or if the strain is more favorable in brown dog ticks. To answer this, PCR was used to amplify rickettsial DNA in samples extracted from ticks collected off canines by the Midwestern Mobile Veterinary Clinic. Previously, we demonstrated that rOmpA is an excellent marker for the presence of R. rickettsii but is too highly conserved to be of use for phylogenetic analysis. Therefore, we analyzed two intergenic regions (RR0155 and RR1240) that have much higher variability for phylogenetic analysis. We designed primers to perform a semi-nested PCR reaction for each of the intergenic regions, to ensure highly enriched and pure DNA, as samples contain both tick and rick- ettsial DNA. Phylogenetic analysis of the intergenic regions shows that rickettsial strains do vary within the region and are correlated with human case rates. This suggests that while the reasons behind the spread of RMSF in the region may be multi-factorial, rickettsial strain is a contributing factor.

Research Grant: Midwestern University Student One Health Research Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

2021 National Veterinary Scholars Symposium • 291 Identification of tick salivary gland antigens recognized by protective immune sera using a cDNA library

Bridgette M. Rogers, Sammuel Shahzad, Stefan H. Keller, Kyle J. Cacciatore and Roger W. Stich

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO

Among vectors of our domestic animals, ticks are responsible for the greatest occurrence of disease transfer worldwide. Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is the most widespread vector-borne dis- ease of cattle. Decreased weight gain, diminished milk production, abortion and high mortality associated with this disease cause significant production losses. In addition, once cattle are exposed to this pathogen, they typically remain carriers for life. Although subclinical, carriers maintain the ability to spread the disease to naive hosts through infection of ticks. Some vaccines can control acute clinical anaplasmosis in cattle but, to the best of our knowledge none of these vaccines induce sterile immunity to A. marginale. This means that exposed cattle still serve as reservoirs of disease. As an intensely studied vector-borne disease, the bovine anaplasmosis model offers a tractable system for the study of tick-pathogen-ver- tebrate interactions. Identification of seroreactive tick molecules is an important next step in the creation of a vaccine that will block transmission of A. marginale to the host. Therefore, the objective of this study is to identify tick salivary gland antigens that are uniquely seroreactive to putatively protective immune sera. Using RNA extracted from the salivary gland tissue of Dermacentor andersoni ticks, we will create a cDNA expression library. Resulting clones will be screened with putatively protective bovine serum. Clones identified as positive may have the potential to become vaccine candidate antigens for the control of this prototypical pathogen as well as other closely related tick-borne disease agents of livestock, companion animals and people

Research Grant: USDA NIFA 17-67015-26630 Student Support: an endowment established by IDEXX-BioAnalytics

Detection and characterization of isolates from natural lakes and waters in Alabama

Chloe Rogers, Yesutor Soku, Abdelrahman Mohamed

Department of Biomedical Sciences, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama

Edwardsiella tarda is a Gram-negative bacterium that infects both cultured and wild species of fish worldwide. Serious economic loss is associated with E. tarda due to the bacteria infecting economically important species of fish such as the Japanese eel, Japanese flounder, tilapia, and channel catfish. In the USA, commercial channel catfish farms in Alabama, Mississippi, and Louisiana have reported E. tarda infection within the last decade. This infection is called emphysematous putrefactive disease, which may further lead to muscular necrosis. The hypothesis of this study is, if isolates of E. tarda are found in natural bodies of water, then commercial fisheries that house certain species of fish (channel catfish) will most definitely be exposed toE. tarda due to the presence of this bacteria in water bodies. E. tarda is mostly masked amongst many other bacteria in natural bodies of water making isolation difficult.Therefore, different media agents of both broth and agar are utilized to in isolating E. tarda which is followed by various biochemical tests. Confirmation ofE. tarda will then lead to the determination of an antibiotic susceptibility pattern for these bacterial isolates. Ultimately, the aim of this study is to identify and characterize environmental isolates of E. tarda and to determine the antibiotic susceptibility profile for these isolates.

Research Grant: Research grant number: NI20AHDRXXXXG025 Student Support: Tuskegee Veterinary Scholars Program

292 • 2021 National Veterinary Scholars Symposium Prevalence of TB and FMDV Among Animals Selected from Slaughterhouses in Mbarara District in Western Uganda

Braiam Rosado-Ramos, Peter Kalumba, Robinah Sarah Nakabuye, Andrew Rwotomara, Susan Kerfua, Celsus Sente, Cooper Brookshire, Margaret Khaitsa

Dept of Pathobiology and Population Med (Rosado-Ramos, Brookshire, Khaitsa), College of Vet Med, Mississippi State University, Starkville, MS, College of Vet Med,Animal Resources and Bio-security (Kalumba, Nakabuye, Rwotomara, Sente), Makerere University, Kampala, Uganda, National Agricultural Research Organization Lab (Kerfua), Kampala, Uganda

Bovine tuberculosis (TB) is a public health concern particularly in developing countries, such as Uganda. Mycobacterium bovis, has an extremely wide host range, spread by ingestion or inhalation of animal bioproducts like meat and/or milk. The lack of a control policy in Uganda and the livestock-wildlife interface raises concerns about other diseases of great economic or public health concern, such as Foot and Mouth Disease (FMD). FMD affects all cloven-footed animals, trans- mitted via aerosols, fomites and body fluids. The objectives of this study are to investigate the potential of using slaugh- terhouses in Uganda as bio-surveillance points for TB and to conduct FMD surveillance. To estimate the prevalence of TB among cattle, sheep, and goats slaughtered in Mbarara District, we utilized evaluation of gross lesions, Ziehl-Neelsen (ZN) staining, and PCR surveillance. Fifty animals from Mbarara district abattoirs were randomly selected, and lung/ lymph node smear impressions were performed for ZN analyses. Blood, lung, and lymph node tissues were collected for PCR analyses. The gross lesion evaluation and ZN staining revealed a single positive case of TB from a goat sampled at the Rubindi slaughterhouse in both lymph node and lung tissues. PCR analyses are currently pending. Forty-seven appar- ently healthy Ankole beef cattle from the Nshara Ranch and 15 beef cattle presenting for slaughter at the Mbarara City abattoir were tested for FMD seropositivity utilizing a FMDV 3ABC ELISA test. Seropositivity rates of 93.6% (95% CI; 0.83-0.98) and 53% (95% CI; 0.3-0.75) were detected from the ranch and slaughterhouse, respectively. None of the 20 sheep and goats tested at the Mbarara City slaughterhouse were seropositive for FMD.

Research Grant: Boehringer Ingelheim Veterinary Scholars Program and Mississippi State University College of Veterinary Medicine Student Support: None

Identification of signaling protein RNA in the sea star egg transcriptome

April Rosales, David Carroll

Department of Biochemistry and Molecular Genetics, College of Graduate Studies, Midwestern University, Glendale, AZ

One requirement for a successful egg activation event is Ca2+ release at the conclusion of the initial fertilization signal- ing pathway. This occurs in all plants and animals that have been studied. Currently, only a few proteins are known to be involved in the fertilization signaling pathway, including two Src family kinases (SFK1 and SK3) and phospholipase Cg in the sea star. This project utilized the T cell receptor signaling pathway as a model for identification of RNA for specific proteins present in the Patiria miniata egg RNA transcriptome. The T cell receptor signaling pathway shares similarities with the fertilization signaling pathway in Patiria minata, such as Ca2+ release and initiation of DNA synthesis. The sea star has benefits as a model system, such as genome and RNA transcriptome availability, and synchronization of cells in the ovary. Twenty proteins from the T cell receptor signaling pathway were analyzed, and RNA from 18 of these pro- teins were identified in thePatiria miniata egg transcriptome. The identified proteins included two growth factor recep- tor-bound protein 2, three tyrosine protein kinases, and four mitogen-activated protein kinases, among others. Results with a strong homology (> 40% identity) were considered to demonstrate expression in Patiria miniata eggs, and percent identities ranged from 35% to 76%. Overall, the identification of these proteins in the sea star egg transcriptome are signif- icant because of the potential to expand our understanding of the fertilization pathway. These findings are also applicable to mammals, due to similarities in the signaling pathway at fertilization.

Research Grant: Midwestern University Start-Up Funds Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 293 Effects of Porcine Intestinal Normothermic Machine Perfusion and Cold Storage on Epithelial Barrier Integrity

Natalia L. Rosa-Padilla, Elsa K. Ludwig, John Freund, Amy S. Stewart, Hannah Jones, Liara M. Gonzalez

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Intestinal failure (IF) is a loss of intestinal function resulting in an inability to absorb nutrients. IF can be due to patholo- gies such as inflammatory bowel disease, vascular thrombosis or short bowel syndrome. Intestinal transplantation is the final therapeutic option available for IF patients. Cold storage (CS) is the current gold-standard storage method necessary for preservation prior to transplant. However, CS results in epithelial damage which causes impairment of mucosal barrier function, bacterial translocation, sepsis, and graft rejection. Tight junction proteins (TJ) maintain intestinal epithelial in- tegrity and their loss has been associated with CS in other organs. Normothermic Machine Perfusion (NMP) is an alter- native preservation method that has been demonstrated to be less damaging than CS to kidney and liver grafts, however intestinal NMP has not been described. Our objective was to compare the effects of intestinal CS and NMP on epithelial barrier integrity with an emphasis on structural proteins. We hypothesized that epithelial barrier integrity would be more preserved using NMP compared to CS. Seven porcine intestines were preserved with CS or NMP for 6hrs. Jejunal biop- sies were obtained prior to and after CS or NMP storage. Immunofluorescence for apoptotic cells (CC3) was performed and immunofluorescence for tight junction proteins claudin-3 and claudin-4 is underway. Data was compared between conditions using 2-way ANOVA. Cells undergoing apoptosis were significantly greater in both CS (P = 0.0026) and NMP (P = 0.0061) compared to control, but no significant differences were found between NMP and CS. Completion of clau- din-3 and claudin-4 immunofluorescence is required before a conclusion can be made.

Research Grant: U.S. Department of Defense PR181265, NIH K01OD010199 SERCA, NIHP30 DK034987 Student Support: NIH-T-35 Interdisciplinary Biomedical Research Training Program (IBRTP)

Characterization of immunoreactive cDNA expression library clones of Baylisascaris procyonis.

Max Rowley, Vishnu Manikantan, and Sriveny Dangoudoubiyam

Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, Purdue University, West Lafayette, IN

Baylisascaris procyonis infection is an emerging zoonosis in North America, Europe, and Asia. Adult B. procyonis are found in the small intestine of raccoons and parasitic eggs are shed in their feces. Accidental ingestion of the eggs can result in larva migrans in humans and over 150 different species of mammals and birds. MigratingB. procyonis larvae travel through somatic tissues (visceral larva migrans), the brain (neural larva migrans - NLM), and eyes (ocular larva migrans - OLM), inflicting serious damage. While several cases occur as covert disease, infection can lead to irreversible vision loss, encephalitis, and death. Currently, there is a lack of understanding of B. procyonis larval virulence factors that play a role in the pathogenesis of this parasite. This study aims to characterize the immunoreactive clones that have been identified via screening of the larval cDNA expression library. Sequencing and bioinformatic analyses were performed on the previously identified and isolated cDNA clones, and their putative identities were established. A few select clones were over-expressed in a bacterial expression system and the recombinant proteins were purified using metal-affinity chromatography to further establish their sero-reactivity and to generate antisera. Experiments are ongoing to establish their differential expression between variousB. procyonis life cycle stages to identify a larva-specific antigen. Overall, this study is part of a larger project aimed at discovery of B. procyonis larval effector molecules that play a role in host-patho- gen interaction during the migratory phase of the parasite in the abnormal human host.

Research Grant: Departmental Start-up Grant, Purdue University Student Support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

294 • 2021 National Veterinary Scholars Symposium Evaluating the relative survivability of Mannheimia haemolytica in various environments

Robert P. Ruffin, Sara D. Lawhon, Brian V. Lubbers, Sarah F. Capik

College of Veterinary Medicine and Biomedical Sciences (Ruffin) and Department of Veterinary Pathobiology (Lawhon, Capik), Texas A&M University, College Station, TX 77843, College of Veterinary Medicine (Lubbers), Kansas State University, Manhattan, KS, Texas A&M AgriLife Research (Capik), Amarillo, TX

Bovine Respiratory Disease (BRD) is a multifactorial disease that poses significant challenges for the beef and dairy in- dustries. Of the clinically relevant bacterial pathogens, Mannheimia haemolytica is most commonly isolated from clinical cases of BRD. The objective was to evaluate the survival of M. haemolytica under various environmental parameters on metal, wood, and plastic. Temperature and humidity were monitored in microenvironments created using plastic contain- ers maintained at approximately 38C, 228C, and 368C. The average humidity of the three environments was approximately 84%, 52%, and 57%, respectively. An inoculum of M. haemolytica in Brain Heart Infusion broth (2.5x104 CFU/mL) was used to inoculate the surface of 6.35 mm diameter spheres made of stainless steel, polypropylene, and wood which were then placed in each microenvironment. At 0, 2, 6, 24, and 48 hours after inoculation, each material was cultured for the presence of M. haemolytica and identification was confirmed using MALDI-TOF mass spectrometry. Stainless steel mate- rials were culture negative at all timepoints in all environmental conditions. Mannheimia haemolytica survived the longest (up to 24 hours) on plastic at 38C and survived for up to 6 hours on both wood and plastic at 228C. Bacterial survival was negatively impacted by increasing temperature and decreased humidity with all materials culture negative at 2 hours in the 368C environment. Our results demonstrate that M. haemolytica survivability is impacted by both material and environ- ment. Better understanding the roles of these factors in M. haemolytica survivability will allow for improved biosecurity protocols for both beef and dairy operations.

Research Grant: None Student Support: Boehringer Ingelheim VSP, Texas A&M College of Veterinary Medicine & Biomedical Sciences

Chemotactic and adhesion properties of equine CD172a+ peripheral blood eosinophils

Joshua T. Russek, Jack Guinan, and Margaret M. Brosnahan

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Eosinophils are granular leukocytes with various functions, but most notably play a role in parasitic infections and allergic inflammation. To further grasp these various functions, understanding the mechanisms of eosinophil degranulation and homeostasis are important first steps. One important surface molecule is CD172a, an inhibitory receptor signal regulatory protein that contributes to eosinophil homeostasis. CD172a appears to prolong eosinophil survival by inhibiting degranu- lation, thus preventing eosinophils from contributing to an inflammatory response.We have demonstrated that stimulation of equine eosinophils with IL-5 upregulates expression of CD172a, but the functions of this molecule in the context of equine disease have not been investigated. Due to the horse’s large blood volume, isolation of these rare cells is possible and may serve as a model for comparative eosinophil research. Our lab isolates equine peripheral blood eosinophils using discontinuous density centrifugation and further purifies the sample using cell sorting. Eosinophils are then stained with CD172a and passed through a magnetic-activated cell sorting column to separate CD172a+ and CD172a- cells. These two populations of eosinophils are analyzed via adhesion and chemotaxis assays, which are measured by fluorescent intensity. This project will serve as a prelude for further investigation of eosinophils obtained from tissue digestion and other bodily fluids, such as bronchoalveolar lavage. Equine eosinophil research will help further our understanding of the roles that eosinophils play in disease and may serve as a model for humans with common diseases like asthma.

Research Grant: Midwestern University College of Veterinary Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 295 Colorectal cancer in rhesus macaques and the hunt for hypermethylation

Declan Ryan, Simon Deycmar, Brendan Johnson, Gregory Dugan, David Caudell, Shane Sills, Mark Cline

School of Veterinary Medicine, University of California Davis, Davis, California, USA (Ryan); Department of Pathology, Section on Comparative Medicine, School of Medicine, Wake Forest University, Winston-Salem, North Carolina (Decymar, Johnson, Dugan, Caudell, Sills, Cline)

Colorectal carcinoma (CRC) is the second leading cause of cancer deaths in humans and the most frequent malignancy in rhesus macaques. A subset of human CRCs exhibit hypermethylated CpG islands in the promoter regions of specific genes, demonstrating the so-called CpG island methylator phenotype (CIMP). However, this epigenetic phenotype has not been investigated in rhesus. The aim of this project is establish the CIMP status of spontaneous rhesus CRCs. We designed rhesus-specific primers and methylation-specific qPCR probes for bisulfite converted genomic DNA to investigate the methylation status of the 5’-UTR CpG islands of MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1. Methylation independent primers and probes targeting ACTB will be used as a reference. To establish this method, we will spike-in artificially methylated rhesus control DNA (from peripheral leukocytes) into untreated DNA and prepare serial dilutions. Then, we will purify tumor DNA from biobanked FFPE sections and determine the CIMP status. Establishing the CIMP status of rhesus tumors will contribute to the development of an exceptional preclinical model and pave the way for “liq- uid biopsies” in rhesus as performed in human medicine.

Research Grant: Roche Oncology Student Support: NIH T35 OD010946

Standardized fecal capsulation for canine fecal transplants does not alter microbial community structure

Matthew Salerno, Hannah Klein, Jenessa Winston

Department of Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

Fecal microbiota transplantation (FMT) transfers feces from a healthy donor into a diseased recipient to confer a health benefit. While common in human medicine, the utilization of FMT in canine medicine is expanding. Historically, canine FMT procedures were performed via enema or nasogastric tube administration of fecal slurries. Recently the use of oral FMT capsules is growing. Though the microbial community of canine fecal samples has been proven to stay viable for months while stored at -80C with 10% glycerol, the processing of stored samples into fecal capsules for oral FMT admin- istration is unexplored. The purpose of this study was to investigate if standardized capsulation of canine fecal material impacts the microbial community structure. Banked feces from 4 canine fecal donors at The Ohio State University’s Com- panion Animal Fecal Bank were processed into FMT capsules using a standardized protocol. Samples from each donor were pulled from specific steps during the capsulation process and underwent DNA extraction and Illumina sequencing of the V4 region of the 16S rRNA gene. Amplicon analysis was performed using MOTHUR. Preliminary analysis re- vealed differences in alpha-diversity (Shannon and Chao1 indices) and beta-diversity (NMDS) measures between donors. However, no significant differences in microbial community structure (AMOVA) were detected within individual donors throughout the capsulation protocol. Results indicate that a standardized capsulation process does not significantly alter the microbial composition of feces from healthy donors, further promoting the practicality of oral FMT administration in canine medicine. Additional studies evaluating microbial viability are required.

Research Grant: None Student Support: NIH T35 Training Grant T35OD010977

296 • 2021 National Veterinary Scholars Symposium Characterization of immune responses to LPS in bone marrow derived dendritic cells from the chickens

HermanMax O. Salvosa-Chua, Yvonne Drechsler, and Theros Ng

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA

Dendritic cells (DCs) are key innate immune cells, bridging the innate and adaptive immune systems. DCs have not been adequately characterized in chickens. The innate immune system recognizes broad evolutionarily conserved antigens. The adaptive immune system reacts to specific antigens, especially upon a second encounter. This memory is the basis of a more effective immune response and the basis of vaccine efficacy. Newer research demonstrated that the innate immune system does have a memory, which is not specific for a particular antigen, but rather enables an enhanced innate immune response to a variety of pathogens. This has been demonstrated in macrophages and DCs of mammalians, as well as carp, and has been named “innate memory” or “trained immunity.” Studies show a correlation exists between trained immunity (TI) in activated macrophages and energy metabolism. Activated macrophages display increases in mTOR and HIF1-a glycolytic pathways. DCs are professional antigen presenting cells like macrophages, can be induced to differentiate under different culturing conditions from monocytes. We hypothesize that expressions of mTOR and HIF1-a will be increased in chicken DCs treated to undergo TI. Chicken bone marrow-derived DCs were collected from chicken femurs and tibias. Monocytes were grown in culture with granulocyte-macrophage colony stimulating factor and interleukin-4 to induce DC growth and proliferation. RT-PCR of cell marker mRNA levels were used to analyze DC induction. TI response was evaluated by RT-PCR of mTOR and HIF1- a expressions after secondary LPS challenge. This study will provide evidence that chicken dendritic cells exhibit trained immunity.

Research Grant: USDA Animal Health and Disease Research Student Support: USDA Animal Health and Disease Research

Increased Infiltration of Innate Immune Cells Associates with AVF Maturation Failure

Gunimat Samra, Vikrant Rai, Devendra K. Agrawal

College of Veterinary Medicine (Samra), College of Osteopathic Medicine (Rai), Research of Biotechnology (Agrawal), Western University of Health and Sciences, Pomona, CA

Arteriovenous fistula malformation is a clinical and financial burden in hemodialysis treatments.This surgical procedure is used to gain access to blood vessels for continuous usage by causing minimal tissue damage. The success for treatment is heavily dependent on a patent AVF. AVF failure is the body’s immune system and inflammatory response responding a foreign junction between artery and vein. This causes remodeling of tissue which is conducted by innate and adaptive arms of the immune system. The role of innate and adaptive immune cells in atherosclerosis and neointimal hyperplasia, the underlying pathologies in AVF maturation or failure, have been documented, however, the immune cell regulation in AVF maturation or failure has not been studied in detail. Macrophages, dendritic Cell, and T regulatory cells are main role players that have subsets which can tip the scales between inflammatory and anti-inflammatory response. Success ofAVF maturation is dependent on several factors ranging from the patient, history, surgery, and post-surgical treatment. In this study, AVF was surgically created in swine model and treated with TAK242 and LR12. The protein and gene expression of innate immune cells were evaluated via immunostaining and RT-PCR. Increased expression of immune cells in femoral artery and vein involved in AVF was revealed compared to contralateral femoral artery and vein after 4 months. Angiog- raphy and optical coherence tomography (OCT) of these animals showed stenosed or blocked femoral artery on AVF side. These findings suggest that increased innate immune cell recruitment may mediate chronic inflammation andAVF failure and regulating this recruitment might be novel therapeutics.

Research Grant: R01 HL144125 and R01HL147662 to DKA from the National Heart, Lung, and Blood Institute, National Institutes of Health, USA Student Support: Boehringer Ingelheim

2021 National Veterinary Scholars Symposium • 297 The impact of TP53 missense and truncating mutations on protein function and tumor progression in canine OSA

Brittney Sanfacon, Rupa Idate, and Dawn L. Duval

Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, Colorado

The transcription factor P53 is well known for its role in cancer development across many species. P53 mutations have been identified in approximately 80% of canine osteosarcomas (OSA), but recent studies suggest that not all p53 mu- tations have equal effects on protein function and response to chemotherapy. An exon-6 truncating mutation found in a subset of canine OSAs, known as P53-psi may be unable enter the nucleus and regulate DNA damage responses. Instead, this truncated P53 is directed to the mitochondria where it may alter membrane pore permeability. The goal of this study was to synthesize the canine P53-psi protein using Wild type (WT)-P53 template DNA and PCR technology, and assess its function when expressed in P53-null cell lines. Another goal of this project was to determine sensitivity in canine OSA cell lines with either WT P53 or various missense P53 mutations to the standard of care treatments, Doxorubicin and Carboplatin. It has been suggested that cancers with P53 mutations have greater sensitivity to DNA damaging chemother- apy and longer disease-free intervals after treatment. Canine OSA cell lines Gracie (WT), HMPOS (R226H) and OS2.4 (I284T) were tested for their sensitivity to both drugs using a resazurin-based bioreductive fluorescent assay to assess cell viability. Initial results indicate that the HMPOS cell line exhibits the most sensitivity to doxorubicin, however studies are ongoing.

Research Grant: Flint Animal Cancer Center Student Support: Boehringer-Ingelheim Veterinary Scholar

Deliver CRISPR Cas9/sgRNA to respiratory epithelial cells

Xiaoyang Sang, Joseph Whitley, Chenming Ye, Rohit Katti, and Houjian Cai

Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy (Whitley, Ye, Katti, Cai), College of Veteri- nary Medicine (Sang), University of Georgia, Athens, GA

The CRISPR/Cas9 system has empowered efficient genome editing of human cells in culture. It has the potential as a therapeutic tool for treating human diseases such as cancers and other infectious diseases. Efficient and reliable delivery of Cas9/sgRNA in vivo, however, still remains the primary challenge to implement due to delivery efficiency, immune response as well as technical requirement. Extracellular vesicles (EVs) -phospholipid bilayer vesicles secreted by both and eukaryotes during normal life cycle of cells- on the other hand have a promising potential as DNA, RNA, protein, and lipid delivery vehicle while eliciting less immunogenicity, increasing target-specific efficiency and preventing rapid clearance by recipient cells upon administration. The purpose of this study is to generate EVs encapsulating geneti- cally engineered Cas9/sgRNA ribonucleoprotein particles (RNPs) complex, and further integrate an envelop protein into EVs membrane to enhance EVs transfection efficacy and specificity. Genetic fusion of an octapeptide to the N-terminus of Cas9 promotes its myristoylation, subsequently encapsulation of Cas9/sgRNA into EVs in EVs-producing cells. Further- more, a viral envelope protein was also incorporated onto EVs membrane based on the EVs biogenesis. To proof of princi- ple, the created EVs encapsulating Cas9/sgRNA-GFP (targeting GFP gene in recipient cells) were administered to A549 cells expressing GFP, an adenocarcinomic human alveolar basal epithelial cell line. Our study has provided a technology with a promising therapeutic potential for gene therapy in many respiratory diseases.

Research Grant: Federal Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

298 • 2021 National Veterinary Scholars Symposium Presence and Quantity of Influenza Virus-Specific Natural IgM Antibody in Human Umbilical Cord Blood

Kate E. Sarkan, Martha A. Alexander-Miller

College of Veterinary Medicine, University of Georgia, Athens, GA (Sarkan); Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC (Alexander-Miller)

The neonatal immune system varies greatly from adults. These changes leave infants at increased risk of infection. Influ- enza virus in particular poses a significant threat to newborns. Previous studies have demonstrated the presence of neona- tal innate immune cells called B-1 cells that are capable of producing natural IgM antibodies to pathogens in the absence of prior exposure. However, little research in humans exists documenting the reactivity of natural IgM antibodies to influenza-virus, and none within the context of the neonatal immune system.We hypothesize that influenza virus-specific natural IgM is present in human umbilical cord blood in quantifiable levels and that there is broad reactivity of these IgM antibodies to HA proteins from heterologous strains of influenza virus. To test this, total IgM within umbilical cord blood was quantified by ELISA. These samples were evaluated for the presence of IgA to ensure limited contamination by ma- ternal blood. IgM antibody was detected in all cord blood samples, with threshold titers ranging from 1:12800 to 1:51200. The presence of influenza-specific IgM was assessed by ELISA using HA coated plates. Initial studies using A/Califor- nia/07/2009 (H1N1) influenza strain showed reactivity with titers ranging from 1:100 to 1:800.Assessment of reactivity to other HA proteins is ongoing. Increasing our understanding of the infant’s capacity to produce influenza-specific IgM antibodies will contribute to our working knowledge of the neonate immune system and benefit future research aimed at bolstering neonate immunity to influenza virus.

Research Grant: None Student Support: T35OD010946

Evaluating systemic exposure of drug detection dogs to narcotics while performing their duties

Lakshmi K. Sastry, Cynthia Otto, Joanne Haughan, and Mary A. Robinson

Department of Clinical Studies - New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA; Working Dog Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; Pennsylvania Equine Toxicology and Research Laboratory, West Chester, PA

Narcotic detection dogs are working canines trained in scent-detection. Exposure to narcotics during training is necessary to assist handlers in locating illegal substances. It is hypothesized that individual or combined exposures of drug detection dogs to heroin, cocaine, or both during training will result in detectable levels in their urine and topical samples. Pennsyl- vania K9 units participate in training days to maintain skills in environments such as auto junkyards and abandoned build- ings. These training days replicated authentic working scenarios. 134 total samples were retrieved from 18 individual dogs and environments on 33 separate training days. 15 dogs participated in two training days, one dog participated in three days, and two dogs participated in one day. Samples will be collected at two more training days. Urine and topical sam- ples were retrieved from each dog before and after each search. Urine was retrieved as free-catch. Topical samples were retrieved by wiping all four paws with one 4x4 gauze square dampened with 2-3 mL of 0.9% NaCl solution. This topical method was also used to sample the environment before and after the narcotics were hidden. Recent narcotic exposure prior to training days was documented via questionnaire. 72.7% of dogs had a known exposure to at least heroin, cocaine, or another, unknown substance. 27.3% of dogs had exposures to drugs that were not heroin or cocaine. Levels of detect- able cocaine and heroin in urine and topical samples will be analyzed using Liquid chromatography coupled with mass spectrometry. The effect of exposure time and time to post-exposure sample collection on amount of drug detected will be evaluated using appropriate statistical methodology.

Research Grant: Pennsylvania State Horse Racing Commission Student Support: NIH T35 OD010919

2021 National Veterinary Scholars Symposium • 299 Crocodylus niloticus penile glans malformation: a case study for consideration of breeding soundness

Nicole Scarberry, Mark Does, Jan G. Myburgh, and Brandon C. Moore

College of Veterinary Medicine, University of Missouri, Columbia, MO (Scarberry, Moore); Vanderbilt University School of Medicine, Nashville, TN (Does); Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa (Myburgh); School of Health Sciences, Stephens College, Columbia, MO (Moore)

As part of a larger-case series, here we present an anatomical evaluation of a malformed phallic glans from a juvenile male Nile crocodile, Crocodylus niloticus. Upon ex vivo glans inflation of the spongiform tissue, a bisection of the glans ridge showed a pronounced functional problem in achieving a normal copulatory shape. We utilized magnetic resonance imaging to produce a 3D reconstruction of the glans internal structures and histologic analysis to provide tissue level-res- olution of the malformation. We posit that the malformation on the bell-shaped glans affects the ability for the male to inflate the glans into a normal copulatory morphology that excludes water from the site of insemination.Therefore, the potential reproductive success for this animal is adversely affected. Individuals with similar malformations may not be the best candidates for breeding stock. Understanding the form and function of interacting copulatory organs provides insight into species-specific reproductive mechanisms, however little is known about crocodylian taxa. Malformations of male crocodylian reproductive organs may have a direct impact on the copulatory function and thus could be a determining factor of the reproductive success of the animal. Therefore, when considering captive management of breeding stock, it is vital that male animals chosen to breed can effectively inseminate.As part of a male physical exam in a captive breeding program, examination for abnormalities of the phallus should be routine.

Research Grant: None Student Support: Stipend for Nicole Scarberry is supported by an endowment established by IDEXX-BioAnalytics.

Dietary Omega-3 Fatty Acid Attenuates Silica-Triggered Autoimmune Disease in16-Week-Old Lupus-Prone Mice

Tasha Scarlett, James Wagner, Lauren Heine, James Pestka, and Jack Harkema

Department of Pathobiology and Diagnostic Investigation (Scarlett, Wagner, Harkema), Department of Pharmacology and Toxicology (Heine), Department of Food Science and Human Nutrition (Pestka), College of Veterinary Medicine, Michigan State University.

Systemic Lupus Erythematosus (Lupus) is an autoimmune disease affecting five million people worldwide.Though a ge- netic disease, occupational exposures to crystalline silica dust (cSiO2) are associated with lupus flares.We have found that dietary supplementation with an omega-3 fatty acid (docosahexaenoic acid, DHA) prevents cSiO2-triggered autoimmuni- ty and pathology in lungs (ectopic lymphoid structures, ELS) and kidneys (glomerulonephritis) of 8-week-old lupus-prone mice. The present study was designed to test the hypothesis that older mice have even greater pathology after repeated pulmonary exposures to cSiO2, and with less dietary DHA protection. 16-week-old, female, NZBWF1 mice were intrana- sally instilled once a week for 4 weeks with: 1) saline-vehicle and fed a control diet (VEH/CON); 2) 1mg cSiO2 and fed a control diet (cSiO2/CON); and 3) 1 mg cSiO2 and fed a DHA supplemented diet. Animals were sacrificed 1 or 5 week(s) after the last instillation. Lung and kidney tissues were processed for light microscopic examination and morphometric analysis. VEH/CON mice had no pulmonary or renal histopathology. cSiO2/CON mice at 5 weeks post-instillation had marked pulmonary ELS and glomerulonephritis, that were more severe than after 1-week. cSiO2/DHA mice had conspic- uously less lung ELS and glomerulonephritis than cSiO2/CON mice. 16-week-old mice developed pathologies that were comparable in character and severity to what we have previously observed in similarly exposed younger mice. Likewise, dietary DHA effectively ameliorated cSiO2-triggered lupus in these older mice. This suggests that age might not be a fac- tor in DHA prevention of occupationally triggered lupus flares.

Research Grant: NIH R01 ES027353 (J.P.) and Albert C. and Lois E. Dehn Endowment (J.H.) Student Support: NIH grant R25HL103156, BRUSH Summer Research Program, CVM, Michigan State University, MI

300 • 2021 National Veterinary Scholars Symposium Circulating tumor DNA analysis in canine osteosarcoma patients

Max Schermacher, Patricia Favaro, Bradon McDonald, Muhammed Murtaza, Xuan Pan, Nathan Van Asselt

Medical Sciences (Schermacher, Pan, Van Asselt), University of Wisconsin School of Veterinary Medicine, University of Wisconsin, Madison, WI; Surgery (Favaro, McDonald, Murtaza), University of Wisconsin School of Medicine and Public Health, University of Wisconsin, Madison, WI

Shallow whole genome sequencing (sWGS) of circulating tumor DNA in blood has been utilized as a novel method for diagnosis of cancer and evaluation of minimal residue disease during cancer treatment. Osteosarcoma (OSA) is a great comparative model to human disease due to its prevalence within canines and a genetic similarity to human tumors. OSA in canines is an aggressive cancer that can results in amputation. OSA metastasizes quickly, usually requiring adjunctive therapy. Current therapies like chemo or radiation are expensive and progress is hard to evaluate until the end of treatment. By comparing ctDNA data from canine cancer patients to healthy dogs, we could detect and evaluate treatment response of cancers. Blood samples were collected from OSA and HSA dogs at UW Madison Veterinary Care. The plasma was isolated and used for ctDNA detection and sWGS. Samples were drawn before, during, and after chemotreatment course. Healthy dogs with no history of cancer were used as a control group to create a map of normal canine cell-free DNA. We have collected samples from 25 OSA patients and 55 healthy patients. Sequencing and statistical analysis are ongoing with Dr. Murtaza’s Lab. We expect to see that dogs with cancer will have a higher ctDNA fraction in their blood compared to healthy dogs. Additionally, we expect the ctDNA fraction to decrease as the patient goes through therapy. We also hope to develop a normal cfDNA fragmentation map in canines and figure out the sequence of the DNA fragments in the healthy and sick patients. The data can be used to develop a liquid biopsy test for diagnostic purposes. It would be a quick and cost-effective tool to detect cancer and evaluate patients throughout treatment.

Research Grant: None Student Support: UW-Madison School of Veterinary Medicine

Role of canine hemangiosarcoma-associated extracellular vesicles in promoting tumor angiogenesis

Samantha Scott, Joey LaMastro, Dawn Meola, Vicky Yang

Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts

Extracellular vesicles (EV) are small membrane bound vesicles secreted by cells for intercellular communication, and contents are selectively packaged according to the cell of origin. EVs from cancer cells have been implicated in primary tumor survival by inducing angiogenesis, as well as establishing premetastatic niche. Hemangiosarcoma (HSA) is an ag- gressive canine cancer with a malignant endothelial phenotype. We hypothesize that EVs from HSA (HSA-EV) will pro- mote endothelial cell activation and induce angiogenesis in normal canine endothelial cells. Canine aortic endothelial cells (CnAOEC) were incubated with EV-containing conditioned media (EV-CM) from 3 HSA cell lines. EV-CM was prepared by first culturing HSA cells with HSA growth media containing 10% FBS until 50-70% confluency, then switched to non- FBS containing media. After 24 hrs, the EV-CM was collected. Two different EV-CM concentrations (50% and 100%) were tested compared to the control non-FBS containing media. Markers of endothelial cell activation were evaluated by reverse transcriptase quantitative PCR to evaluate mRNA expression after exposure to EV-CM. ICAM (P = 0.0148, fold change 1.860.3 for 50% EV-CM, 361 for 100% EV-CM) expression was significantly increased and vWF (P = 0.0229, fold change -1.360.2 for 50% EV-CM, -2.360.7 for 100% EV-CM) was significantly decreased. CnAOEC proliferation and viability evaluated with an MTT assay were significantly increased when cultured in EV-CM (P = 0.0058). Induction of CnAOEC tube formation in Matrigel as a measure of angiogenesis in vitro showed no significant effect.These results suggest that HSA-EVs may be involved in HSA progression, and further work with isolated EVs will be performed.

Research Grant: Morris Animal Foundation Student Support: Morris Animal Foundation

2021 National Veterinary Scholars Symposium • 301 Hydrogel Templates as Cardiac Matrix Mimic for Tissue Engineering

Sharona Sedighim, Finosh Thankam

Western University of Health Sciences, College of Veterinary Medicine (Sedighim), Western University of Health Sciences, Department of Translational Research (Thankam)

Heart failure (HF) is a clinical syndrome that occurs as a secondary mechanism to various cardiac diseases in animals, such as bacterial endocarditis, mitral valve disease, and dilated cardiomyopathy. Current treatments for HF typically involve pharmacological and nutritional support, neither of which are permanent solutions. This study aims to develop hydrogel scaffolds for cardiac tissue regeneration following myocardial infarction.Two superabsorbent hydrogels were developed following interpenetration chemistry: CAHA2A contained alginate, carboxymethyl cellulose, (hydroxyethyl) methacrylate (HEMA), and acrylic acid, and CAHA2AP was CAHA2A interpenetrated with polyvinyl alcohol. Hydro- gels displayed excellent equilibrium swelling ratio, which was 0.893 for CAHA2A and 0.878 for CAHA2AP. Both the hydrogels were biocompatible as evident by direct contact assay and MTT assay using H9c2 cardiomyoblasts revealing the viability of 106.20% for CAHA2A and 99.77% for CAHA2AP. The hydrogels promoted cell migration where the migrated cells attained confluency within a week of initial seeding.Also, the cell infiltration facilitated by the hydroges were assessed by z-stack analysis revealing the penetration of H9c2 cells to 180mm in both the gels. Both the hydrogels are suitable for cardiac tissue engineering and further validations are ongoingDo we want specific amounts of these? Not sure if needed in the abstract.

Research Grant: WU startup funds for Dr. Thankam Student Support: Boehringer Ingelheim, USDA

Characterization of vendor-dependent differences in the juvenile and adult mouse ileal lumen and mucosa

Nikki Seilhamer, Erin Donovan, Amber Russell, Kevin Gustafson, Aaron Ericsson

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

The gut microbiome is known to influence overall health and play a role in many human diseases.Additionally, the gut microbiome influences disease development and progression in laboratory mice used as models for human diseases. Research suggests that the source of mouse is the largest determinant of the gut microbiome, resulting in vendor-depen- dent differences in physiology and disease susceptibility. Many studies have been done to characterize the functional and compositional differences in the feces; however, the mechanisms by which the different gut microbiomes are affecting changes in the host are still unclear. The vendor-dependent differences occurring upstream in regions of dense mucosa-as- sociated lymphoid tissue remain unknown. The objective of the present study is to characterize the mucosal and luminal microbiome in the ileum of juvenile and adult mice from Jackson Laboratory and Envigo. Luminal contents and mucosal scrapes will be collected from adult dams and 10-day old pups with either microbiome, and DNA will be extracted. The MU DNA and Informatics Cores will generate and sequence 16S rRNA libraries to identify microbial populations and PERMANOVA will be used to evaluate diversity and richness. Based on pilot studies, we hypothesize that mice from Envigo will have a richer ileal microbiome than the Jackson mice, specifically having segmented filamentous bacteria and more in their ileum. These results will offer a better understanding of different vendor-origin gut microbio- ta, providing insights to their influence on mouse models of disease.

Research Grant: MU Metagenomics Center Student Support: MU Metagenomics Center

302 • 2021 National Veterinary Scholars Symposium Calcium metabolism and associated patterns of milk constituents in early lactation dairy cows

Jackson Seminara, Jessica Anne Allerton McArt, Dave Barbano

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York (Seminara, McArt) and Department of Food Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York (Barbano)

As dairy cows transition into early lactation, physiological demands for calcium dramatically increase. As a result, more than 50% of dairy cows experience some form of hypocalcemia in the early postpartum. Though clinical hypocalcemia is presently well managed through prepartum dietary changes and established treatment protocols, subclinical hypocalcemia (SCH) remains a major challenge for dairy farmers due to its negative impact on production and its role as a risk factor for other adverse health events. SCH is difficult to assess at the herd level as two separate blood samples at 1 and 4 days in milk (DIM) are required for definitive diagnosis. The aim of our prospective cohort study was to investigate a method for diagnosing SCH using Fourier transform mid-Infrared spectroscopic analysis of milk samples from the first 10 days in milk. We collected serum from 384 multiparous dairy cows at 1 and 4 DIM to determine calcium status and milk was collected from 3 through 10 DIM. Serum samples will undergo analysis for total calcium, and cows will be divided into one of four dynamic calcium-metabolism response groups. Patterns of milk somatic cell count, lactose, protein, fat, urea nitrogen, and relative fatty acid percentages will be compared for the first 10 DIM to discover if significant differences- ex ist for each parameter between calcium groups. Analysis and identification of differences of the levels of one or more milk constituents between calcium groups might provide a novel non-invasive method for assessing SCH at the herd level.

Research Grant: USDA-NIFA grant no. 2019-67015-29446/project accession no. 1018890 Student Support: NIH T35 OD010941, Cornell University College of Veterinary Medicine

Prevalence of Heterobilharzia americana in normal dogs from Louisiana

Katelyn M. Minchew Setzer, Priscilla A. Devora, Brooke A. Delcambre, Chin-Chi Liu, Adriano F. Vatta, Nathalie Rademacher, Andrea N. Johnston

Veterinary Clinical Sciences (Setzer, Devora, Liu, Rademacher, Johnston) and Pathobiological Sciences (Delcambre, Vatta) School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Heterobilharzia americana is a trematode parasite that lives in the mesenteric vessels of various wild and domestic mam- mals. The natural hosts of the trematode parasite Heterobilharzia americana include the racoon and nutria in an expanding range of geographic locations primarily involving the Gulf Coast and south Atlantic states. The clinical effects ofHetero - bilharzia americana infection in dogs are nonspecific and can range from no apparent clinical signs to death due to wide- spread distribution of eggs within organs. Most commonly affected organs include small intestines, liver, large intestine, pancreas, lung, lymph nodes, spleen and stomach. The infection has been associated with periportal fibrosis in people and glomerulonephritis in dogs as well as with a variety of concurrent neoplastic diseases. Veterinarians rarely add this disease to their list of differentials, even in endemic areas and annual fecal examinations are not routinely done. SinceHeterobil - harzia americana is probably more widespread in endemic areas, this study aims to identify the prevalence of infection in healthy dogs in Louisiana and check for potential changes on abdominal ultrasound to advocate for annual fecal examina- tions as part of a routine work up in our geographic location.

Research Grant: 2021 Comparative Gastroenterology Society/IDEXX Veterinary Student Summer Scholar Award and the School of Veterinary Medicine Louisiana State University Student Support: 2021 CGS/IDEXX Veterinary Student Summer Scholar Award and School of Veterinary Medicine LSU

2021 National Veterinary Scholars Symposium • 303 Deslorelin-induced infertility affects expression of selected antioxidant enzymes in the testis of dogs

Brigid Sexton, Duygu Yaman Gram, Narin Liman, Linda Muller, Aykut Gram*, Orsolya Balogh*

Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA (Sexton, Balogh); Erciyes University, Kayseri, Turkey (Gram DY, Liman, Gram A); University of Veterinary Science, Budapest, Hungary (Muller); *equal contribution

Spermatogenesis takes place in a hypoxic environment, and reactive oxygen species are normally produced during sper- mato- and steroidegenesis. Testicular antioxidant enzymes protect germ and somatic cells from free radical-mediated damage. The slow-release GnRH agonist deslorelin 4.7 mg implant induces temporary, reversible suppression of en- docrine and germinative testicular function in male dogs. We hypothesized that the expression of testicular antioxidant enzymes changes following deslorelin treatment as a result of induced infertility in dogs. The purpose of this study was to investigate the expression of superoxide-dismutase (SOD1, SOD2), catalase, glutathione-peroxidase (GPx1), and gluta- thione-disulfide reductase (GSR) in the downregulated canine testis 16 weeks after deslorelin treatment. Gene expression was evaluated by qPCR and protein expression by immunohistochemistry in five deslorelin-treated dogs (DES), and compared to untreated adult (CON, n = 7) and prepubertal (PRE, n = 8) dogs. We found that in DES, SOD1 gene expres- sion was significantly lower and GPx1 higher than in CON, and SOD2 was higher than in PRE. Expression of all except SOD2 mRNA differed between CON and PRE. Protein expression pattern differed by cell type and/or between groups. In DES, strong immunoreactivity for SOD1 and GPx1 protein appeared in spermatogonia and Sertoli cells; Leydig cells also stained. GSR signals seemed strong in spermatogonia and were weak in Sertoli and Leydig cells. These findings show the presence of a functional antioxidant system in the DES testis that may protect spermatogonia, Sertoli and Leydig cells from oxidative stress during temporary infertility, likely helping to ensure reversibility of testicular suppression.

Research Grant: None Student Support: Virginia-Maryland College of Veterinary Medicine

Lysine demethylase inhibition decreases proliferation in canine osteosarcoma

Kathryn Shaffert, Heather Gardner

Tufts Cummings School of Veterinary Medicine

Osteosarcoma is the most common neoplasia of bone in dogs, known for its invasive and metastatic behavior. Recent research has identified recurrent mutations inSETD2 in canine osteosarcoma samples. The SETD2 gene encodes the sole histone methyltransferase responsible for the addition of a third methyl group to histone 3 lysine 36 (H3K36) to form H3K36me3. In human cancers, inactivating mutations in SETD2 that result in loss of H3K36me3 are associated with more aggressive behavior. Furthermore, upregulation of lysine demethylase (KDM4) enzymes is reported in osteosarcoma, also resulting in loss of H3K36me3 activity. Therefore, it is hypothesized that canine osteosarcoma cells will exhibit loss of H3K36 trimethylation and increased sensitivity to KDM4 inhibition. To test these hypotheses, a series of experiments was performed on Abrams, Gracie, and D17 canine osteosarcoma cell lines. To understand the downstream effects of SETD2 on H3K36 trimethylation, protein was isolated from cell lines using complete lysis buffer and quantified using a Bradford assay. Western blotting was performed using standard techniques. Membranes were probed for SETD2 (CST23486), H3 (CST9717), and H3K36me3 (CST9763). To investigate the effects of KDM4 inhibitor treatment on proliferation, cells were plated in triplicate in 96-well plates and treated with increasing concentrations of JIB-04 for 72 hours. Cells were fixed and stained with SRB, and proliferation was calculated as a percent of untreated controls. H3 and H3K36me3 were expressed in all cell lines and JIB-04 decreased proliferation of canine osteosarcoma cells at nanomolar concentrations. Correlation of proliferation and H3K36me3 expression after exposure to JIB-04 is underway.

Research Grant: Morris Animal Foundation Student Support: Anivive

304 • 2021 National Veterinary Scholars Symposium Variation of in-vitro bronchiolar attachment of zoonotic H1 influenza A viruses of swine

Laura E. Shaw, LaRhonda K. Sobchishin, Susan E. Detmer

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Due to similarities in both sialic acid receptor type and distribution along the respiratory tract, influenzaA viruses (IAV) can be transmissible between swine and humans. In 2013, an H1N2 virus was identified in Manitoba pigs with a unique 2AA deletion in the hemagglutinin gene and was later named Alpha3 as a large subclade was further identified. This virus has since been found in pigs coast-to-coast across the United States and Canada with the first human case identified in Minnesota in 2016. During the recent COVID-19 pandemic, this virus has been identified in 3 people in Manitoba and a variation without the 2AA deletion, termed Alpha3a, was found in a person in Alberta. Given that the deletion is at site 130 where human H1 viruses have no AA, this virus may be more adapted to humans than most swine H1 viruses and may also have a different pattern of virus attachment (PVA) compared to other zoonotic H1 IAVs. Using phylogenetics and antigenic sites, 12 representatives of the H1 subclades: a1, a3, a3a and pdmH1N1, were selected for assessment of PVA using virus histochemistry. The amount of virus attachment within bronchioles of four healthy pig lungs was mea- sured and compared using pixel counts with ImagePro software. The results of this study will be compared to the actual replication of some of these viruses in inoculated pig tissues to help translate the results of this in-vitro study into data that will help us understand the pathogenesis of emerging IAV strains in pigs.

Research Grant: Saskatchewan Agricultural Development Fund and The Natural Sciences and Engineering Research Council of Canada Student Support: Boehringer Ingelheim Veterinary Scholars Program

PPAR induction of Angiopoietin-like 4 in osteoblasts and its interaction with hypoxia

Emily Shelton, Alice Wong, Clare Yellowley

Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, 1285 Veterinary Medicine Drive, Davis, CA

Bone has the incredible capability to adapt to physiologic needs through bone remodeling. However, osteoporosis, a common bone pathology, disrupts normal homeostatic mechanisms and leads to bone loss and increased risk of fracture. Therefore, it is important to find pathways that can be manipulated in order to promote anabolic bone formation. Osteo- blasts play a major role in bone formation and regulate the activity of bone resorbing osteoclasts. Our previous studies demonstrated an upregulation of Angiopoietin-like 4 expression (Angptl4) during osteoblast differentiation and high expression of Angptl4 in mineralizing osteoblasts in the fracture callus. We have shown that Angptl4 expression in osteo- blasts can be upregulated by peroxisome proliferator-activated receptor b/d agonist and by hypoxia. PPARs are ligand-ac- tivated transcription factors with major roles in energy homeostasis and hypoxia can occur in bone under pathologic con- ditions such as bone fracture due to disruption of vasculature. Published studies suggest that the hypoxia response element and PPAR response element lie in close proximity to each other and that this facilitates crosstalk to amplify downstream signaling. Therefore, the goal of this study is to determine whether activation of PPARb/d under hypoxic conditions syner- gistically enhances Angptl4 signaling in osteoblasts. Murine osteoblastic MC3T3-E1 cells were treated with the PPARb/d agonist GW0742 in normoxia (21%) and hypoxia (1%) and Angptl4 mRNA expression was measured by qRT-PCR. We expect that MC3T3-E1 cells exposed to the PPARb/d agonist in the presence of hypoxia will show enhanced expression of Angptl4 when compared to cells exposed to the agonist or hypoxia alone.

Research Grant: None Student Support: STAR NIH T35 Grant

2021 National Veterinary Scholars Symposium • 305 Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and Promotes Egress of VP40 VLPs

Ariel Shepley-McTaggart, Michael P. Schwoerer, Cari A. Sagum, Mark T. Bedford, Chaitanya K. Jaladanki, Hao Fan, Joel Cassel and Ronald N. Harty

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, Department of Epigenetics & Molecular Carcinogenesis, University of Texas, Smithville, TX, Bioinformatics Institute, National University of Singapore, Molecular Screening Facility, The Wistar Institute, Philadelphia, PA

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high mortality rates. The matrix proteins of EBOV and MARV, called eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and VP40 can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack spe- cific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus-cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40-SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of key host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections.

Research Grant: T32-AI070077 Training grant Student Support: T32-AI070077 Training grant

Bat activity and diversity across three microhabitats on a university campus in North Grafton, Massachusetts

Rachel C. Sherinian, Marjorie Bercier

Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA

Bat species worldwide have seen dramatic population declines due to human activity and habitat loss in recent decades. Hibernating, cave-dwelling bat populations in North America have been decimated by the additional threat of White Nose Syndrome. Not only are bats critical to the ecosystems that they populate, they are also of particular interest to epidemi- ologists as a potential source of zoonotic spillover. Using stationary acoustic monitors, we measured native chiropteran diversity (number of species present) and activity levels (number of echolocating calls) on the Tufts Cummings School of Veterinary Medicine (TCSVM) campus in North Grafton, Massachusetts between June and August of 2021. We recorded the ultrasonic, echolocating calls of bats across three microhabitats: forest, cropland and institutional (school buildings). Our study detected six of the nine bat species that are known to be native to Massachusetts, including two species that are listed as endangered at the state level. Preliminary analysis suggests that bat activity and diversity is significantly lower near the institutional buildings when compared to either forest or cropland. While the big brown bat (Eptesicus fuscus) was most commonly identified at the forest and cropland locations, hoary bats Lasiurus( cinereus) were the dominant species detected near institutional land.

Research Grant: Faculty Development Grant, Cummings School of Veterinary Medicine at Tufts University Student Support: Summer Research Teaching Program, Cummings School of Veterinary Medicine at Tufts University

306 • 2021 National Veterinary Scholars Symposium MiteBusters: The Survival, Control, and Identity of Respiratory Mites (Halarachnidae) in Sea Otter Facilities

Morgan Shields, Risa Pesapane, Tara Roth, and Melissa Miller

Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio State University, Columbus, OH (Shields, Pesapane), San Mateo County Mosquito and Vector Control District Burlingame, CA (Roth), Marine Wildlife Veterinary Care and Research Center, California Department of Fish and Wildlife, Santa Cruz, CA (Miller)

Marine mammals are hosts for several parasitic nasal mites (Halarachnidae) which impair respiration by way of muco- purulent exudate, bronchitis, and turbinate lysis. Infestations are common in rehabilitated sea otters (Enhydra lutris) and negatively impact survival post-release. Despite disinfection in marine mammal care facilities, infestation is associated with captivity, suggesting that current husbandry practices are inadequate. The risk of environmental transmission and disinfectant efficacy is unknown. To examine whether environmental transmission represents viable risks, and the abil- ity of disinfectants to control mites, this study will analyze survival under various environments and disinfectants. Live mites will be exposed to common disinfectants as well as microcosms that mimic the temperature and salinity of enclosures. Mites will be stimulated at regular intervals to evaluate survival and mortality rate survivorship curves will be created. The results will determine whether mites can persist environmentally, potentially infesting naive animals, as well as the efficacy of disinfectants. Future mite-specific protocols can then be developed to improve animal welfare at marine mammal institutions. Additionally, characterization of mite-host associations has been complicated by repeated revisions to halarachnid mite and reclassification of misidentified specimens. Species identification currently requires multiple keys, knowledge of revisions to species classification through time, and training in acarology, which is impracti- cal for marine mammal clinicians. A simple pictorial key to species of halarachnids based on published keys was generat- ed to aid in future identification of mite species.

Research Grant: The Sea Otter Foundation & Trust Student Support: Unknown

The Role of Advanced Glycation End Products in Neuroinflammation and Neurodegeneration

Grace Shinn and Yiling Hong

College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA

Advanced glycation end products (AGEs) are a family of diverse chemical compounds that are the products of nonenzy- matic reactions between reducing sugars and proteins, lipids, or nucleic acids. The accumulation of AGEs can be intro- duced from both endogenous and exogenous sources. Endogenous sources of AGEs can come from glycation, polyol pathways, and glycoxidation. Exogenous sources of AGEs come from high fat foods sweetened with sugars and cooked at high temperatures in dry heat. Adverse effects of AGEs on tissues occur through nonreceptor- and receptor-medi- ated mechanisms. In the receptor-mediated mechanism, interaction of AGEs with the receptor for advanced glycation end-products (RAGE) elicits an array of biologic responses. However, little is known about the impact of AGEs on mental health. The aim of this study was to determine the various effects of AGEs on the neuronal network, which contained neurons, astrocytes, and microglia that were generated from induced pluripotent stem cells with our stem cell neuronal differentiation platform. Immunostaining of the network with neuron, microglia, and astrocyte markers was conducted and quantitatively measured with confocal microscope images. Results from this study indicated that neuron population and neurite outgrowth were reduced in a dose dependent manner in response to different concentrations ofAGE exposure when compared to the control group. Furthermore, AGE exposure increased RAGE expression, induced microglia and astrocyte activation, and activation of NLRP3/Caspase1 inflammasomes.These results suggested that microglia activation and neuroinflammation were the key contributors to AGE-mediated neurodegeneration.

Research Grant: Western University intramural funds Student Support: Boehringer Ingelheim summer research program

2021 National Veterinary Scholars Symposium • 307 Barriers to female fertility in xylosyltransferase II deficient mice

Katelyn E Siegel, Myron E Hinsdale

Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

Placental insufficiencies can be the cause of early pregnancy loss or preterm labor that impacts approximately 10-15% of human pregnancies. Due to the hemochorial nature of human and murine discoid placentas, the mouse can be used to model the human placenta to help create better outcomes for pregnancies at risk of placental insufficiency. Proteoglycans, such as heparan sulfate, are glycoproteins with glycosaminoglycans (GAGs) attached to a core protein and are commonly considered integral to connective tissue structure and function; despite their high expression by cells of the placenta and ovarian follicle, little is understood of their role in oocyte development and in embryo implantation and placentation. We have a unique mouse model where significant decreases in glycosaminoglycans (GAGs) occur in multiple tissues and organs due to the loss of xylosyltransferase II. We have observed that female xylosyltransferase II deficient mice (Xylt2- /-) fail to produce litters. Therefore, we hypothesize that the GAGs of the proteoglycans are pivotal to oocyte develop- ment, ovulation, or to implantation. Using Xylt2-/+ and Xylt2+/+ mice as controls, female mice will be bred naturally to a fertile Xylt2+/+ male. Ovarian and uterine tissue will be harvested at 10-14 days post vaginal plug for gross and histologic assessment of ovulation and implantation. Serum will also be collected for steroid hormone analyses. Our present data shows normal oocyte and follicular development and ovulation in the ovary of Xylt2-/- females. Current studies are inves- tigating implantation and placenta development in the Xylt2-/- mice. Once completed these studies will reveal the critical role that proteoglycans have in female uterine function.

Research Grant: Unknown Student Support: Boehringer Ingelheim Veterinary Scholars Program

Characterizing non-target bacteria from animal carbapenemase-producing Enterobacterales surveillance specimens

Nicole A. Silverstein, Stephen D. Cole, Jaclyn Dietrich

Department of Pathobiology at the School of Veterinary Medicine at the University of Pennsylvania

One of the most urgent antimicrobial resistance threats is carbapenemase-producing Enterobacterales (CPE) which harbor carbapenemase genes on plasmids that can spread among other bacteria such as Pseudomonas aeruginosa and Acineto- bacter baumanii. Previously, during an outbreak of CPE at a veterinary hospital, animals were screened for asymptomatic CPE carriage by culture on chromogenic medium. These media often grow non-target organisms (non-Enterobacterales). There is value in the characterization of these isolates since they may serve as reservoirs of antimicrobial resistance genes and lead to opportunistic infections. For this study, 172 banked bacterial isolates were identified using the Vitek2. The minimum inhibitory concentration to imipenem was measured via E-test and production of carbapenemase enzymes deter- mined by modified Carbapenem Inactivation Method (mCIM). Of the 172 banked non-target isolates, 144 (83.7%) were P. aeruginosa, 10 (5.8%) were Pseudomonas putida, 2 (1.2%) of each were , Klebsiella pneumoni- ae and Stenotrophomonas maltophilia and 1 (0.6%) of each was marcescens, Aeromonas sobria, A. baumannii, lapagei, and Burkholderia cepacia. 4 (2.3%) were unable to be identified. 5 isolates (2.9%) were resistant to imipenem and 6 isolates (3.5%) were mCIM positive. The majority of non-target organisms were not carbapenem resistant and did not produce a carbapenemase. Surveillance of microbes from hospitalized patients can help veterinarians to under- stand the types of bacterial infections their patients might acquire, and plan for how they can treat these infections. Further testing by whole genome sequencing will be done to characterize carbapenem resistant isolates.

Research Grant: This work was partially funded as part of a cooperative agreement (1U18FD006669-1) with the Food and Drug Administration's Veterinary Laboratory Investigation and Response Network (Vet-LIRN). Student Support: NIH T35 OD010919

308 • 2021 National Veterinary Scholars Symposium Development of a novel point-of-care prognostic test of neural injury for dogs

Katherine E. Simon, Ashutosh Chilkoti, and Natasha Olby

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Simon, Olby); Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC (Chilkoti)

Acute spinal cord injury (SCI) due to intervertebral disc disease is one of the most common neurological emergencies in dogs. The prognosis for recovery of ambulation is variable and difficult to predict in severe SCI. Two nervous system cytoskeletal proteins, glial fibrillary acidic protein (GFAP) and phosphorylated neurofilament heavy chain (pNF-H) can be detected in the serum of dogs with severe SCI and their levels predict outcome. Currently, these are measured using ELISA tests that are not clinically useful due to cost and need for batching. We hypothesize that a new, inexpensive, rapid immunoassay point of care multiplex test of canine serum GFAP and pNfH (D4POC) will measure both biomarkers with high specificity and sensitivity and predict outcome with high accuracy. The study aims were to bank serum from 100 dogs with severe SCI and known outcome, to measure GFAP and pNfH using both ELISA and D4POC in a subset to de- termine intraclass correlation between tests, and in the remaining samples with D4POC to determine predictive accuracy for outcome. Thus far, 239 samples have been banked of which 88 dogs reportedly recovered ambulation. Testing of the samples using ELISA and D4POC is about to start. We anticipate that the D4POC will be as specific and more sensitive than the ELISA tests and will predict outcome with high accuracy. If correct, the D4POC can be used in the clinic to deter- mine the severity of SCI in paraplegic dogs at time of presentation, assisting owners and veterinarians in decision making and optimal patient triage.

Research Grant: Morris Animal Foundation Student Support: NIH Interdisciplinary Biomedical Research Training Program T35-T35OD011070

Evaluating three-dimensional kinematics of bipedal chimpanzees near the walk-run transition speed

Amanda Sinclair, Matthew O’Neill

College of Veterinary Medicine (Sinclair), Department of Anatomy, College of Graduate Studies (O’Neill), Midwestern University, Glendale, AZ

Qualitative differences of bipedal walking in chimpanzees compared to humans have been noted for more than 75 years. However, recent quantitative comparisons have greatly expanded our understanding of the similarities and differences in the 3-D kinematics of the pelvis and hind/lower limbs of these species at moderate walking speeds. While human 3-D kinematics are well characterized across a wide speed range, comparable data are only available for a single walking speed in bipedal chimpanzees. This study aimed to collect new 3-D kinematics of the pelvis and hind limb to understand the musculoskeletal limits on bipedal movement near the walk-run transition in bipedal chimpanzees, using an integrative experimental and modeling-simulation approach. The initial data collection phase was conducted between 2012 and 2015, and consisted of three male common chimpanzees, each of whom walked across a level runway instrumented with four force plates. Each subject was fitted with reflective, nontoxic anatomical markers, and datasets based on 3-D marker -lo cations were digitized using Xcitex ProAnalyst software. Walking speeds were calculated as the average forward velocity of four markers over a stride. The average dimensionless velocity (v’) and Froude number (Fr) were 0.65 6 0.07 and 0.42 6 0.09, respectively, for the three subjects over twelve trials, which closely approximates the human walk-run transition in dimensionless form (i.e., v’ = 0.7; Fr = 0.5). 3-D marker data will be integrated with an existing chimpanzee musculo- skeletal model to determine 3-D pelvis and hind limb segment and joint angles. These results will be compared to existing data from moderate walking speeds in bipedal chimpanzees.

Research Grant: NSF BCS 0935327 and 2018436 Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 309 Immunomodulatory activity of mesenchymal stem cell derived extracellular vesicles on canine mononuclear cells

Nicole M Sipple and Jennifer L Johns

Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR

Mesenchymal stem cells (MSC) are used for immunomodulatory therapy, but have drawbacks including potential em- bolism and variability of product. MSC-derived extracellular vesicles (MSC-EV) are a potential alternative therapy with easier storage and administration, decreased risk (vs. injecting MSC), and a more uniform product. MSC-EV contain numerous molecules involved in cell signaling pathways, and may have immunomodulatory activities similar to the parent MSC. The purpose of this study is to evaluate the immunomodulatory effects of MSC-EV on canine peripheral blood mononuclear cells (PBMCs). We have found that MSC-EV cause increases in viable canine PBMCs. Our hypothesis is that MSC-EV increase cell proliferation of stimulated canine PBMCs and this change can be measured via an ATP assay. Further characterizing MSC-EV induced changes via flow cytometry, qRT-PCR and multiplex analysis of secreted prod- ucts are important next steps to understanding immunomodulatory effects of MSC-EV on canine PBMCs.

Research Grant: Research Grant information: Funding support through internal award from Department of Biomedical Sciences, Oregon State University. Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program

Development of Anti-TNFa VHHs for immunotherapy of chronic inflammatory diseases in humans and canines

Lorena Sistig, Charles Shoemaker, Abhineet Sheoran

Veterinary Student (Sistig), and Department of Infectious Disease and Global Health (Shoemaker, Sheoran), Tufts Cummings School of Veterinary Medicine, North Grafton, MA

Tumor necrosis factor alpha (TNFa) plays a major role in inflammatory autoimmune diseases such as RheumatoidArthri - tis (RA), Crohn’s disease, and ulcerative colitis. Treatment with monoclonal antibodies (MAbs) to block TNFa activity exist for humans, while animals almost exclusively rely on pain management. In human medicine, MAbs have shown great results in people suffering from chronic autoimmune inflammatory diseases, but these molecules have some draw- backs. Not only is the production of MAbs very time consuming and costly, but MAb also only target one epitope. This has led to the search for a smaller, more effective antigen-binding agent. Variable regions of heavy-chain-only antibodies (VHH) of camelids are smaller (~14 kDa) and more effective antigen-binding agents than MAbs. Unlike MAbs,VHHs can be functionally expressed in microbes and are cheaper to manufacture. The Shoemaker lab has identified and ex- pressed 9 unique VHHs against human TNFa. They recognize human TNFa in enzyme linked immunosorbent assays, but their TNFa-neutralizing ability is unknown. The goal of this research is to use ELISA, cell cytotoxicity assay, and immunoblotting to determine the ability of the 9 VHHs to neutralize human TNFa. We will also include 12 VHHs against mouse TNFa to analyze their ability to cross-neutralize human and/or canine TNFa. This research will provide new infor- mation on neutralization of both canine and human TNFa via these VHHs. These findings would be very important in the development of less costly immunotherapy for canine and human patients with chronic inflammatory diseases. Likewise, it will give more information on using immunotherapeutic agents for the treatment of inflammatory autoimmune diseases in animals.

Research Grant: Unknown Student Support: Tufts Cummings School of Veterinary Medicine

310 • 2021 National Veterinary Scholars Symposium The use of Oxford Nanopore Technology as a single test to identify and characterize infectious bronchitis

Brittany Skaggs, Miranda Painter, Amro Hashish, Yuko Sato, Mohamed El-Gazzar

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Infectious bronchitis virus (IBV) is a coronavirus that affects the respiratory system of chickens, causing major econom- ic losses within the poultry industry. Like other Coronaviruses, IBV has a high mutation rate resulting in the continuous emergence of variant serotypes. There is little to no cross-protection between IBV serotypes, therefore, serotype determi- nation is necessary for vaccine selection. The most common method of IBV serotype characterization is through sequenc- ing the S1 gene. However, sequence typing is not always possible. Due to the high mutation rate, sequencing primers often fail to anneal and need to be continuously updated. Oxford Nanopore Technology (ONT) is a third-generation sequencing platform that utilizes sequence-independent technology to sequence nucleic acid in its native form in minutes to hours. This study aims to adopt, optimize, and validate ONT for direct sequencing of IBV without the need for specific primers. As a result, this use of ONT can serve as a single test for fast and accurate detection and characterization of IBV. Serial dilutions of different IBV isolates and known positive clinical samples will be used to evaluate ONT’s sensitivity of identification and specificity of characterization. Results from this study are currently pending. If proven effective, identification and characterization of the IBV will be easier, less expensive, and less time-consuming. This will inform the vaccine selection process and facilitate disease prevention and control.

Research Grant: Iowa Livestock Health Advisory Council (ILHAC) Student Support: Iowa Livestock Health Advisory Council (ILHAC)

Assessment of anti-viral and anti-inflammatory effects of Mesenchymal Stem Cells in herpesviral infections

Shannon Skalecki, Valerie Johnson

Michigan State University, Department of Clinical Sciences, College of Veterinary Medicine, East Lansing MI

Elephant Endotheliotropic Herpesvirus (EEHV) is a major threat to African and Asian Elephants. This virus is especially threatening to and accounts for most deaths of young elephants in captivity. Death from EEHV is a result of fatal hemor- rhagic disease or systemic inflammation leading to organ failure. There currently is no vaccine and intensive supportive care is provided to animals as soon as they become viremic. Despite this mortality remains approximately 50% in young elephants who contract this disease. Mesenchymal Stem Cells (MSCs) have been investigated as a therapy for sepsis and other infectious diseases due to their anti-inflammatory and antimicrobial properties. Few studies have been performed to assess the anti-viral effects of MSCs although studies on the anti-viral effect of MSCs in animal models of influenza suggest a beneficial effect. To determine if MSCs or factors secreted by MSCs (MSC-CM) have direct anti-viral effects against herpesvirus equine cells will be infected with EHV-1 then treated with MSCs, MSC-CM or untreated. Viral load will be assessed by qPCR. In addition to direct anti-viral effects MSCs could improve the outcome of this disease through reduction of the systemic inflammation that occurs in this disease process. Commercially available equine ELISAs have been demonstrated to cross react with elephant cytokines. Cytokine levels will be measured in banked serum samples from elephants with EEHV who received or did not receive treatment with intravenously delivered MSCs. Cytokine levels will be compared using a 2-way ANOVA to determine if treatment with MSCs decreases systemic levels of pro-inflamma- tory cytokines and increases levels of anti-inflammatory cytokines in EEHV infection.

Research Grant: Michigan State University Start-up Fund Student Support: Student funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

2021 National Veterinary Scholars Symposium • 311 Senecavirus protective immune response and transmission in replacement gilts on persistently infected farms

Robert Skelly, Pablo Pineyro

Associate Professor, Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University

Senecavirus A (SVA) causes vesicular disease and epidemic transient neonatal losses in swine. Outbreaks of SVA are characterized by an abrupt onset of vesicles that progress to ulcerative dermatitis on the nostrils, coronary bands, and sto- matitis, which affect a high percentage of sows and finisher pigs. Seroprevalence studies indicate that ASV is endemic and highly prevalent in the United States, and it might circulate sub clinically in sows and finisher pigs. In addition, persistent infection studies suggest that sub clinically infected animals play an essential role in carrying and maintaining the virus in persistently-endemic farms. Current epidemiological data support that occasional outbreaks still occur and cause a mul- tifactorial economic burden to the swine industry. This project consists of multiple phases. Phase one consists of identi- fication of sub clinically or persistently SVA infected sow farms. Phase two consists of characterization of SVA immune response and viral transmission dynamics infection in gilts in persistently infected sow farms. Phase three consists of eval- uation of the protective efficacy of the SVA specific antibody induced in field conditions against SVA in an experimental challenge. The goal of this summer is to develop phase one of this multistep project. Thus we will visit multiple sow farms to collect blood, nasal and fecal swabs, and oral fluids for the detection of SVA antibodies or viral antigen.

Research Grant: Iowa Livestock Health Advisory Council Student Support: Iowa State University College of Veterinary Medicine Office of Research and Graduate Studies

Evaluation for anti-erythrocyte and anti-platelet antibodies in healthy dogs administered lokivetmab

Megan Slaughter, Michael Lappin, Nida Chornarm, Jennifer Schissler, Sarah Shropshire

Clinical Sciences Department and Center for Companion Animal Studies, Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

Lokivetmab (Cytopoint ) is a common medication for the management of canine allergic pruritus which acts to neutralize IL-31, a cytokine shown to activate the neuronal signaling pathway associated with itch in dogs. Multiple publications confer the efficacy and safety of lokivetmab for allergic disease when given at the recommended dosages. However, there have been anecdotal concerns that this therapy could trigger an immune-mediated response leading to immune-mediat- ed hemolytic anemia (IMHA) or immune thrombocytopenia (ITP). Anti-erythrocyte antibodies (AEA) or anti-platelet antibodies (APA) can form in both IMHA or ITP respectively. Therefore, the aim of this study is to determine if AEAs or APAs develop following a standard-dose therapy of lokivetmab in healthy dogs. Determining if such antibodies form during lokivetmab therapy would be clinically beneficial for veterinarians who may be evaluating an anemic or thrombo- cytopenic patient shortly after receiving a lokivetmab injection. Whole blood samples were taken from 8 healthy beagles at baseline (day 0) prior to lokivetmab administration and on days 14 and 28 following lokivetmab administration. A complete blood count and flow cytometry to detect AEA and APA were performed at all time points. No dogs exhibited anemia or thrombocytopenia and all were negative for AEA and APA at baseline. Day 14 and day 28 data collection are currently ongoing. Future work includes a clinical trial for the evaluation of anemia, thrombocytopenia, AEAs, and APAs in client-owned allergic dogs receiving lokivetmab.

Research Grant: Colorado State University Veterinary Summer Scholars Program and the Kenneth W. Smith Professorship in the Center for Companion Animal Studies Student Support: Colorado State University College of Veterinary Medicine and Biomedical Sciences Dean's Office

312 • 2021 National Veterinary Scholars Symposium Grape toxicosis in dogs: in vitro studies

Alaunie Smiley, Arthur Armstrong, Stephen B Hooser

Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN

In 2001, the ASPCA Animal Poison Control Center (APCC) reported that a review of calls revealed cases of acute renal failure in dogs that ingested grapes or raisins. These cases were rare, and while very few incidences of grape or raisin ingestion result in acute renal failure, there were sufficient confirmed cases to warrant a warning letter to the veterinary community in JAVMA. Grapes and raisins were analyzed for known renal toxins, but none were identified. In 2021, the ASPCA APCC reported two cases in which dogs developed acute renal failure following ingestion of large amounts of tar- taric acid (cream of tartar). Tartaric acid can be present in some grapes and raisins in widely varying amounts, and in some instances is not present at all. The current study aims to evaluate if canine kidneys can be adversely affected by tartaric acid. Our hypothesis is that MDCK cells, a canine kidney cell line, will be adversely affected by exposure to tartaric acid in vitro. MDCK cells were plated in 96 well culture plates and grown to near confluency. The cells were dosed with PBS (vehicle), tartaric acid (100 or 10mM), valproic acid (positive control: 100 or 10mM), or malic acid (negative control: 100 or 10mM) and incubated at 378C. At 24hrs, the cells in each well were examined microscopically, and, in each well, an LDH cytotoxicity assay and an MTT cell viability assay were performed. Preliminary results indicate that MDCK, canine kidney cells, are sensitive to the toxic effects of tartaric acid.We conclude that tartaric acid, if present in sufficient amounts in grapes/raisins, or cream of tartar, may contribute to acute renal failure in dogs following its ingestion.

Research Grant: None Student Support: Boehringer Ingelheim and Purdue University College of Veterinary Medicine

Developing bioresorbable orthopaedic implants to apply increasing loads throughout healing

Anna N. Smith, Ana P. Peredo, Richa Gupta, Sarah E. Gullbrand, Robert L. Mauck, and Michael W. Hast

Department of Orthopedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA (Smith, Peredo, Gupta, Gullbrand, Mauck, Hast) and Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA (Gullbrand, Mauck, Hast)

Veterinary orthopaedic interventions, ranging from fracture repair to ligament reconstructions, are currently reliant upon metallic implants to bear mechanical loads during healing. However, the load-bearing requirements of these implants typically decreases to zero as healing progresses, leaving metal implants within the body permanently. 3-D printed biore- sorbable polymer components have tremendous potential for clinical use, as the lattice architecture can be tuned to change mechanical strength and alter degradation kinetics. However, longitudinal mechanical properties during biodegradation are relatively unknown. The aim of this study is to determine the degradative kinetics of novel, 3-D printed porous lat- tices created with poly lactic-co-glycolic acid (PLGA) at time zero and at clinically relevant time points (4-8 weeks). We hypothesized that there would be a 10% decrease in mechanical properties at the 4-week timepoint, and a 30% decrease in mechanical properties at the 8-week timepoint. Test coupons were fabricated with two variables of interest: lattice densi- ty (40, 80, 100%) and infill pattern (rectilinear, gyroid, triangle). In vitro (immersion in fetal bovine serum) and in vivo (subcutaneous implantation in a rat) degradation assays were performed. Preliminary results suggest substantial decreas- es in mechanical strength at 4 weeks for 40% infill with no substantial differences in the other groups. Results from this study will guide the design and development of patient-specific PLGA devices that may shift the paradigms of veterinary orthopaedic surgery.

Research Grant: NIH K25AR078383, VA RR&D I01 RX002274 Student Support: NIH T35 OD010919

2021 National Veterinary Scholars Symposium • 313 Effects of exercise on gastrointestinal tissue in an accelerated aging murine model

Emma L. Smith, Colby Erickson, Tom L. Broderick, Minsub Shim, and Layla Al-Nakkash

College of Veterinary Medicine (Smith), Arizona College of Osteopathic Medicine (Erickson, Al-Nakkash), Department of Physiology (Broderick, Al-Nakkash), Department of Biochemistry (Shim), College of Graduate Studies, Midwestern University, Glendale, AZ

Senescence accelerated mouse-prone 8 (SAMP8) mice display a phenotype of accelerated aging and are used to study the mechanisms and physiologic effects of aging. The aim of this study was to determine whether or not exercise benefits intestinal tissue in SAMP8 mice. SAMP8 female mice (8-9 weeks old) were randomly assigned to one of the following groups (n = 5-6/group): sedentary SAMP8 or runner SAMP8. Age-matched wild type mice and 12-month aged SAMP8 mice were used as controls. Exercise consisted of wheel running on a voluntary basis seven days per week for the entire study duration of 12 weeks. The total distance ran each week was tracked. SAMP8 females had comparable weights at the end of the study, regardless of exercise training, while age-matched controls gained significantly more weight. We are currently determining total protein expression of key markers of inflammation and gut function, including SLC26A4,Akt, p-Akt, and pH2AX in colonic tissue using standard western blot techniques. Current trends indicate increased SLC26A3 expression in SAMP8 runners compared to sedentary SAMP8 and control SAMP8 groups. Other targets to be evaluated include COX-2 and TLR-4.

Research Grant: Midwestern University Arizona Alzheimer's Consortium Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program

Comparison of the fecal microbiome of feral and domesticated cats

Katelyn Smith, Amie Burling, Milan Piva, and Craig Franklin

Departments of Veterinary Medicine and Surgery (Burling), and Veterinary Pathobiology (Franklin), College of Veterinary Medicine, University of Missouri, Columbia MO

There is growing evidence that differences in the diversity and composition of the gut microbiome (GM) can have a profound impact on general fitness and disease. For example, in humans, lowered GM diversity has been associated with diseases such as obesity. Moreover, in mice, it has recently been shown that housing in pristine research environments has resulted in lowered GM diversity and associated changes in immune system development when compared to mice housed in more diverse pet store environments. Feral cats represent a population exposed to a markedly diverse and changing en- vironment when compared to domestic cats, and it is conceivable that their GM has greater diversity that, if pathogen-free, may improve overall fitness. The aim of this study is to begin to characterize the GM of the feral cat population, with the hypothesis that this population will have a more diverse and richer fecal microbiome (FM) than cats residing in a more do- mestic environment. Either rectal swabs or voided samples were collected from 9 solitary cats housed in a common shelter environment and 10 feral cats. DNA will be extracted from these fecal samples and the FM richness, diversity, and com- position determined by next generation sequencing. At the completion of this project, we expect to find that feral cats have a statistically significant, greater FM richness and diversity compared to cats from a domestic environment. Moreover, assessment of composition will reveal key differences in microbial species that may serve as future targets for probiotics designed to enrich domestic cat GMs and improve overall fitness.

Research Grant: This work is supported by Franklin research incentive funds . Student Support: Stipend for Katelyn Smith is supported by Franklin research incentive funds

314 • 2021 National Veterinary Scholars Symposium The pharmacokinetics of a non-invasive (needle-less) administration of flunixin meglumine in grower pigs

Lilly Smith, Emily Wiloch, Hiroko Enomoto, Laura Neumann, Ronald Baynes, Kristen Messenger

Center for Chemical Toxicology Research and Pharmacokinetics (Smith, Wiloch, Enomoto, Neumann, Baynes) Department of Molecular Biomedical Sciences (Messenger), College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Flunixin meglumine is a nonsteroidal anti-inflammatory drug approved by the FDA for use in swine for the treatment of pyrexia associated with respiratory disease by intramuscular (IM) injection. It is commonly used extralabel to treat pain associated with multiple conditions. A method of flunixin administration that is easily and non-invasively administered on farms could have a major impact on the animal welfare and economics of the swine industry. The objective of this study is to determine the pharmacokinetics of a novel, non-invasive route of administration of flunixin compared to intramuscular injection. In a randomized crossover study, six grower pigs each received both routes of administration of flunixin at 2.2 mg/kg. Plasma samples were collected for up to 60 hours post administration and were analyzed by ultra performance liquid chromatography with tandem mass spectrometry to determine drug concentrations over time. Noncompartmental analysis was used to obtain pharmacokinetic parameter estimates. Median (range) pharmacokinetic parameter estimates for the IM (control) and non-invasive (experimental) routes, respectively, were area-under-the-curve 6.39 (4.99-7.63) and 2.98 (2.84-4.74) hr*mg/mL; maximum concentration 3.92 (2.90-4.09) and 2.55 (2.11-2.75) mg/mL, which occurred at time 0.17 (0.17-0.33) hrs for both routes. Terminal half-life was 7.46 (6.48-7.75) and 6.83 (6.33-7.91) hrs. Relative bio- availability of the novel administration method compared to IM administration was estimated to be 46.59 (39.13-95.02)%. In conclusion, the novel non-invasive method of flunixin administration to pigs appears to be relatively well absorbed and with plasma levels corresponding to analgesic efficacy in other species.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

A citizen science pilot study: results from a survey about urbanized parrots in Point Loma, San Diego

Nicole Smith, Madelaine Steevens, Curtis Eng, Rhea Hanselmann

College of Veterinary Medicine, Western University of Health Sciences, Pomona, California

Parrots are vibrant colored, non-territorial birds who thrive as canopy dwellers of tropical or subtropical habitats (Brightsmith & White, 2012). Their endearing characteristics have led to legal and illegal trading of parrots, resulting in decimation of many wild parrot populations and contributed to the establishment of wild parrot populations in non-native lands (Garret, 1997). Knowledge about the species that have populated Southern California and their role in the local ecosystem is limited due to complex reasons including their secretive natural behaviors of flocking and canopy dwelling and lack of monitoring by government agencies (Brightsmith & White, 2012). To learn more about the ecology of the naturalized parrots and understand how these birds interact with the highly urbanized landscape of Southern California, we surveyed residents of Point Loma, San Diego, CA, where parrots are abundant throughout the summer months. In July 2020, an electronic 49 question survey was distributed to these residents via multiple online formats with weekly remind- ers. During the four weeks that the survey was active, over 400 Point Loma residents shared observations and opinions on the parrots inhabiting their neighborhood. The information collected with this survey identified the most common parrots noted, their effects on the native habitat and threats to their existence in a non-native land.All of this significant- ly increases our understanding of the behavior of the parrots that inhabit this coastal community and reveals interesting findings about how these birds interact with their non-native environment.The methodology of using a lay person survey to describe wild birds also posed challenges to data validity.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 315 The role of invG in Salmonella enterica adherence to and invasion of human intestinal epithelial cells

Olivia Smith, Ananta Arukha, Subhashinie Kariyawasam

University of Florida College of Veterinary Medicine (Smith) and Department of Comparative Diagnostic and Population Medicine (Arukha, Kariyawasam) University of Florida College of Veterinary Medicine, University of Florida, Gainesville, FL

Nontyphoidal Salmonella is a common cause of foodborne illnesses in the United States and Europe. Animals, such as poultry, serve as a reservoir of these salmonellae serovars indicating a One Health approach to mitigate these foodborne zoonoses. Of the nontyphoidal salmonellae, Salmonella Enteritidis, is one of the most common serovars implicated in foodborne salmonellosis and most of these outbreaks have been associated with consumption of shell eggs and egg prod- ucts. We recently identified a gene called invasion G invG( ), which is overexpressed by S. Enteritidis when grown in egg yolk. In order to research if invG is involved in S. Enteritidis adherence and invasion of the intestinal epithelial cells, we constructed an isogenic mutant of S. Enteritidis lacking invG and tested in vitro on Caco-2 cells along with the wild-type and mutant complemented strains. Our results show a significant reduction in adherence and invasion with theΔinvG mutant in Caco-2 cells as compared to the wild type parental strain. Complementation of the mutant restored Caco-2 cell adherence and invasion to the wild-type levels. These results support the function of invG in the adherence and invasion of intestinal epithelial cells by S. Enteritidis.

Research Grant: USDA National Institute of Food and Agriculture, Food Safety grant #2020-03308 Student Support: Boehringer Ingelheim, USDA-NIFA, Florida Veterinary Scholars Program

Broadening the spectrum of exotic animal species neoplasia: an analysis of Rollins Laboratory cases

Sidney Smith, Leigh Duke, James Trybus, Tara Harrison

Department of Clinical Sciences (Duke, Harrison), College of Veterinary Medicine (Smith), NC State University, Raleigh, NC; ESCRA Database (Duke, Harrison); Rollins Animal Disease Diagnostic Laboratory (Trybus), Raleigh, NC.

Currently, there is a lack of readily available data regarding neoplasia in exotic species. The Exotic Species Cancer Re- search Alliance’s (ESCRA) mission is to increase published data in this area through the use of their exotic tumor data- base. Our goal was to broaden the spectrum of neoplasia commonly found in exotic species using pathology reports from Rollins Animal Disease Laboratory. Rollins has over 6,000 cases during January 2011 - March 2019, and 416 of these are histopathologically confirmed cases of neoplasia in exotic animal species.This data was organized into preselected cat- egories including species, age, sex, tumor type, location of tumor, tumor behavior, and treatment. We found that of these 416 cases, there were 4 animal orders, over 50 species, and more than 200 different histologic diagnoses within 12 differ- ent tumor classifications. The majority of tumors found were considered malignant, but only 20% of those tumors metas- tasized. This information will contribute to the ESCRA database to be able to gain a better understanding of prevalence and survival of exotic animals with cancer. By publishing more information about cancer in exotic species we hope to lay groundwork for future studies, as well as increase publicly available data regarding treatment, prognosis, and common types of neoplasia in exotic species.

Research Grant: None Student Support: NC State VSP and BIVSP

316 • 2021 National Veterinary Scholars Symposium Novel vaccine inactivation method using UV and riboflavin preserves major influenza surface antigens

Savannah E Snell, Izabela K Ragan, Lindsay M Hartson, Raymond P Goodrich

Department of Biomedical Sciences (Snell, Ragan), Department of Microbiology, Immunology and Pathology (Goodrich), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO. Infectious Disease Research Center (Hartson, Goodrich), Colorado State University, Fort Collins, CO

Infectious diseases are within the top ten causes of death globally. Vaccines have proven to be an effective method for combating infectious diseases, such as influenza and coronaviruses, especially on an epidemic and pandemic scale. Chem- ical treatments are commonly used in vaccine preparation to inactivate whole pathogens by modifying nucleic acid and protein structures, which prevents the pathogen from replicating and being infectious. However, such methods use harsh chemicals and risk damaging key antigenic epitopes that are needed to stimulate a robust immune response. Our objec- tive is to test the efficacy of a novel photochemical inactivation method, which uses riboflavin and UV light to irrevers- ibly damage viral nucleic acid. We hypothesize that this method will enhance vaccine immunogenicity by preserving the quantity and quality of viral antigens while also decreasing vaccine production time and waste. Using influenza virus as a virus model, we compared the outcome of this inactivation method against traditional chemical methods (formalin, BPL), specifically in terms of the effect on preserving major antigenic proteins. Our work to date has found that UV+riboflavin inactivation preserves key influenza proteins needed for a robust immune response. Specifically, UV+riboflavin was better at preserving proteins like hemagglutinin compared to formalin treatment and equally as effective compared to BPL.This inactivation method could increase the immunogenicity and efficacy of influenza vaccines, while also decreasing vaccine risk, production costs, and production of harmful waste by-products. The knowledge gained from this study can then be applied to other infectious pathogens with significant public health impacts.

Research Grant: NIH/NIAID, BARDA, Office of the Vice President of Research (OVPR) at Colorado State Universi- ty, influenza virus stock provided by the Bowen Laboratory at Colorado State University Student Support: Boehringer Ingelheim

Determining correlations between blood glucose, saliva glucose, and pH in small and large animal patients

Bryanna Snipes, Athema Etzioni

Department of Pathobiology, College of Veterinary MedicineTuskegee University, Tuskegee Institute, Alabama

Determining glucose levels in small and large animal patients currently requires venipuncture to obtain blood to use in a glucometer or to run a full panel of bloodwork or a surgically-placed glucose meter. Because of this, most canine and feline diabetic patients must be routinely brought to the veterinarian to have glucose levels checked. When it comes to equine and bovine patients, nutritional status and oral health can be assessed using glucose levels, but obtaining blood is not always practical, especially in field situations. In both small and large animal patients, salivary pH levels can be a good indicator of oral health and could correlate to the glucose levels in the saliva. The aim of this study is to determine if a correlation exists between blood and salivary glucose levels and saliva pH levels in hopes of developing an alternative, less invasive method of obtaining glucose levels in veterinary patients.

Research Grant: Tuskegee University College of Veterinary Medicine Student Support: Boehringer Ingelheim

2021 National Veterinary Scholars Symposium • 317 Estrogens and Their Association with Illness in Hospitalized Foals

Hailey Snyder, Jacob Swink, Jamie Summers, Julia Horton, Hannah Kinsella, and Ramiro Toribio

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

During late pregnancy the equine placenta acquires steroidogenic functions including the synthesis of androgens and estrogens using fetal progestogens as precursors. Previous studies have shown that abnormally high concentrations of progestogens and androgens are linked to sepsis and neonatal maladjustment syndrome. Therefore, endocrine dysregula- tion contributes to mortality in critically ill foals. Estrogens (17b-estradiol, estrone, total estrogens) have not been inves- tigated in sick foals. We hypothesize that during hospitalization, critically ill foals will have higher estrogen concentra- tions that will be proportional to disease severity. In addition, foals with persistent elevation of estrogens will have more severe laboratory abnormalities due to organ dysfunction and an increased risk of mortality. Prospective, multicenter, and descriptive study in hospitalized (n = 80; 50 septic, 30 sick non-septic) and healthy (n = 20) foals of < 3 days of age at 4 institutions. A power analysis was performed. Progress: Blood samples have been collected at 0, 24, 48, and 72 h for estrogen measurement via immunoassays (ELISA, RIA). Clinical information will be retrieved from the medical records for disease severity classification (ongoing). Measurement of estrogen concentrations is in progress. Data will be assessed for normality and analyzed by parametric and non-parametric statistical methods. Results from this study will provide additional details on the steroid profile of healthy and sick foals (e.g. steroid clearance) and enhance our understanding on the role of estrogens (and other sex hormones) in the immediate post-partum period.

Research Grant: Effects of prepartum progestagen administration to mares on the health of their foals (Equine Research Funds) Student Support: NIH T35 Training Grant T35OD010977

Diagnostic and prognostic markers of canine peripheral nerve sheath tumors

Jaclyn M. Soulas and Molly E. Church

School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (Soulas) and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (Church)

Peripheral nerve sheath tumors (PNSTs) arise from supporting cells of the nerve (i.e., perineurial cells, fibroblasts, Schwann cells) and may be classified as either benign or malignant. Malignant PNSTs (MPNSTs) are aggressive neo- plasms that pose both diagnostic and therapeutic challenges, especially due to subjective interpretation of these tumors and their overlapping histologic appearance with benign PNSTs (BPNSTs) and other soft tissue sarcomas. Determining specific histologic and immunohistochemical characteristics of BPNSTs and of MPNSTs will aid in definitive diagnosis and prognosis, which is crucial for veterinary oncologists to be able to effectively treat these entities. In general, BPNSTs are considered to have an excellent prognosis with complete excision while tumors classified as MPNSTs are thought to have poor prognoses given that they tend to be locally infiltrative, recurrent, and have metastatic potential, rendering treatment and long-term survival oftentimes unsuccessful. However, explicit histologic characterization (i.e., mitotic in- dex, amount of necrosis, degree of differentiation) with an established grading scheme, and specific immunohistochemical staining characteristics (i.e., SOX10) of BPNSTs from MPNSTs is lacking. By examining the histologic and immunohisto- chemical profiles of 40 BPNSTs and 40 MPNSTs we will establish clear diagnostic criteria for these currently ambiguous- ly diagnosed tumors. Finally, by correlating histologic and immunohistochemical criteria with clinical outcome data (via Kaplan-Meier analysis), we will be able to provide long sought-after prognostic information for these frequently recurrent tumors in dogs.

Research Grant: McCabe Research Grant, Perelman School of Medicine Student Support: NIH T35 Training Grant T35OD010919

318 • 2021 National Veterinary Scholars Symposium Assessment of viral load and cytokine gene expression in lung tissues from SARS-CoV2-infected mice

Caroline Spangenberg, Rachel Olson, Craig Franklin, James Amos-Landgraf

UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4 (Spangenberg), Department of Veterinary Pathobiology, College of Veterinary Medicine (all), Laboratory for Infectious Disease (Olson), Mutant Mouse Resource & Research Center (Franklin, Amos-Landgraf), University of Missouri, Columbia, Missouri

Emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on global health, quality of life, and the economy, but little is known about the pathogenesis of disease, or why there is an extreme disparity in morbidity and mortality rate. There is a need for additional research on the pathogenesis of the virus to aid in the development of potential therapies and improve clinical outcomes of infected patients. The most severe forms of infection correlate with an exaggerated pro-inflammatory cytokine response.The aim of this study is to continue the on- going investigations on the pathogenesis of disease by associating levels of the following cytokines, TNF-a, IFN-g, IFN-a, IFN-b, IL-6, IL-4, and IL-10 with changes in viral load and histologic disease severity. The studies comprise mice that were genetically engineered to express the human ACE2 (hACE2) receptor for SARS-CoV-2 and were harboring three distinct gut microbiomes of differing diversity associated with varying susceptibility to disease.The microbiomes were designated as GM1 (low richness), GM4 (high richness) and GM1 + SFB (low richness plus segmented filamentous bacte- rium, a commensal bacterium known to modulate mucosal immunity). Mice from each group were intranasally inoculated with doses of SARS-CoV-2 and consecutively euthanized six days post-inoculation, with lung tissue collected for cyto- kine gene expression, viral load, and histologic analysis. By using this mouse model to explore host immune interactions, we will be able to closely reflect the pathogenic outcomes of infection in humans and ultimately gain a better understand- ing of the pathogenesis of Covid-19.

Research Grant: NIH grant U42 OD 010918-21S1, Optimization of Murine Models of COVID-19 through gut microbiota manipulation Student Support: American Society of Laboratory Animal Practitioners and IDEXX-BioAnalytics

The effect ofFusarium spp. on cold-stunned Kemp’s ridley sea turtles

Jessica F. Sparks, John Thomason, Angela Knight, Debra Moore, Stephen Reichley, Christa Barrett, Hossam Abdelhamed, Michelle Banes, and Mark L. Lawrence

College of Veterinary Medicine, Mississippi State University, Starkville, MS

Fusarium is a filamentous fungus found worldwide that can cause animal and human infections. In sea turtles, it causes sea turtle egg mortalities and infects juveniles and adults. We hypothesized that Fusarium spp. cause respiratory infections that have not cleared after administration of antibiotics and carapace lesions in cold-stunned Kemp’s ridley sea turtles (Lepidochelys kempi). In December 2020, twelve cold-stunned Kemp’s ridley sea turtles were rescued and transported to the Institute for Marine Mammal Studies in Gulfport, Mississippi. Of the twelve turtles, two were unresponsive to anti- microbials administered to treat respiratory inflammation; one of these also had a circular, well-demarcated shell lesion. To determine whether Fusarium spp. is associated with respiratory infection and carapace lesions, as well as to determine a baseline of the normal flora found on Kemp’s ridley sea turtles, fungal cultures were collected from five different sites of these two turtles. The five sites included bronchoalveolar lavage (BAL), blood, carapace, cloaca, and nasal cavity. The samples taken were inoculated onto three different types of media. For 25 days, fungal growth was monitored macro- scopically by phenotypic identification and microscopically by morphology of the conidia.Fusarium spp. was identified from the carapace lesion of one turtle, but it was not isolated from BAL. To confirm the identification of the fungus, DNA extraction and PCR were conducted, and sequencing is being conducted. Other fungal species were isolated, with Penicil- lium being the most common species from all sites. Fungal cultures from ten other turtles are pending.

Research Grant: Mississippi Marine Mammal and Turtle Conservation, Recovery, and Monitoring Program; National Fish and Wildlife Foundation under Mississippi Department of Environmental Quality Agreement No. 18-00081 Student Support: Mississippi Department of Environmental Quality Agreement No. 18-00081

2021 National Veterinary Scholars Symposium • 319 Describing the Geospatial Distribution of Antimicrobial Resistance Genes in the Boston Urban Rat Population

Aubrey G. Specht, Nichola Hill, Joseph D. Madison, Ashlee M. Earl, Marieke H. Rosenbaum

Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA (Specht, Hill, Rosenbaum), Department of Biology, University of Massachusetts Boston, Boston, MA (Madison) and Broad Institute of MIT and Harvard University, Cambridge, MA (Earl)

For centuries, rats have been vectors of infectious diseases that impact humans, from pestis, responsible for the infamous Black in the Middle Ages, to leptospirosis, an endemic, yet neglected, disease that causes febrile illness in humans and animals. Due to their interactions with humans and with environmental sources of antimicrobial resistance (AMR) genes, we hypothesize that urban rats also harbor and shed microbial genes encoding resistance to drugs used to treat bacterial infections in human and animals. With rodent numbers increasing due to the rise in urbanization and inef- fective pest management strategies, there is a need to understand the role rats may play in the circulation of AMR genes within an urban community, especially among populations of people experiencing homelessness, who face an elevated risk for AMR. This study seeks to characterize the resistome of urban rats in the city of Boston by focused trapping in areas with a large homeless population. One hundred rats are being trapped using the RATMO detection device through- out the city. Using the Omega MagBind Universal Pathogens kit, DNA and RNA are extracted from fresh fecal samples, obtained via necropsy. Illumina MiSeq Next Generation Sequencing will be used to detect genes encoding AMR and to describe the rat lower intestinal microbiome. The geospatial distribution and characterization of AMR genes found in rats may further imbue urgency to responsible antimicrobial stewardship from a One Health perspective and inform pest con- trol strategies as well as public health policy.

Research Grant: Tufts Clinical and Translational Science Institute Mentored Career Development 1KL2TR002545 NIH/ NCATS Student Support: AVMA/AVMF 2nd Opportunity Summer Research Scholarship

Measuring Axonal Damage in Drosophila Model of TBI

Justine Spiker, Lori Buhlman

College of Veterinary Medicine, Midwestern University, Glendale, Arizona (Spiker, Justine), Biomedical Sciences Program, Midwestern University, Glendale, Arizona (Buhlman, Lori)

Traumatic brain injury is a major contributor to neurologic deficits and mortality in humans. In addition to the primary injury, a significant component to the neurodegeneration seen is caused by activation of the secondary cellular signaling pathways leading to chronic neuroinflammation. The proposed triggering mechanism is release of damage associated molecular patterns from degenerating axons. We hypothesize that axonal degeneration will be observed following TBI, and that degeneration will increase in aged Drosophila exposed to TBI. Groups of eight 30-356 day old flies expressing membrane-bound green fluorescent protein in a subset of neurons are placed in a tube and injured using a High Impact Trauma Device to induce a mild TBI, evident by ataxia and incapacitation. Sham flies are loaded into the device without being hit. Flies are then either placed in an activity monitor or their brains are dissected at 30m, 60m, 24h, and 7d post-in- jury. The activity monitor measures the effect of injury on average height climbed, total height climbed, and number of climbing attempts to gain insight on their climbing ability and motivation. Brains are imaged on a confocal microscope and axon length and surface area to be measured as an indication of axonal damage. . The results of this study will shed light on whether axonal degeneration contributes to TBI-induced neurodegeneration and if advanced age exacerbates injury outcomes.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program

320 • 2021 National Veterinary Scholars Symposium Effect of chemotherapy agents on the diversity of the fecal microbiome of canine lymphoma patients

Eric Spilker, Hinako Terauchi, and Linda Mansfield

Department of Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Chemotherapy-induced gastrointestinal disease (CIGD) has been associated with chemotherapy treatment resulting in clinical signs of nausea, vomiting and diarrhea. CIGD can decrease quality of life and is a common reason for discontinu- ation of potentially life-saving chemotherapy. We hypothesized that CHOP protocol chemotherapy agents would decrease the diversity of the canine fecal microbiome and create dysbiosis of gastrointestinal bacteria in dogs with lymphoma. Nine canine lymphoma patients undergoing cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) che- motherapy had fecal samples collected weekly before and throughout treatment.16S rRNA gene sequencing analysis was performed on DNA from fecal samples to determine the composition and relative abundance of bacterial taxa in the mi- crobiome. From each dog, 16S sequencing data from ten longitudinal samples were analyzed using QIME2 and bacterial taxa assignments made using the SILVA database. During intervals of CHOP treatment, changes in the relative abundance of bacterial taxa were observed using heatmaps. These plots provided evidence that CHOP chemotherapy administration caused dysbiosis within the canine gastrointestinal tract. The specific bacterial taxa affected suggested that commensal bacteria decreased and opportunistic pathogens increased. Analysis will focus on determining if a significant association exists between the GI microbiota changes observed and clinical signs of CIGD. We will also test the secondary hypothesis that CHOP chemotherapy causes an increase in pro-inflammatory GI bacterial taxa that favor a pathological phenotype of CIGD. Future analyses will be performed using 16S sequence data from 15 additional canine lymphoma patients.

Research Grant: Endowed Research Fund grant RT082792, College of Veterinary Medicine, Michigan State University; Albert C. and Lois E. Dehn Endowment; University Distinguished Professor Endowment Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

Preliminary investigation of deleterious alleles in peregrine falcons, golden eagles, and zebra finches

Kelsey Springer, Sian Durward-Akhurst, Jonah Cullen, James Mickelson, Molly McCue

Department of Veterinary Population Medicine (Springer, McCue), Department of Veterinary Clinical Sciences (Durward-Akhurst, Cullen), Department of Veterinary and Biomedical Sciences (Mickelson), College of Veterinary Medicine, University of Minnesota, Saint Paul, MN

Genetic variants computationally predicted to have a detrimental effect on phenotype in healthy individuals have been es- tablished in many domesticated species but have not been widely explored in wildlife. Avian species have dramatically re- duced genomes compared to mammalian species. The distinctly sparse genomes of birds pose unique obstacles concerning how avian species are functionally compensating for gene variance and loss, as well as management of genetic diversity in threatened species. We seek to (1) build catalogs of genetic variation for peregrine falcons (Falco peregrinus), golden eagles (Aquila chrysaetos), and zebra finches Taeniopygia( guttata), (2) determine the frequency of deleterious mutations in highly conserved genes, and (3) compare the the frequency of deleterious alleles in wild birds to that of domestic birds (turkeys/chickens) and domestic mammals (horses/dogs). We hypothesize that our target avian species will exhibit lower tolerance to damaging variants compared to mammalian species with larger genomes. To date, we have collected publicly available genomes (fastq format) of 10 peregrine falcons, 4 golden eagles, 10 zebra finches, and 56 chickens.A modified GATK pipeline was developed to map raw reads to appropriate reference genomes and perform joint genotyping for iden- tification of variants. This project is expected to serve as preliminary data for the development of a larger project focused on comparative molecular disease in threatened avian species, particularly raptors.

Research Grant: Morris Animal Foundation D20EQ-013 Student Support: MAF D21ZO-610, Boehringer-Ingelheim VSS Program, College of Veterinary Medicine UMN

2021 National Veterinary Scholars Symposium • 321 Platelet-rich plasma lysate as a treatment for biofilm associated placentitis

Bryan C. Stallings, Jessica M. Gilbertie, Megan E. Jacob, Lauren V. Schnabel, Sara K. Lyle

Department of Clinical Sciences, College of Veterinary Medicine, NCSU, Raleigh, NC (Stallings, Schnabel, Lyle), Comparative Medicine Institute, NCSU, Raleigh, NC (Gilbertie, Jacob, Schnabel) Department of Population Health and Pathobiology, College of Veterinary Medicine, NCSU, Raleigh, NC (Jacob)

Placentitis-induced pre-term fetal loss has caused hardships and financial losses to the equine breeding industry for de- cades. While traditional multi-modal treatments including a combination of gentamicin, penicillin, altrenogest, firocoxib, and pentoxifylline, have shown some success, treatment failures still occur. The understanding of potential biofilms in these reproductive infections has found a pitfall in traditional treatment options, in which the antimicrobials are ineffec- tive at penetrating and killing biofilms. Platelet-rich plasma lysate has begun to be a widely accepted approach as a new treatment option for biofilm associated infections across species. It has been proven to be successful in treating equine synovial fluid biofilm infections withStaphylococcus aureus, especially when combined with an aminoglycoside. With these promising results, this study aims to look at different isolates ofEscherichia coli from the equine uterus. Biofilm formation was tested amongst 10 clinical isolates followed by treatment with PRP-L, using a crystal violet assay. On av- erage, a 60% reduction of in vitro biofilm formation occurred as a result of the PRP-L treatment. For some isolates, nearly all biofilm formation was eliminated. AlthoughE. coli was the bacteria used in this study, it is expected that similar results will be found from other gram negative, as well as, gram positive bacterial infections found in the equine reproductive tract. Moving forward, our laboratories aim to compare traditional multi-modal therapy for placentitis, with multi-modal therapy augmented with PRP-L.

Research Grant: None Student Support: North Carolina State University Veterinary Scholars Program

Co-localization of interleukin (IL)-17 and CD3 expression in equine (Equus caballus) supporting limb laminitis

Miranda P. Starr, Julie B. Engiles, Andrew van Eps, and Hannah Galantino-Homer

Department of Clinical Studies (Starr, Engiles, van Eps, Galantino-Homer), New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA and Department of Pathobiology (Engiles), School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Supporting limb laminitis (SLL) is a debilitating and often fatal complication of equine severe lameness. Altered limb loading and decreased tissue perfusion compromise the hoof lamellae resulting in failure of digital suspension within the hoof capsule. The tissue damage triggers inflammation and lesions with similarities to human psoriasis. Our previous study showed activation of the interleukin (IL)-17 pathway, the major pro-inflammatory pathway in psoriasis, in SLL. IL-17, the effector cytokine of this pathway, is produced primarily by Th17 cells (a subset of CD3+ T cells) and other inflammatory cell types. We have previously shown CD3+ T cell infiltration of lamellae correlates with severity of SLL, but the IL-17 status of these cells was unknown. Here, we use dual indirect immunofluorescence to identify IL-17+ cells and determine if IL-17 expression localizes to CD3+ T cells. Severe SLL cases and controls were co-stained using CD3 and IL-17 primary antibodies and fluorophore-tagged secondary antibodies. Dual positive cells were imaged using epi- fluorescence and confocal microscopy and quantified as a proportion of all CD3+ cells. Morphology and distribution of CD3-/IL-17+ cells were also noted. Initial results indicate that severe SLL is associated with increased CD3+/IL-17+ cells relative to controls. We also found that severe SLL is associated with increased IL-17 expression by other cell types, such as keratinocytes and CD3- leukocytes, suggesting that these cells might also contribute to IL-17 pathway activation in SLL. These data improve our understanding of the pathogenesis of SLL and its parallels with human psoriasis, suggesting new approaches for improved diagnosis, treatment, and prevention of SLL.

Research Grant: School of Veterinary Medicine intramural funds (Raker/Tulleners Fund), Grayson-Jockey Club Re- search Foundation Student Support: NIH T35 Training Grant OD010919

322 • 2021 National Veterinary Scholars Symposium Relationship between fecal microbiota and Cryptosporidium shedding in calves

Madison Staves, Clemence Chako

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Cryptosporidium parvum has been highlighted as an organism of interest within veterinary medicine due to the parasite’s zoonotic potential, diverse range of subspecies, and ability to persist within the environment in an infective oocyst form. Its subspecies are waterborne coccidian protozoa that act as an opportunistic parasite, which causes parasitic gastroenteri- tis and diarrheal disease predominantly in dairy calves. The aims of this study are threefold. The first aim is to enumer- ate Cryptosporidium oocysts within fecal samples using a hemocytometer and record clinical signs presented by calves. Secondly, to isolate and sequence Cryptosporidium parvum DNA based on the 18S rRNA and subsequently perform PCR analysis. The final aim is to determine the relationship between fecal microbiota and shedding ofCryptosporidium oocysts. Ten healthy newborn dairy calves from Paul Rovey Dairy in Glendale, AZ are used for sample collection. Fecal samples will be taken from the dairy calves on days 3, 7, 14, 21, and 28. Fecal flotation, hemocytometer enumeration, DNA isolation, DNA amplification with PCR are techniques used in this study.

Research Grant: None Student Support: Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Involvement of the CCR9-CCL25 axis in CD8+ T cell migration into the brain

Zachary T. Hilt, Megan A. Steinhilber, Brian D. Rudd

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY

Congenital human cytomegalovirus (CMV) infection can induce severe neurologic disease in newborns, resulting in hearing loss, seizures, and other neurodevelopmental deficits. Because human congenital CMV is hard to study, we use a mouse model to study the dynamics of the immune response. While it is known that CD8+ T cells infiltrate the brain during congenital CMV infection, it is still unknown the mechanism by which CD8+ T cells enter the brain. CD8+ T cells in the brain express high level of chemokine receptor 9 (CCR9), a known gut-homing mechanism. Chemokine ligand 25 (CCL25) is the specific ligand to CCR9. The aim of this study was to investigate if T cells utilize the CCR9-CCL25 axis to enter the brain. The hypothesis of this study was that blocking any of the steps in this signaling pathway would reduce trafficking of T cells to the brain; specifically, administration of anti-CCL25 blocking antibody would reduce immune cell trafficking to the brain as anti-CCL25 antibody treatment has been shown to reduce trafficking of immune cells to the gut in various immune-associated gastrointestinal conditions in humans. Mice were infected with MCMV-gB and trans- genic CD8+ T cells that respond to gB were transferred into a congenic host at birth (D0). Pups were given anti-CCL25 antibody (5 ug), administered intraperitoneally every 3 days, while controls received an IgG antibody. At 17 days post-in- fection, the experimental anti-CCL25 antibody treatment group had significantly fewerT cells in their brain compared to the IgG control. Current plans for continued evaluation of the importance of this pathway in T cell trafficking to the brain involve future use of CCR9 knock-out and dominant negative retinoic acid receptor mice.

Research Grant: R21142382 Student Support: Cornell University College of Veterinary Medicine, NIH T35 OD010941

2021 National Veterinary Scholars Symposium • 323 Factors affecting vitrification of canine epididymal spermatozoa

Serena Stoddard, Jean Linn, Alemayehu Lemma, Gemechu Wirtu

Department of Biomedical Sciences, College of Veterinary Medicine (CVM), Tuskegee University, Tuskegee (TU), AL

Vitrification is an emerging method of sperm cryopreservation that provides advantages over slow freezing, including the ability to perform the procedure with minimal equipment. Hence, there is a growing interest in understanding the factors that affect sperm survival during vitrification & devitrification.We evaluated the effects of selected cryoprotectant agents on the cooling & cryosurvival of canine epididymal spermatozoa after vitrification.Testis (n = 5 testis) were collected from the Alabama Animal Alliance & TUCVM. Semen samples were extracted from the cauda epididymis in Tris based extender (Pipan et al 2020), centrifuged, resuspended, cooled, & frozen in the following treatments: 1) Tris/sucrose (TS), 2) TS + 1 mg/ml myo-inositol (MI), 3) TS + 1% poly-vinyl alcohol, 4) TS + egg yolk (TSEY), 5) TSEY + 1 mg/ml MI, 6) TSEY + 0.5% glycerol, 7) TSEY + 1.5 % glycerol, & 8) TSEY+ 2.5% glycerol. After resuspension, samples were cooled to 4°C for 30 mins, & pre-freeze motility was determined using IVOSII Computer-Assisted Sperm Analysis. Samples were then vitrified by dropping 50 mL into liquid nitrogen from 11 cm & pellets were stored for > 1 day. Pellets were de- vitrified in 200m l of extender at 428C. Pre-freeze and post-thaw treatment effects on percent motility were analyzed using ANOVA in SPSS. Treatments had no effect (P = 0.876) on pre-freeze percent total motility (range: 47.4 to 66.3%); how- ever, they affected (P = 0.05) the post thaw total motility [range: 0.7% (Trt 3) to 7.5% (Trt 5)]. We conclude that supple- mentation of vitrification media with egg yolk but not low concentration of glycerol has beneficial effects on the post-thaw survival of canine epididymal spermatozoa.

Research Grant: HRSA #D34HP00001, TU-CBR/RCMI #U54MD007585 Student Support: HRSA #D34HP00001

Effects of receptor tyrosine kinase inhibition on canine glioma cell linesin vitro

Brianna Stoyle, Kevin Woolard

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California

Dogs develop spontaneous glioma brain tumors at a similar incidence and with remarkable histologic similarity to hu- mans. Currently, no standard-of-care exists for canine gliomas, and surgery and radiation are the only available options for treatment. Recent work reveals that genomic amplification of receptor tyrosine kinases (RTKs) are some of the most common events driving tumor formation in dogs, particularly platelet derived growth factor alpha (PDGFRA). Toceranib phosphate (Palladia ) is a tyrosine kinase inhibitor (TKI) that blocks autophosphorylation of RTKs through competitive in- hibition of ATP and preferentially inactivates PDGFRA. In this study we characterize the activity of several RTKs in four canine glioma cell lines, determine IC50 dose-response curves for toceranib in canine glioma cell lines, and assess tocer- anib’s effect on canine glioma cell proliferation and RTK activity in vitro. We hypothesize that PDGFRA activity drives proliferation in a subset of canine glioma cells, and that treatment with toceranib inhibits cell growth. This highlights a potential adjuvant therapy for for canine glioma patients that is more accessible and financially feasible than surgery and radiation.

Research Grant: CCAH 2019-16-F: Tyrosine Kinase Receptor Inhibition in Canine Glioma. Center for Companion Animal Health. University of California Davis School of Veterinary Medicine. Student Support: Students Training in Advanced Research (STAR) Program, funded by UC Davis SVM Endowment funds

324 • 2021 National Veterinary Scholars Symposium Vocal characteristics of an Atelopus zeteki breeding colony

Stephanie L. Straw, Alan Ziegler, Tobias Riede

Department of Physiology, College of Veterinary Medicine, Midwestern University, Glendale, AZ and The Maryland Zoo in Baltimore, Baltimore, MD

Frog populations are rapidly declining worldwide due to habitat loss, climate change, and pathogens such as the chytrid fungus. The Panamanian golden frog (Atelopus zeteki) has not been seen in its native habitat since 2009 and can be stud- ied only in human care. The investigation of physiology and behavior of its remaining populations is critical for success- ful breeding. Male A. zeteki use conspicuous forelimb movements, as well as mating calls to communicate visually and acoustically. The purpose of this study was to better understand the importance of vocal signals in A. zeteki. Analyses of variance were performed on five call properties (call duration, pulse interval, primary and secondary dominant frequency, and fundamental frequency) measured in 20 calls from 13 male frogs (6 from a highland and 7 from a lowland popula- tion) housed at the Maryland Zoo in Baltimore. We quantified individual- and population-specific differences in male mating calls. Acoustic features were correlated with body size, which was measured as body mass and snout-vent length. Four acoustic properties varied significantly between individuals and three call properties showed significant population differences, providing robust cues to differentiate the two populations.Two spectral properties varied depending on body size. The field of conservation bioacoustics can elucidate useful properties for our understanding and protection of imper- iled species. Here, we were able to differentiate individual calls from two frog populations. Body-size related differences represent a source of between-male differences. In sum, these properties have the potential to play a role in the acoustic discrimination by competing males or potential female mates.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Determining the threshold of serum progesterone to diagnose pregnancy in moose using ROC Analysis

Madeline S. Struck, William J. Severud, Seth A. Moore, and Tiffany M. Wolf

Department of Veterinary Population Medicine (Grotz, Severud, Wolf), College of Veterinary Medicine, University of Minnesota, Saint Paul, MN; Grand Portage Band of Lake Superior Chippewa (Moore), Grand Portage, MN

Pregnancy determination is necessary for sound management of animals and understanding population dynamics. While rectal palpation is the gold standard test in large animals, false negatives are possible if the pregnancy is very early. It can be especially challenging to get a veterinarian on site to perform it if the target is a free-ranging animal, as is the case with the moose population of Grand Portage, Minnesota. Serum progesterone quantification is also frequently used in many species to determine pregnancy status. A threshold of 2 ng/ml of progesterone in serum has been widely used by moose biologists to indicate pregnancy but has not been rigorously investigated. In order to develop a more precise threshold, we examined relationships between serum progesterone and pregnancy and calving data. We analyzed archived serum samples from 87 moose with capture dates spanning from 2010 to 2020 from 23 males and 64 females. Pregnancy was confirmed via rectal palpation of females (n = 25), necropsy (n = 2), calf observation (n = 25), or a characteristic pre-calv- ing movement (n = 6), with a total of 58 moose determined pregnant and 29 moose not pregnant (6 cows and 23 males). We utilized a receiving operator characteristic (ROC) analysis to quantitatively determine the optimal threshold of 0.48 ng/mL with an accuracy of 0.93 (95% confidence limits 0.86, 0.97). Progesterone levels were significantly higher in cases of multiple births than in singleton births (P < 0.05). Developing a reliable method for determining pregnancy status via serum progesterone levels will allow wildlife managers to assess pregnancy rates and fecundity of moose without devot- ing substantial time and resources to palpation and calf monitoring.

Research Grant: Van Sloun Foundation, U.S. Fish and Wildlife Service Tribal Wildlife Grant, U.S. EPA Great Lakes Restoration Initiative, and the Bureau of Indian Affairs Endangered Species Program Student Support: University of Minnesota, College of Veterinary Medicine, Office of Graduate Programs

2021 National Veterinary Scholars Symposium • 325 Optimizing the detection of persistent parvovirus infections in zoo-housed red pandas (Ailurus fulgens)

Eric L. Stubbs, Charles E. Alex, Kenneth A. Jackson, Patricia A. Pesavento

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA

Persistent (life-long) Amdoparvovirus infections are common in many carnivore species. We have established that 50% of US zoo-housed red pandas (Ailurus fulgens) are infected with the recently discovered Red Panda Amdoparvovirus (RPAV). Host-specific APVs can be pathogenic in other species, so the high prevalence of RPAV in this endangered species is concerning. We hypothesize that RPAV causes persistent infections that can be associated with disease. An accurate, sensitive, and reliable method of detection is needed to understand the population-level health impact of RPAV in red pandas. We developed and validated a SYBR Green-based quantitative PCR (qPCR) assay targeting a 121-nt-long fragment of the viral capsid gene. Initial applications of this assay have so far supported two studies. The first study used a single cohort of infected animals in a prospective analysis of persistent infection and shedding. A 4-year longitudinal fecal shedding survey demonstrated consistent viral shedding from two clinically normal animals for the duration of the study period. We also demonstrated fecal shedding from two previously RPAV-negative red pandas after they were introduced to an infected cohort. The second study is a retrospective analysis of RPAV-associated disease. In this study, qPCR is used to detect RPAV infections in archived red panda necropsy tissues, and followed with cell and tissue distribution studies by in situ hybridization (ISH). Combined results demonstrate RPAV is present in putative sites of asymptomatic persistence (spleen, intestine) and in association with significant lesions including myocarditis, tubulointerstitial nephritis, pharyngitis, and interstitial pneumonia.

Research Grant: This research is funded in part by a philanthropic grant from the McBeth Foundation via the San Diego Zoo Wildlife Alliance Student Support: UC Davis School of Veterinary Medicine Students Training in Advanced Research (STAR) Grant

Evaluating basal arrhythmia incidence in a colony of telemetered non-human primates

Jessica C. Suarez, Nicholas D. Ether, Derek J. Leishman, Marc B. Bailie, and D. Adam Lauver

Department of Pharmacology and Toxicology, College of Veterinary Medicine, Michigan State University, Lansing, MI

Sudden death caused by ventricular arrhythmias comprises 15-20% of all global deaths. In the 1990s and early 2000s, the FDA removed several drugs from the market due to increased risk of ventricular arrhythmias like Torsades de Pointes related to prolongation of the QT interval. As a result, new drugs must undergo an extensive preclinical evaluation to determine their effect on the electrocardiogram (ECG). The focus of our work is to improve preclinical cardiovascular safety studies by examining the incidence of arrhythmias after the administration of dofetilide, a class III antiarrhythmic drug that causes QT prolongation. We hypothesize that the incidence of ventricular arrhythmia will increase as the dose of dofetilide increases, correlating to the pharmacokinetic plasma exposure curve. To test our hypothesis, we analyzed 48- hour ECG recordings of eight cynomolgus monkeys at four different doses of dofetilide for irregular ECG waveforms us- ing the data analysis software Ponemah. The recordings were separated into one-hour bins, and we examined the quantity of ventricular arrhythmia per bin for each animal. The incidence of arrhythmia was compared for each dose. We observed a dose-dependent increase in ventricular arrhythmia in dofetilide-treated primates. The data suggest increased plasma concentrations of dofetilide induce more ventricular arrhythmias and that the data from these preclinical studies accurately recapitulates the clinical response of dofetilide in causing arrhythmias. Future studies are designed to evaluate the degree of QT interval prolongation in these subjects.

Research Grant: None Student Support: NIH Grant 5R25HL103156-11 to Michigan State University

326 • 2021 National Veterinary Scholars Symposium Analysis and culture results of free-catch urine samples in dogs

Monica M. Suero, Wendy W. Mandese, Sarah S. Beatty, Subhashinie Kariyawasam, Francesca C. Griffin, Penelope S. Reynolds

Department of Small Animal Clinical Sciences (Suero, Mandese, Griffin) & Department of Comparative, Diagnostic & Population Medicine (Beatty, Kariyawasam), College of Veterinary Medicine, University of Florida, Gainesville, FL; Department of Anesthesiology (Reynolds), College of Medicine, University of Florida, Gainesville, FL

Urinalyses are a common diagnostic procedure in small animal practice; however, the degree of contamination from free catch collection lacks rigorous study. Results from free catch urinalyses are susceptible to diagnostic uncertainty due to contamination. The gold standard for urinalysis is a cystocentesis, due to reduced contamination. As such, it’s valuable to know if this benefit outweighs the benefits associated with free catch.This study sought to assess degree of bacterial con- tamination between two methods of free catch collection: cleaned and uncleaned. Dogs were randomized to either the con- trol group or experimental group; the peri-vulvar or preputial region of dogs in the experimental group was cleaned with gauze soaked in sterile saline prior to urine voiding. A midstream, free catch sample was collected, analyzed, and cultured. The samples were cultured on MacConkey and blood agars to assess for different bacterial growth. Patients with UTI were excluded from contamination analysis. Preliminary results include 47 samples: 24 patients were allocated to the experi- mental cohort and 23 were randomized to the control. Preliminary results show no difference in pH between the groups (P = .18), no difference in the presence of bacterial colonies between the experimental and control groups (P = 1.00), and no difference in contamination in dry mount analysis (P = 0.75). Contamination between cleaned and uncleaned cohorts was statistically insignificant. Therefore, sterile saline preparation before free catch urine collection confers no benefit. Contin- uation of this study to increase sample size-amplifying statistical power-and exploration of additional methods to reduce contamination in free catch collection are recommended.

Research Grant: Fear Free Research Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program

Evaluating Ovine Models for Human Reprometabolic Syndrome

Elizabeth Sullivan, Dilyara Murtazina, Jeremy Cantlon, Kelly Kirkley, Terry Nett, Colin Clay, Christianne Magee

Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

Irregularities in body condition and metabolic state can negatively impact the reproductive capability of both domestic animals and humans. Obesity-related reproductive dysfunction in women, recently termed Reprometabolic Syndrome, is characterized by reduced secretion of gonadotropins and sex steroids during the menstrual cycle. It is hypothesized that this is a result of reduced pituitary sensitivity to Gonadotropin Releasing Hormone (GnRH), leading to blunted and delayed secretion of downstream gonadotropins, specifically luteinizing hormone (LH). Evaluating sensitivity to GnRH at the level of the pituitary gland is not feasible in humans. The strength of using an ovine model is the ability to measure the LH surge in vivo in real time and to utilize primary ovine pituitary cells in vitro to begin to determine the mechanism by which gonadotropes are less sensitive to GnRH. The ovariectomized (OVX) ewe offers a unique system to control for estradiol (E2) while studying how obesity and obesity-related factors, such as increased circulating free fatty acids (FFA) and insulin, modify pituitary sensitivity to E2 and GnRH. Our group has demonstrated that obesity delays and blunts the E2 induced LH surge in OVX ewes. In an ongoing study, we mimick the obese state by infusing normal weight OVX ewes with FFA (0.15g/kg/hr), insulin (4mU/kg/min) and evaluate the LH response after GnRH (10 mg iv). Additionally, we are quantifying the in vitro effect of FFA on gonadotrope-specific gene expression in both primary ovine pituitary and immortalized murine pituitary cells. These data set the stage for identifying the mechanism by which obesity interferes with normal E2 and GnRH signaling both in vivo and in vitro.

Research Grant: CVMBS College Research Council Grant - Magee, PI Student Support: USDA Animal Health and Disease Scholarship

2021 National Veterinary Scholars Symposium • 327 Vitamin D and parathyroid hormone during hospitalization in critically ill foals

Jamie Summers, Laura Hostnik, Hannah Kinsella, Hailey Snyder, Julia Horton, and Ramiro Toribio

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

Sepsis is a major cause of foal mortality and responsible for disorders of mineral homeostasis. Vitamin D is essential for calcium and phosphorus regulation, musculoskeletal health, and immunity. However, little is known about its role in disease progression and mineral dysregulation in critically ill foals. This project seeks to determine the dynamics of 25-hydroxyvitamin D and parathyroid hormone (PTH) concentrations over time in hospitalized versus healthy foals while associating metabolite changes with laboratory variables (e.g. calcium and phosphorus), disease severity and mortality. We hypothesize that during hospitalization, critically ill foals will have low 25(OH)D3 levels, increased PTH concentra- tions, and that persistent hypovitaminosis D will be linked to disease severity and an increased risk of mortality. Blood samples were collected from healthy, septic and sick non-septic foals of # 72 hours of age (n = 60) at 0 (admission), 24, 48, and 72h. Concentrations of 25(OH)D3, PTH, calcium, and phosphorus concentrations were measured using immuno- assays (ELISA) and chemistry/hematology analyzers. Data will be assessed for normality and analyzed by parametric and non-parametric statistical methods. Plasma 25(OH)D3 and PTH analysis is ongoing, with results and statistical analysis pending. Preliminary statistical evaluation of calcium and phosphorus concentrations between the septic, sick non-septic, and healthy foals have shown to be largely not significant. However, we have seen that the phosphorus concentrations in septic foals is lower than sick non-septic foals. This was determined using the Mann-Whitney rank sum test. Ongoing data analysis is necessary.

Research Grant: Dynamics of Vitamin D Metabolites in Healthy and Hospitalized foals (Equine Research Funds) Student Support: NIH T35 Training Grant T35OD010977

Effect of mRNA-expressed antibodies on in vitro attachment of T. foetus to bovine preputial keratinocytes

Ella Swales, Lauren Ellison, Merrilee Thoresen, E. Heath King, Darcie Sidelinger, Daryll Vannover, Hannah Peck, Philip J. Santangelo, Amelia R. Woolums

Department of Pathobiology & Population Medicine (Swales, Ellison, Thoresen, King, Sidelinger, Woolums), College of Veterinary Medicine, Mississippi State University, Starkville, MS; Wallace H Coulter Department of Biomedical Engineering (Vannover, Peck, Santangelo), Georgia Institute of Technology & Emory University, Atlanta, GA

The protozoal parasite Tritrichomonas foetus (Tf) infects the preputial epithelium of bulls. Infected bulls are asymptomat- ic carriers that cause recurring infections in cows, leading to costly reproductive failure. TF1.17, a Tf cell surface antigen, is integral for attachment to bovine preputial epithelium. We hypothesized that transfection of preputial epithelium with synthetic mRNA encoding antibodies against TF1.17 could help bulls resist or clear Tf infection. To establish conditions for this novel treatment, the objectives were 1) optimize fluorescent staining ofTf, 2) determine the optimal number of Tf supporting accurate quantification in cell attachment assays, and 3) assess attachment ofTf to primary preputial keratino- cytes (PPK) transfected with mRNA encoding antibody (IgG1) to TF1.17. Tf grown to 24-hour logarithmic phase were stained with CellTrace CFSE or ViaFluor 405 SE fluorescent stains at 5m M, 10 mM,15 mM, or 20 mM. Fluorescence was assessed at 3, 6, 12, and 24 hours. Zero, 250,000, 500,000, or 1,000,000 stained Tf were added to bovine kidney (BK) cells or transfected PPK in triplicate wells of 6-well plates. At 6, 12, 24, and 48 hours unattached Tf were washed off and attached Tf in 6 fields of each well were counted. Tenm M CellTrace CFSE provided the best staining for the necessary duration with least cytotoxicity. 500,000 Tf per well was optimal for attachment assays. Tf attachment to transfected cells was not clearly decreased, but attachment variability complicated assessment. Work is ongoing to optimize assessment of the effect of expressed antibody on Tf attachment.

Research Grant: CRIS Grant Funding (MSU-CVM, USDA Section 1433 Formula Funds) Student Support: NIH 5T35OD010432

328 • 2021 National Veterinary Scholars Symposium Monoclonal antibody optimization and nanoparticle vaccine development for Streptococcus pneumoniae

Adrianna K. Szostek, Aaron D. Gingerich, Jarrod J. Mousa

Center for Vaccines and Immunology (Szostek, Gingerich, Mousa) and Department of Infectious Diseases, College of Veterinary Medicine, Athens, GA (Mousa); Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, Athens, GA (Mousa)

Streptococcus pneumoniae (Sp), a pathogenic bacterium, causes otitis media, meningitis, bacteremia, and fatal pneumo- nia worldwide. 100 serotypes of Sp have been identified, however, commercially available vaccines only cover 13 or 23 serotypes. The bacterial capsule is a major virulence factor and a main target of vaccine development efforts. Antibiotic resistant strains and the increased incidence of non-vaccine serotypes necessitates more effective therapies and prevention strategies. Monoclonal antibodies (mAbs) that target conserved surface antigens of Sp have been shown to be protective, including pneumococcal histidine triad protein (PhtD). We have previously shown that the human mAb PhtD3 was protec- tive in a septic and a pneumococcal pneumonia mouse model against multiple serotypes. To further study mAbs to PhtD, large quantities of the mAb must be generated in the laboratory. First, we aimed to optimize antibody production in the laboratory by cloning the heavy and light chains of mAb PhtD3 into pcDNA3.4 plasmid. Validation from past experiments demonstrating the protective benefits of targeting PhtD and other pneumococcal antigens opens the door to test new vac- cines. Secondly, we aimed to create a nanoparticle vaccine candidate by using the SpyTag/SpyCatcher technology adorned with immunogenic proteins from Sp. The advantages of the SpyTag/SpyCatcher system include high yields, high thermal stability, and previous success displaying antigens from SARS-CoV-2, influenza A virus, and HIV. The pneumococcal pro- teins include pneumococcal surface protein A (PspA), pneumolysin (Ply), and PhtD3. By developing therapies that target prevention and treatment, we can reduce the incidence of invasive pneumococcal disease.

Research Grant: American Lung Association Student Support: Boehringer Ingelheim, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

Epitope based vaccine design: targeting HIV fusion peptide and membrane proximal external region

Tiare M. Takaesu, Ling Niu, Michael W. Cho

Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Elicitation of broadly neutralizing antibodies (bnAbs) against HIV-1 is a long-standing goal in HIV vaccine development. Currently substantial effort has been given to devise immunogens composed of HIV fusion peptide (FP) and membrane proximal external region (MPER) as various bnAbs targeting these two vulnerable sites have been isolated from pa- tients, including VRC34 and 10E8 respectively. An immunization schedule including FP based nano-particles and gp140 has been reported to induce bnAbs, however, the neutralization titer of sera is extremely low. Until now no research has reported that MPER based immunogens elicited bnAbs. In this study, we developed a simple peptide immunogen named FP-PADRE-C, which is composed of FP, a T cell epitope and C-terminal cysteine. Our results show that the immunization schedule including FP-PADRE-C and gp140 can induce strong antibody response against FP, however, the immune sera only showed low neutralizing activity against glycan deleted HIV pseudovirus, implicating that improved immunization strategy to steer the response to FP with more native conformation is needed. Protein complex is an important type of immunogen that can be used to generate required response, however, dissociation may occur after the complex is injected. In this study, we designed a fusion protein that includes MPER and a modified 10E8 single-chain variable fragment with greatly improved solubility, we call this protein MPER-v4. MPER-v4 is expressed in bacteria as an inclusion body, we are currently working on refolding and crystallizing MPER-v4. Once we obtain the crystal structure, the immunogenicity of MPER-v4 will be evaluated in mice and rabbits.

Research Grant: None Student Support: NIH T35 Training Grant

2021 National Veterinary Scholars Symposium • 329 Markers of endothelial injury in canine trauma patients: a pilot study

Jordan D. Tarbutton, Claire Tucker, and Kelly Hall

Critical Care Services, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado

The endothelial glycocalyx is a complex network of glycoproteins and proteoglycans on the surface of cells lining blood vessels throughout the body. When injury occurs, this glycocalyx surface sheds, releasing molecules such as syndecan-1, heparan sulfate, hyaluronan, and ve-cadherin. The extent of endothelial glycocalyx damage may play a role in the devel- opment of systemic inflammatory processes and multiple organ failure, causing long term complications and a worsening prognosis in trauma patients. Additionally, studies suggest the initial fluid resuscitation methods we commonly use today - such as large volume, rapid crystalloid boluses - may cause an increase in glycocalyx shedding and, ultimately, a poor- er outcome. The purpose of this pilot study is to determine whether these molecules can be measured in canine trauma patients and utilized as biomarkers of glycocalyx shedding. We hypothesize that in measuring these molecules, we can utilize them as biomarkers of glycocalyx degradation in canine trauma patients. Canine patients presenting to the Colora- do State University Urgent Care service for a recent traumatic injury will have these molecules measured at various time- points after injury. They will be classified into mild, moderate or severe injury based on a validated trauma injury score. This information will inform the design of future studies to compare various resuscitation methods and their effects on the glycocalyx leveraging translational aspects of trauma patient care.

Research Grant: Internal Funding Student Support: CSU Clinical Sciences

IgE and sCD14 as Potential Biomarkers for Culicoides Hypersensitivity in Icelandic Horses

Justine Tarsillo, Ellie Larson, Heather Freer, Bettina Wagner

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY

Culicoides hypersensitivity is a common insect allergy in horses that causes pruritus, alopecia, and skin irritation. During an allergic reaction, chemoattractant proteins called chemokines are secreted by cells. Detection of specific chemokines and other molecules in the bloodstream can act as biomarkers in early response stages. Soluble CD14 (sCD14) is a protein that is cleaved from the surface of monocytes and macrophages during early immune responses, causing a pro-inflam- matory response. Cul hypersensitivity causes a widespread inflammatory response and thus we hypothesized that sCD14 cleavage may be increased in allergic horses. IgE is another biomarker candidate for Cul allergy because it is an IgE me- diated type I hypersensitivity. Some studies have described allergy detection using total serum IgE, but others have found little difference in total serum IgE between allergic and healthy horses. This study aims to identify possible biomarkers and characterize the role of specific chemokines in the pathogenesis ofCul hypersensitivity. A bead based immunoassay was used to measure IgE and sCD14 levels in serum and plasma samples of 8 severely allergic, 2 mildly allergic, and 12 non-allergic Icelandic horses over 18 months. sCD14 levels were not elevated in allergic horses, suggesting that sCD14 activation is not a major immunological response to Cul allergy. We also surveyed 41 Icelandic horses that were exposed to Cul midges at different ages to investigate the environmental component ofCul allergy development. Horses exposed to Cul allergens as adults developed allergy at a higher rate than horses exposed as neonates. We conclude that environmen- tal factors strongly contribute to Cul allergy development in horses.

Research Grant: None Student Support: Cornell University College of Veterinary Medicine and Boehringer Ingelheim

330 • 2021 National Veterinary Scholars Symposium Temporal relationship of fever and positivity on nasal and blood PCR in a 2016 outbreak of EHM in California

S.L. DeNotta, K. Flynn, K. Hatch, A. Pelzel-McCluskey, D.S. Tartaglia, J. Traub-Dargatz

College of Veterinary Medicine (DeNotta, Tartaglia), University of Florida Gainesville, FL; Department of Agriculture (Flynn), Kentucky State University, Frankfort, KY; California Department of Food and Agriculture (Hatch), Sacramento, CA; USDA-APHIS Veterinary Services (Pelzel-McCluskey), Fort Collins, CO and College of Veterinary Medicine and Biomedical Sciences (Traub-Dargatz), Colorado State University, Fort Collins, CO

Outbreaks of equine herpesvirus myeloencephalopathy (EHM) are difficult to mitigate, and biosecurity strategies rely on clinical monitoring of at-risk horses through twice daily temperature recording and PCR testing of whole blood and nasal swabs at the time of initial fever or neurologic disease detection. To release horses from quarantine as soon as possible, horses are often sampled at the first sign of a fever very early in the course of disease. Health officials involved in miti- gating EHM outbreaks have indicated they observed initial tests to be negative and have required follow-up samples be tested. This retrospective investigation will be conducted by investigating records obtained by the California Department of Food and Agriculture. These records were reviewed to obtain information regarding the number of horses on the prem- ises, individual exposure, quarantine duration, timing of initial fever, sample collection, and diagnostic test results. Of the 725 horses housed on the affected premise, 330 horses were considered exposed and subsequently quarantined. Fifteen horses were confirmed positive for EHV-1 on PCR of blood and/or nasal swab at least at one timepoint. Of the 15 positive horses, 8 had signs consistent with EHM. Six horses were negative on PCR of nasal swab and/or whole blood samples at the time of initial fever detection, and positive on retest 24-72 hours later. Main limitations stem from the retrospective nature of the study of one outbreak incident. Our goal is to collect similar data from multiple outbreaks to determine if this observation is repeatable. Despite these restrictions this investigation illustrates the unique temporal relationship EHM has on diagnostic assays.

Research Grant: Florida Veterinary Scholar Program at UFCVM, Boehringer Ingelheim Veterinary Scholar Program, and Dr. Sally Anne DeNotta Student Support: None

Epidemiology and risk factors for Sarcocystis neurona infection in juvenile California sea lions from 2012-17

Anna Taylor, Karen Shapiro, Andrea Packham, Magdalena Plancarte, Patricia A. Conrad, Woutrina A. Smith, Cara Field

School of Veterinary Medicine, University of California Davis, Davis, California (Taylor, Shapiro, Packham, Plancarte, Conrad, Smith), The Marine Mammal Center, Sausalito, California (Taylor, Field)

Sarcocystis neurona is a protozoan parasite that can infect a wide range of terrestrial and aquatic mammals. Sometimes sarcocystosis manifests as encephalopathy; however, in California sea lions (Zalophus californianus), S. neurona has been noted to manifest as polyphasic rhabdomyositis. Studies of sea otters suggest that marine mammals may be exposed to S. neurona through land-to-sea transmission of sporocysts shed by the definitive host, theVirginia Opossum (Didelphis virginiana). A previous study reported a low (1.2%) seroprevalence to S. neurona in stranded California sea lion pups and yearlings from 1998-2009. However, The Marine Mammal Center (TMMC, Sausalito, CA) has observed an increase in cases of sarcocystosis in stranded California sea lions along the Northern and Central California coast in recent years. This study aimed to investigate whether exposure to the parasite has increased during the past decade and to evaluate potential risk factors associated with S. neurona infection. Archived serum samples from stranded California sea lions were strati- fied by age-sex class and tested for antibodies toS. neurona using indirect fluorescent antibody testing (IFAT). Preliminary results show a marked increase in crude prevalence of exposure to S. neurona in stranded California sea lion pups and yearlings admitted to TMMC between 2012 and 2017 compared to the previous study period (1998-2009). Demographic, environmental (algal blooms, river flow), and climatic (sea surface temperature, precipitation) risk factors for exposure to S. neurona are currently being evaluated.

Research Grant: None Student Support: Students Training in Advanced Research (STAR), UC Davis School of Vet Med Endowment Fund

2021 National Veterinary Scholars Symposium • 331 Bone’s black hole: comparing the osteolytic mechanisms of hemangiosarcoma of the bone with multiple myeloma

Corrine E. Thomas, Bahaa A. Fadl-Alla, and Timothy M. Fan

College of Veterinary Medicine (Thomas, Fadl-Alla, Fan) and Cancer Center at Illinois (Fan), University of Illinois at Urbana-Champaign, Urbana, IL

Multiple myeloma (MM) is a well-studied, aggressive cancer of plasma cells (a type of white blood cell) that can prolif- erate in the bone marrow of humans. In canines, hemangiosarcoma (HSA) of the bone presents similarly to MM, being predominantly osteolytic in nature. HSA has a high mortality rate and is a common, invasive cancer of endothelial cells (a type of cell that lines blood vessels) that can also proliferate in the bone. Clinically, both bone cancers compromise the structural integrity of the affected bone resulting in similar signs of spiral bone fractures and/or osteolytic lesions seen on radiography. Given their similar osteolytic presentation, canine HSA of the bone may adopt a MM-like mechanism of action. This study uses literature on the mechanism of MM in humans to focus analysis of canine HSA of the bone on spe- cific features of the mechanism. Receptor Activator of Nuclear Factor-kB-Ligand (RANKL) activates osteoclasts, which are cells that break down bone, while Osteoprotegerin (OPG) inhibits RANKL to create a balance of bone remodeling in healthy bones. In severe cases of MM, the RANKL/OPG ratio is increased to favor bone resorption through increased osteoclast activation. The RANKL/OPG ratio for canine HSA cells was analyzed, using total RNA extraction followed by RT-PCR, and calculated. In MM, interleukin (IL)-1beta, IL-3, and IL-6 are responsible for osteoclast activation while IL-7 is responsible for osteoblast inhibition, and together these effects favor osteolysis.These interleukins were analyzed using sandwich ELISA tests to determine their expression from canine HSA cells. This information may lead to new treatments and/or earlier methods of detection of canine HSA of the bone.

Research Grant: Internal funding Student Support: Office of the Director, NIH, T35 OD011145

Protein modification by oxidative fatty acid degradation products in a mouse model of Alzheimer’s Disease (AD)

Haley Thomas, Paul H. Axelsen

Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, PA

Oxidative stress is a prominent feature of Alzheimer’s Disease (AD), but its role in disease pathogenesis is poorly un- derstood. It has been suggested that lipids and proteins both contribute to the pathogenesis of AD, and specifically that the oxidative degradation products of docosahexaenoic acid (DHA) are neurotoxic. These products may cause neuronal loss without amyloid (Ab) fibril formation. We hypothesize that the Ab peptides that aggregate as fibrils in AD cause an increase in the oxidative degradation of DHA, leading to an increase in production of 4-hydroxy-2-hexenal (HHE), a DHA oxidative degradation product. We aim to test this hypothesis by characterizing the rate of oxidative DHA degradation in transgenic mice that accumulate aggregated Ab peptides. U-14C-radiolabeled DHA was prepared by growing Crypthe- codinium cohnii in U-14C- glucose. Purified DHA will be injected into a lateral ventricle of the mouse brain, and the distribution of radiolabel will be examined 3 days later to quantify the extent to which U-14C-radiolabeled DHA has been degraded into oxidation products and the extent to which proteins have become radiolabeled by U-14C- DHA degradation products.

Research Grant: None Student Support: NIH T35 OD010919

332 • 2021 National Veterinary Scholars Symposium Renal effects of diltiazem continuous infusion in healthy dogs

D. Angela Thompson-Butler, Megan Kelley, Matthew Tanner, Justin Thomason, William Whitehouse

Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

There are several medications available for patients with acute kidney injury (AKI) that optimize glomerular filtration rate (GFR) and urine output (UOP); however, many fail to have positive effects on both GFR and UOP in dogs or their use is cost prohibitive. Diltiazem may offer an inexpensive alternative treatment forAKI, especially in states of oliguria or anuria, but has not been adequately studied in dogs. The objective of this study is to determine if an intravenous (IV) continuous rate infusion (CRI) of diltiazem increases GFR, UOP, and fractional excretion of sodium (FeNa). A prospec- tive, randomized, crossover study was used with a study population consisting of 5 healthy dogs at least 1 year of age. Baseline screening diagnostics including GFR measurement by iohexol clearance was performed within 30 days of the first infusion. Dogs were randomized to receive diltiazem 0.24 mg IV loading dose followed by a CRI at 6 mg/kg/min or equal volume 5% dextrose in water (D5W) for 300 minutes followed by the opposite treatment one week later. GFR was measured after diltiazem was at steady state for 60 minutes. Samples for FeNa were taken immediately before and during the last hour of the CRI. GFR and FeNa did not change significantly from baseline after receiving diltiazem or D5W, nor did they differ significantly between the diltiazem and D5W groups. UOP did not change significantly with D5W; -how ever, there was a significant increase in UOP with diltiazem (P = 0.0391). An increase in UOP is suggestive of beneficial renal effects of diltiazem; however, a larger study population is needed.

Research Grant: ACVIM Resident Research Grant, Kansas State University Mark Derrick Research Fund Student Support: Morris Animal Foundation Veterinary Student Scholars Program

Developing a model of aldosterone producing adenomas for ablation therapy to eliminate hyperaldosteronism

Sarah Timmerman, J. Sebek, TB. Shrestha, MA. Highland, I. Mali, M. Payne, G. Zia, N. Zeiali, SH. Bossmann, M. O’Halloran, MC. Dennedy, P. Prakash, Matthew Basel

Depts of Anatomy and Physiology (Timmerman, Shrestha, Basel), Clinical Sciences (Highland), Electrical and Computer Engineering (Sebek, Zia, Prakash), Chemistry (Payne, Mail) Kansas State Univ, Manhattan, KS; Univ of Kansas Med Ctr (Bossmann), Lawrence, KS; National Univ of Ireland Galway (O’Halloran, Dennedy), Galway, Ireland

Aldosterone-producing adenomas are benign adrenal tumors responsible for half of primary aldosteronism cases. Standard care for unilateral disease is adrenalectomy, but bilateral disease requires medical management with significant side ef- fects. Targeted microwave ablation may destroy the adenomas while sparing healthy tissue leading to better outcomes. 12 nude mice (nu/nu) were injected subcutaneously with 6 million HAC15 cells into the right hind flank and allowed to grow to 5 mm. Mice were separated into test and control groups. Both groups were anesthetized but only the test group was ablated. A 14 Tesla MRI was used to align an experimental microwave probe and monitor volumetric temperature profiles during ablation (defined as 558C as measured at 4 points within the tumor by MRI thermometry). 24 hours post-ablation, angiotensin II injections were given to stimulate aldosterone production and blood was collected 4 hours later, post-eu- thanasia. The tumor was harvested, stained with TTC and H&E, and analyzed blindly by pathologists. TTC viability stain suggested that 5/6 mice were successfully ablated. Blinded verification by H&E stain indicated that of the 6 ablations: 4 were complete, 1 was partial, and 1 was unsuccessful, likely due to poor targeting. Functional effects of the ablated tumors will be determined by serum aldosterone levels currently being analyzed by Birmingham University (UK). Results show that when treated with microwave ablation at a temperature of at least 558C for one minute, the tumors are successfully ablated resulting in complete necrosis. Work is ongoing to create a more homogenous and predictable HAC15 model by increasing the number of cells injected and using matrigel as a cell-matrix.

Research Grant: NIH R01EB028848 Student Support: Boehringer Ingelheim Veterinary Scholars Program

2021 National Veterinary Scholars Symposium • 333 Interrogating the gene regulatory network underlying pro-T cell fate allocation using CRISPR and scRNA-seq

Victoria Tobin, Boyoung Shin, Tom Sidwell, and Ellen Rothenberg

School of Veterinary Medicine, University of California Davis, Davis, California (Tobin), Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California (Tobin, Shin, Sidwell, Rothenberg)

How cells make fate decisions is a fascinating yet poorly understood process. Using T cell development as a model sys- tem, we hope to better understand the principles of how developmental lineage choices are made. When hematopoietic precursors migrate to the thymus to acquire T cell identity, they initially maintain multipotentiality and express non-T associated genes. As cells progress through development, they commit to the T cell fate and give up alternate options. Bcl11b is a T cell transcription factor that is turned on precisely at T cell commitment. Upon deletion of Bcl11b in pro-T cells, cells maintain some immature features, including high expression of the surface marker cKit, and are more likely to upregulate NK-associated genes. Bcl11b-regulated genes have been identified, including two notable repression targets, Id2 and Zbtb16, which encode transcription factors that promote innate lymphoid cell development. Our goal is to dissect how Bcl11b repression of Id2, Zbtb16 and Kit affects the developmental trajectory of pro-T cells. We used CRISPR to delete Bcl11b by itself or together with Id2, Zbtb16 or Kit in the same cells, and are analyzing their developmental states by flow cytometry and single-cell RNA sequencing (scRNA-seq). Flow cytometry results suggest that upon deletion of Bcl11b, fewer cells stay on the T-pathway when compared to wild type cells, especially when deleted in combination with Kit. As expected, Bcl11b deficient cells also maintain high expression of cKit. Ongoing work using scRNA-seq will reveal changes in the developmental trajectories at finer resolution. Together, this work will offer insight into the mechanisms by which Bcl11b repression plays a role in acquiring T cell identity.

Research Grant: NIH R01AI135200, Cancer Research Institute Irvington Postdoctoral Fellowship CRI.SHIN Student Support: NIH T35 OD010956

Surveillance of Borrelia burgdorferi in white-footed mice (Peromyscus leucopus) in Pennsylvania

Trey Tomlinson, Erica Machtinger, Brian Herrin

Department of Diagnostic Medicine/Pathobiology (Tomlinson, Herrin), College of Veterinary Medicine, Kansas State University, Manhattan, KS; Department of Entomology (Machtinger), Pennsylvania State University, University Park, PA

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent vector-borne disease found in the United States. Direct disease transmission to humans and other species occurs through the feeding of infected Ixodes ticks. Due to the ongoing expansion of its tick vector, there is a need for robust surveillance programs to continually monitor B. burgdorferi prevalence in wildlife reservoirs as this directly impacts disease incidence and influences control programs.To aid in this effort, this study focuses on identifying the prevalence ofB. burgdorferi in wild Peromyscus leucopus, a known Lyme reservoir, from an endemic area in Pennsylvania. A total of 378 blood and 272 tissue samples will be analyzed for the presence of the bacterium using a nested polymerase chain reaction (nPCR) targeting Flagellin B gene. Positive sam- ples are sent for sequencing and those sequences compared to known B. burgdorferi references on GenBank. Currently, 12 out of the 112 blood samples have shown to be PCR positive giving an initial prevalence of 10.7%. Positive sequences have most closely matched B. burgdorferi sensu stricto. More samples are continually being processed with tissue samples soon beginning the nPCR phase of the protocol. When compared to previous studies, this study offers the advantage of being able to compare the detection differences seen in blood and tissue samples obtained from the sample population of P. leucopus to determine the ideal sample for surveillance.

Research Grant: Unknown Student Support: NIH T35 Training Grant T35OD029981

334 • 2021 National Veterinary Scholars Symposium The pathology and incidence of building-caused bird mortality

Alanna Tonelli-Raylove, Christopher Olson, and Jason D. Struthers

Department of Pathology and Population Medicine, College of Veterinary Medicine (Tonelli-Raylove, Struthers), Midwestern University, Glendale, AZ; Department of Physiology, College of Graduate Studies (Olson), Midwestern University, Glendale, AZ

Window collisions are a major cause of mortality among wild birds. To understand window-caused mortality and increase successful treatment outcomes, we determined the pathology from window collisions and the incidence of building-caused bird mortality on Midwestern University’s Glendale campus. We found that window-killed birds have characteristic gross pathology, most of which results in death. Eighty-four window-killed birds, from 18 species, were collected over 15 weeks in 2021, yielding 77 reliable postmortem examinations to record sustained lesions and determine cause of death. We found that birds die from injuries of blunt force trauma sustained by and/or translated through their pectoral girdle/muscle area. Specifically, 83% had lower airway injury, 81% had hepatosplenic injury, and 47% had cardiovascular pathology. Skele- tal fractures were also common, specifically pectoral fractures (49%). In contrast, only 14% of birds had cranial injuries, suggesting that cranial injury is less common. Sixty (78%) of the examined birds had fatal pathologies from two or more of the aforementioned categories and, in five cases (8%), it was primary skeletal pathology that caused fatal visceral inju- ries. Understanding the cause of death of window-killed birds will improve treatment of injured birds admitted to wildlife rehabilitation centers, as not all window collisions are immediately fatal. The incidence of window collisions to identify troublesome areas among large buildings will also provide further insight into future remediation. Thus, subsequent work will investigate the role and design of large buildings and their effect on avian mortality.

Research Grant: 2021 Intramural Student One Health Research Grant Award Student Support: Boehringer Ingelheim Veterinary Scholars Program

Modulation of chicken macrophage function by microbial catabolites of tryptophan

Jessica Topka, Solomon O. Odemuyiwa

Veterinary Medical Diagnostic Laboratory, Dept. of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO

In mammals, catabolites of the essential amino acid tryptophan, collectively called kynurenines (KYN), play a crucial role in the development of regulatory T cells. Similarly, microbial metabolism of tryptophan generates other immunoactive catabolites that modulate cytokine secretion by acting as endogenous ligands of aryl hydrocarbon receptors (AhR). Macro- phages react to perturbations of tissue environment, including tryptophan catabolites, by secreting appropriate cytokines that orchestrate adaptive immune response. The objective of this study is to determine whether tryptophan catabolites gen- erated by members of the genus , an abundant group of bacteria in the chicken gut, will affect the transcription of cytokine genes in chicken macrophages. The HD-11 chicken monocyte-macrophage cell line will be treated with differ- ent concentrations of TDD, a synthetic ligand of AhR. Cells will also be treated with the following tryptophan catabolites: FICZ, indole-3-propionic acid (I3Pr), and indole-3-aldehyde (I3Al) for 4, 8, 16, or 32 hours. Expression of mRNA specific for AHRR, CYP1A1, CYP1B1, IL4iL, IFN-alpha, IL-1, IL-6, and IL-10 will be measured using real-time PCR. We expect that treating the macrophages with these catabolites will alter mRNA expression for these targets. Such results would indicate that microbiota-derived tryptophan catabolites have immunomodulatory effect on chicken macrophages.Thus, chemical manipulation of tryptophan catabolism in the gut could be studied further as a potential approach to recapitulate the immunomodulatory and growth-promoting effects of probiotics in chickens.

Research Grant: None Student Support: University of Missouri College of Veterinary Medicine Office of Research

2021 National Veterinary Scholars Symposium • 335 Bead enrichment of prion protein using RT-QuIC for diagnosis of Chronic Wasting Disease in live animals

Ashley N. Torres Saez, Estela Kobashigawa, and Michael Z. Zhang

Department of Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO

Chronic Wasting Disease is a progressive and non-treatable disease affecting cervids in North America, Korea, and Scan- dinavia. It is a member of the Transmissible Spongiform Encephalopathies caused by misfolded proteins called prions that spread through contaminated bodily fluids, soil, and water. Prions convert the normal host-encoded proteins by promoting a structural change leading to protein aggregate formation in the nervous system. Since the clinical signs are often unde- tected, diagnosing an animal with CWD is done postmortem with immunohistochemistry (IHC) and enzyme-linked im- munosorbent assay (ELISA) on tissues with high levels of prions, such as retropharyngeal lymph nodes (RLN) and obex. However, these samples are difficult to acquire antemortem. As a result, real-time quaking-induced conversion (RT-QuIC) assays are being established to detect prions in live animals. The objective of this research is to further develop a highly sensitive RT-QuIC assay that accurately detects low levels of prions in platelet samples. This is done by RT-QuIC analysis of diluted samples using recombinant peptide beads bound to protein aggregates that are then identified through the fluo- rescent properties of Thioflavin T. The analyzed samples have a postmortem CWD diagnosis by ELISA and IHC testing of RLN. We have obtained results comparable to the IHC and ELISA-positive animals, revealing a higher reaction rate in the 10-2 dilutions with an average of 30 hours and 50 minutes for ThT detection of aggregates above the calculated threshold. Further development of this assay presents as a means of early CWD detection in herds living in prion-infected areas. As such, it could be an essential diagnostic tool for this fatal disease.

Research Grant: Missouri Department of Conservation: Development of a MEMS-Based Biosensor and RT-QuIC Assay for the Detection of CWD Prion Student Support: Endowment established by IDEXX-BioAnalytics

Setting the standard: optimization of diagnostic testing methods for avian bornavirus

Ty Tortella, Caitlin Mencio, Donald J. Brightsmith, Sarah Hamer

Schubot Avian Health Center, College of Veterinary Medicine and Biomedical Sciences, Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas

Avian bornavirus (ABV) is a virus that affects the nervous and digestive systems of many species of birds. Much is un- known about ABV transmission and disease progression as it is difficult to monitor the diseasein vivo. PCR and ELISA are common tests for ABV viral RNA and antibodies, respectively. However, they are often used for a “yes or no” result. The 1st aim of this study was to determine the optimal method for quantitative tracking of virus and antibody production. Secondary aims of the study were to see if bicinchoninic assay (BCA), a protein assay, could be used to screen for ABV and determine if ELISA could be used to examine maternal antibody transmission in egg yolks. PCR and ELISA dynamic ranges were established using serial dilutions of viral plasmid and the most positive serum sample currently available, respectively. For PCR, the lower limit of detection was 0.5 ng of plasmid, and the upper limit was 3.0 ng of plasmid. For ELISA, the lower limit used a 1:512,000 dilution, while the upper limit of detection, an absorbance of 4, required 1:1,000 dilution. The BCA was used on different ELISA positive serum samples to look for correlation between protein quantity and ABV antibody. No correlation was found. IgY was extracted from cockatiel egg yolk using PBS and then tested by ELISA for ABV specific antibodies. A 20% positivity rate was found among eggs (n = 82) collected from breeding cocka- tiels who were negative for ABV by PCR and ELISA at all points tested in life, but share a room with previously positive birds. The impact of this research is the standardization of testing methods for ABV infection and recognizing the need for further research into potential vertical transmission of ABV or its antibodies.

Research Grant: Pat Palmer Foundation and Schubot Center for Avian Health Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Science

336 • 2021 National Veterinary Scholars Symposium Effect of autologous serum on corneal fibrosis in equine corneal stromal fibroblasts

Caroline G. Treadwell, Lynn M. Martin, Praveen K. Balne, Elizabeth A. Giuliano, Rajiv R. Mohan

Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO (Treadwell, Martin, Balne, Giuliano, Mohan) Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO (Mohan) Mason Eye Institute, School of Medicine, University of Missouri, Columbia, MO (Mohan)

Corneal injuries are among the most common equine emergencies often resulting in permanent corneal fibrosis and loss of vision or necessitating enucleation due to secondary infectious keratitis. A common therapeutic strategy used for corne- al ulcers is topical administration of autologous serum to inhibit collagenases that result in keratomalacia. However, the effects of serum on the development and/or prevention of corneal fibrosis are unknown.The objective of this study is to determine if serum applied to equine corneal stromal fibroblasts (ECFs) stimulated withTGF-ð ›½ in vitro alters produc- tion of profibrotic and antifibrotic biomarkers. We hypothesize that treatment with autologous serum will reduce profibrot- ic and increase antifibrotic biomarkers in vitro. Equine serum and corneas will be collected from healthy adult horses from which ECFs will be cultured. Four treatment groups will be studied: naive negative control (CON), TGF-ð ›½ positive control (TGF-ð ›½), autologous serum naive (SER), and TGF-ð ›½ and autologous serum (TGF-ð ›½ + SER). Immu- nocytochemistry will assess known fibrosis biomarkers ð ›¼-SMA and fibronectin while qPCR will assess ð ›¼-SMA, fibronectin, SMAD-2, and antifibrotic biomarker SMAD-7. The expected outcomes include no change of biomarkers in the CON group, an increase of profibrotic biomarkers in the TGF-ð ›½ group, no change of biomarkers in the SER group, and a decrease of profibrotic and increase of antifibrotic biomarkers in theTGF-ð ›½ + SER group. These results would support the effectiveness of autologous serum to prevent or reduce the development of corneal fibrosis as a sequela after complicated equine corneal ulcers.

Research Grant: Ruth Kraeuchi Missouri Endowed Chair Ophthalmology Fund Student Support: Boehringer Ingelheim

Evaluation of 3D printed Tendon Scaffolds for tissue engineering

Payton Tripp, Alberto Gines

Laboratory for 3D Printing and Regenerative Medicine (Tripp, Gines) Department of Veterinary Clinical Sciences (Gines), School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Tissue engineering is a subcategory of regenerative medicine aimed at repairing or replacing damaged and impaired tissues. Currently, in veterinary medicine, there are no biological substitutes for tendons, and when a tendon or ligament sustains a severe injury, the current surgical options involve the use of synthetic implants. The use of bioprinting in tissue engineering is opening new doors for the possible treatment of different pathologies.A 3D printed scaffold should act in place of the target tissues natural extracellular matrix providing some initial strength and a solid platform for cellular inte- gration and future cell culture. The aim of this study is to design and evaluate the mechanical properties of three different scaffold designs using a universal tension testing machine to determine which design, if any, maintains optimal modulus of elasticity (MPa) and maximum load (N). The hypothesis of this study is that of the three different designed scaffolds, the honeycomb modified scaffold will support higher forces to failure compared with the grid and gyroid scaffolds.All three scaffolds will support more than 40% of control construct (based on the infill used to create the scaffolds). Initial descriptive analysis of the results demonstrates that the honeycomb and gyroid scaffolds support a maximum load higher than 40% the control scaffold, 40% infill for honeycomb and gyroid, or mean of 62.06 N and 32.19 N respectively com- pared with the mean 79.83 N of the control scaffold. The modulus of the three scaffold patterns (TO BE CONFIRMED) were within that of a normal tendon. Statistical analysis is pending.

Research Grant: None Student Support: National Institute of Health, LSU Veterinary Summer Scholars Program

2021 National Veterinary Scholars Symposium • 337 Female dorsal raphe serotonin responses to sexual stimuli

Eileen L Troconis, Changwoo Seo, Akash Guru, Caitlin H. Miller, Caleb C. Vogt, and Melissa R. Warden

Department of Neurobiology and Behavior, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY

Brain serotonin-a widely-projecting neuromodulatory system-has been implicated in a variety of emotional brain states and behaviors, including mating. Previous work has shown that pharmacological compounds that interact with the se- rotonin system can modulate female sexual behavior. However, whether female serotonin neurons normally respond to sexual stimuli during mating is unknown. Here, we used fiber photometry to record population calcium activity of dorsal raphe serotonin neurons in the female mouse brain during mating. We found that this neuronal population becomes active in the female at the time of ejaculation. To understand which stimuli during ejaculation trigger this neuronal activity in the female, we developed surgical techniques that perturb various aspects of ejaculatory physiology in males with intact mating behavior. First, we tested the role of ejaculatory fluid release into the vagina by redirecting the male urethra to a stoma in the abdominal wall. Then, we tested the role of penis erectile function by partially transecting a pair of muscles that control penis behavior. We found that the female elevated serotonin neuronal activity at ejaculation disappeared only when her mating partner had undergone both modifications in combination. Moreover, artificial mechanical vaginal stim- ulation was sufficient to trigger serotonin neuronal activity in the female.Together, these results suggest that both penis behavior and release of ejaculatory fluid are important mechanical stimuli to which female dorsal raphe serotonin neurons respond during ejaculation. Detecting ejaculation may be crucial for regulating various aspects of female sexual behavior and reproductive physiology.

Research Grant: NIH Director’s New Innovator Award conferred to Melissa Warden Student Support: NIH Director’s New Innovator Award conferred to Melissa Warden

Optimization of Qubit dsDNA high sensitivity assay for quantification of cfDNA in equine abdominal fluid

Mary-Grace Trogdon, Emily Martin, Kira Epstein, Rosemary Bayless

Department of Large Animal Medicine (Trogdon, Martin, Epstein), College of Veterinary Medicine, University of Georgia, Athens, Georgia; Clinical Sciences (Bayless), College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Neutrophil extracellular traps (NETs) are amalgam of decondensed chromatin, histones, and antimicrobial granules that are rapidly released from neutrophils in times of inflammation and/or infection. Gastrointestinal disease triggers a variable inflammatory response within the abdomen dependent on pathogenesis. NETs in abdominal fluid may serve as a potential early indicator of inflammation associated with gastrointestinal lesions. However, quantification of NETs within equine abdominal fluid has not been reported. The objective of this study is to validate the Qubit double-stranded DNA assay for detection and quantification of cell free DNA (cfDNA) in abdominal fluid for future use in clinical cases of gastrointesti- nal disease as a marker for NET formation. We hypothesize that the assay is an efficient and accurate method to quantify cfDNA in equine abdominal fluid samples. Assay validation was performed by determining coefficients of variability on abdominal fluid samples spiked with known amounts of exogenous double stranded DNA and coefficient of determination on serial dilution of pooled abdominal fluid collected from healthy horses. Coefficients of variability# ( 14.7) and deter- mination (R2$0.98) were both acceptable. Abdominal fluid samples from 6 healthy horses were tested in replicate from a single tube to determine intra-assay variability and from multiple aliquots to determine inter-assay variability (coefficients of variability # 6.7). These results demonstrate that the Qubit is an efficient and accurate method to detect cfDNA in -ab dominal fluid and provide the basis for further analysis in a clinical setting to determine whether a cfDNA-based diagnos- tic test is useful for equine colic.

Research Grant: Equine Colic Research Fund, Department of Large Animal Medicine, UGA College of Veterinary Medicine Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

338 • 2021 National Veterinary Scholars Symposium Role of CT226 in Chlamydia trachomatis infection

Madison Tryzbiak, Hollis C. Holcomb, Jennifer Shaw, and Erika Lutter

College of Veterinary Medicine (Tryzbiak), Oklahoma State University, Stillwater, OK; Department of Microbiology and Molecular Genetics (Holcomb, Lutter), College of Arts and Sciences, Oklahoma State University, Stillwater, OK; Department of Bio-Medical Sciences (Shaw), PCOM South Georgia, Moultrie, GA

Chlamydia trachomatis, an obligate intracellular bacterial pathogen, is the most common cause of preventable blindness and sexually transmitted bacterial infections. Chlamydial infections can lead to chronic conditions, such as pelvic inflam- matory disease, tubal infertility, ectopic pregnancy, and HPV-associated cervical cancer even after treatment. Despite ongoing research there are still major gaps in understanding the host-pathogen pathogenesis of Chlamydia, specifically with regards to immune response. Recent studies, and work in our lab, have shown that a Chlamydial protein, CT226 interacts with key host signaling pathways that regulate the inflammasome. Given the previous information, we hypothe- size that CT226 modulates host immune responses through the inflammasome. Using the murine cervicovaginal infection model established for Chlamydial infections, C3H/HeJ mice (n = 12 per group X 2 groups) were intravaginally infected with C. trachomatis L2 or mutant C. trachomatis L2-ΔCT226 in a dose of 1x108 EBs. Mice were given a dose of 2.5 mg of medroxyprogesterone 7 and 3 days prior to the infection to synchronize estrous. On days 3, 7, 14, 21, 28 and 35 post in- fection, intra-vaginal swabs will be collected, where each swab will be serially diluted and cultured in HeLa cells, stained with Chlamydia specific antibodies, and the quantity of infection forming units will be counted to ascertain if L2-ΔCT226 has a similar infection phenotype to the original L2 wildtype strain. At day 42 the reproductive tracts will be harvested and placed into formaldehyde for eventual histo-pathology analysis to determine if there are differences in inflammation between the strains during infection. No results have been collected at this time.

Research Grant: NIH 1R15AI149439-01 Student Support: OSU Summer Research Training Program Boehringer Ingelheim Veterinary Scholars Program

The effect of peroxynitrite on fibrinogen binding to integrins expressed by THP 1 monocytic cells

Jackson Tucker, Zimu Wei, Dr. James Luyendyk

Department of Pathobiology and Diagnostic Investigation(Tucker, Wei, Luyendyk), Michigan State University, East Lansing, MI.

Fibrinogen is a primary component of blood clots and engages with leukocytes to modify the inflammatory response. Fibrinogen interacts with white blood cells via cell surface b2 integrin receptors. During the blood clotting process, the protease thrombin cleaves fibrinogen to form a fibrin polymer (i.e.,blood clot), a structural change that simultaneously enables b2 integrin engagement. During tissue injury, fibrinogen can be modified by the reactive nitrogen species per- oxynitrite (PN), and. prior studies have shown that high concentrations of peroxynitrite inhibits fibrinogen polymerization. However, the impact of this oxidative modification on fibrinogen-integrin engagement is unknown. I tested the hypothesis that peroxynitrite modification of fibrinogen decreases leukocyte adhesion.To test this hypothesis, tissue culture plates were coated with 10 mg/ml fibrinogen, peroxynitrite-modified fibrinogen, or vehicle (phosphate-buffered saline, PBS), and the adhesion of THP-1 monocytic cells to each surface quantified using a fluorescent DNA-detecting dye (Cyquant) after 25 minutes of incubation. As anticipated, very few cells adhered to vehicle-coated wells, whereas a dramatic increase in THP-1 cell adhesion was observed on fibrinogen-coated wells. Surprisingly, a similar number of THP-1 cells adhered to peroxynitrite-modified fibrinogen when compared to normal fibrinogen..The results indicate that peroxynitrite modifica- tion of fibrinogen has no significant effect on THP1 cell adhesion even at concentrations of peroxynitrite that dramatically reduce fibrin polymerization.

Research Grant: NIH R01 DK120289 and R25 Grant Student Support: BRUSH Program, Office of Undergraduate Research, Michigan State University, East Lansing, MI

2021 National Veterinary Scholars Symposium • 339 A “sub-Lyme” system: Uncovering the protective role of anti-inflammatory NLRX1 against Lyme disease

Juselyn D. Tupik, Mecaila E. McClune, Julianne M. Dressler, Brandon L. Jutras, Irving C. Allen

Department of Biomedical & Veterinary Sciences in the Virginia-Maryland College of Veterinary Medicine (Tupik, Allen) and Department of Biochemistry (McClune, Dressler, Jutras), Virginia Tech, Blacksburg, VA; Department of Basic Science Education (Allen), Virginia Tech Carilion School of Medicine, Roanoke, VA

Lyme disease, caused by the bacterium Borrelia burgdorferi, is an enigmatic disease of pressing concern in the field of immunology. Once transmitted to a host, the bacterium promotes chronic inflammation of the joints inducing Lyme arthri- tis. Immune processes underlying this inflammation have not been fully elucidated, making it important to define these mechanisms to develop new therapies for Lyme disease. Pattern Recognition Receptors (PRRs) serve as the first line of host innate immune defense against pathogens and operate by sensing Pathogen-Associated Molecular Patterns (PAMPs). Intracellular PRRs known as the NOD-Like Receptors (NLRs) have been implicated in the recognition of Borrelia, with pro-inflammatory NLRs instigating inflammation promoting Lyme arthritis. However, the role that anti-inflammato- ry NLRs play in mitigating this inflammation has not been elucidated. Here, we studied the role of anti-inflammatory NLRX1 during Lyme disease by using novel knockout mouse models. We infected wildtype and Nlrx1-/- mice with a mod- erate dose of B. burgdorferi for 30 days. We found that Nlrx1-/- mice exhibited significantly earlier, more severe arthritis than wildtype mice. This suggests that NLRX1 serves to attenuate inflammation during Lyme arthritis. This increased inflammatory phenotype was further reflected inNlrx1 -/- mice and macrophages in response to the Borrelia outer mem- brane layer peptidoglycan (PG) and genomic (g)DNA. These results indicate that Borrelia PG and gDNA could serve as effectiveBorrelia PAMPs for NLRX1. Ultimately, these results indicate that NLRX1 plays a protective role in mitigating Lyme arthritis, warranting a further need to define NLRX1’s mechanism of activation during Lyme disease.

Research Grant: Cohen Foundation and NIH/NIAID R21AI159800 Student Support: Virginia-Maryland College of Veterinary Medicine

Modulation of chicken and equine peripheral lymphocytes using honeybee apitherapy

Jeffrey T. Turn, Joerg Mayer, Koichi Nagata, Kristina Meichner, David J. Hurley, Eric Koslowski, Robert M. Gogal Jr.

Small Animal Medicine & Surgery (Turn, Mayer, Nagata), Department of Pathology (Meichner), Department of Population Health (Hurley), and Department of Biomedical Sciences (Turn, Gogal), College of Veterinary Medicine, University of Georgia, Athens, GA, and ETR Laboratories, Inc., Leominster, MA (Koslowski)

Apitherapy is a form of alternative medicine that utilizes products from the western honeybee (Apis mellifera), including honey, propolis, and bee venom, to improve the health status of patients by modulating host immunity. Major gaps in the data exist regarding the impact of apitherapy on immune cells, and minimal studies exist in veterinary species. In this in vitro pilot study, honey, propolis, and bee venom were cultured with equine and chicken peripheral blood lymphocytes. Blood samples from each species underwent leukocyte enrichment procedures to optimize lymphocyte purity. Enriched lymphocytes were then cultured with honeybee by-products to assess the influence on proliferation, cell cycle, cell viabil- ity/apoptosis, and cell morphology. In chickens, lymphocyte proliferation was significantly increased with honey (0.1 & 0.2%) and venom (0.1 & 1.0 mg/mL) but decreased with propolis (1 & 100 mg/mL). In horses, lymphocyte proliferation was increased with propolis (1 & 10 mg/mL) and venom (0.1, 1, & 10 mg/mL). Cell viability was increased in chicken lymphocytes with venom (0.1, 1, and 10 mg/mL), while the % gated viable cells were increased with propolis (1, 10, and 100 mg/mL) and % gated and viable gated cells were increased with venom (0.1, 1, and 10 mg/mL). Horse lymphocyte cell viability was not significantly affected, however, % gated cells were increased with propolis (10 & 100m g/mL). Cell morphology is currently being evaluated. These preliminary results suggest that honeybee by-products can differentially alter peripheral blood lymphocyte function based on veterinary species.

Research Grant: DOD W911NF-20-P-0022 [or] The Georgia Beekeepers Association [or] The Office of the Dean of UGA College of Veterinary Medicine [or] The Department of Small Animal Medicine and Surgery Student Support: UGA Foundation, Veterinary Medical Experiment Station, UGA College of Veterinary Medicine

340 • 2021 National Veterinary Scholars Symposium Speciation of the etiologic agent of canine leproid granuloma syndrome using PCR on FFPE tissue samples

Skyler Turner, Courtney Meason-Smith, Aline Rodrigues Hoffmann

College of Veterinary Medicine, Mississippi State University, Starkville, MS (Turner) and Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX (Meason-Smith, Rodrigues Hoffmann)

Canine leproid granuloma syndrome (CLGS) has a widely recognized, classic presentation and has been documented around the world. The etiologic agent inciting the condition has yet to be determined beyond suggestion of it belonging to the Mycobacterium genus. This study seeks to confirm that the CLGS agent is a novelMycobacterium species and to characterize it by phylogenetic analyses. Eight previously published PCR primer pairs targeting various regions of the bacterial genome were evaluated and amplicon sequences were compared with those cataloged in the NCBI BLAST da- tabase. These amplicon sequences were also subjected to phylogenetic analyses with better characterized members of the Mycobacterium genus. One pair of PCR primers targeting the mycobacterial ITS-1 gene generated high quality amplicon sequences that allowed for correct identification of agents from several known mycobacterial diseases. PCR using these primers was performed on 35 samples from 26 CLGS cases diagnosed by histology, and 58% (15/26) of cases generated bands of sufficient quality to be sequenced. At least one case sequence exhibited 100% homology with a published but unnamed sequence from a previous CLGS study, and M. simiae was the closest (96%) established species to this CLGS sequence. These results confirm that the CLGS agent is a member of theMycobacterium genus, and the preliminary sequence results suggest that this agent-species is the same as that of previous studies, which likely represents a novel species. Further study will include shotgun sequencing of selected cases to assemble the whole genome of this potentially novel pathogen.

Research Grant: None Student Support: Boehringer Ingelheim VSP, Texas A&M College of Veterinary Medicine and Biomedical Sciences

NRP2 signaling in malignant peripheral nerve sheath tumors

Kyleigh Tyler, Heather Shive

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

Malignant peripheral nerve sheath tumors (MPNST) are soft tissue sarcomas that have a notoriously poor response to con- ventional chemotherapy/radiation. Signaling pathways that contribute to MPNST pathogenesis are not yet fully defined. We are investigating the role for the neuropilin receptor NPR2 in MPNST growth, migration, and invasion. Neuropilins are cell surface receptors that function as co-receptors for various transmembrane receptors and interact with multiple types of ligands. NRP2-associated signaling can promote or repress cancer development depending on available ligands and co-receptors. One such ligand is semaphorins 3F (SEMA3F), which interacts with NRP2 and its binding partners PlexinA1/A2. Semaphorins are extracellular signaling proteins that play a role in many pathways of growth and develop- ment, including tumorigenesis. In carcinomas, SEMA3F interacts with NRP2 and co-receptors PlexinA1/A2 to repress cancer progression. On the other hand, interactions between NRP2 and other receptor-ligand combinations can promote carcinoma progression. The role of NRP2 signaling is unknown for MPNST. We are using human MPNST cell lines to de- termine how NRP2 and SEMA3F modify MPNST phenotypes. We determined that MPNST cell lines and patient samples express NRP2. We generated MPNST cells with stable knockdown of NRP2 and 293TN cells with stable expression of SEMA3F. We are assessing the effect of NRP2 knockdown on proliferation by serial Cyquant assays, apoptosis by cleaved caspase 3/7 assay, and migration and invasion by transwell migration assay. We are also assessing the effects of SEMA3F on MPNST cell migration by co-culture with 293TN cells expressing SEMA3F.

Research Grant: NIH ORIP K01OD021419; OSU CCC startup funds Student Support: NIH T35 Training Grant T35OD010977

2021 National Veterinary Scholars Symposium • 341 Preliminary evaluation of intravenous iron sucrose in healthy cats

Rebecca A. Ullom-Minnich, Abby McKisson, Brandy Kastl, Maria C. Jugan

Departments of Veterinary Clinical Sciences (Ullom-Minnich, McKisson, Jugan) and Diagnostic Medicine/Pathobiology (Kastl), College of Veterinary Medicine, Kansas State University, Manhattan, KS

Iron plays critical roles in oxygen transport and erythropoiesis, yet many diseases impair gastrointestinal iron absorption. Intravenous (IV) iron sucrose is a safe and effective alternative to oral supplementation in humans but has not been stud- ied in cats. This pilot study evaluated IV iron sucrose safety and its impact on hematologic indices in cats. Five healthy research cats were administered iron sucrose (0.5 mg/kg IV) over 30 minutes. Monitoring for acute reactions (tempera- ture, heart rate, respiratory rate, blood pressure) was performed every 5 minutes during injection and every 15 minutes for an additional hour. Hematologic measurements included baseline, 24-hour, and 1, 2, and 3-week post-injection CBC with reticulocyte indices, iron panel, and serum amyloid A (SAA). While no cat experienced an acute reaction, SAA was increased at 24 hours versus baseline (P = 0.016). Transferrin saturation (TSAT) was higher at 24 hours (26%) versus baseline (22%; P < 0.01) but then tended to decrease. Hematocrit (Hct) and hemoglobin (Hgb) were lower at week 3 versus baseline (P < 0.01), with 3 cats developing anemia and functional iron deficiency (FID). Median ferritin was lower on week 2 (169 ng/mL) versus baseline (314 ng/mL; P = 0.003). Hct, Hgb, and reticulocyte Hgb content correlated with serum iron and TSAT. Overall, IV iron sucrose was well-tolerated in healthy cats. Future studies should evaluate efficacy and doses based on iron deficit in sick cats. Despite cumulative blood draw volume below recommended limits, iatrogenic anemia and FID were observed, which has important implications for experimental designs and serial monitoring in sick cats.

Research Grant: Kansas State University College of Veterinary Medicine, Mentored Clinical, Applied, or Transitional Research Grant Student Support: Boehringer Ingelheim Veterinary Scholars Program and the Chet Peterson Family

Isolating canine natural killer cells for development of a novel inhibitory SCT

Christian Urbina, Sarmistha Banerjee, Antonia Rotolo, and Nicola Mason

The Department of Clinical Sciences and Advanced Medicine (Urbina, Rotolo, Mason), School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; The Department of Biomedical Science (Banerjee), School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Missing self theory presents a critical problem for potential universal chimeric antigen receptor T-cells (UCAR-Ts) be- cause they are engineered to lack MHC class I on their surface, thus putting them at risk of destruction by the recipient’s natural killer cells (NK cells). In human UCAR-T studies, one solution being explored is the addition of a single chain trimer (SCT) derived from the non-classical MHC molecule, HLA-E. The SCT aims to deliver an inhibitory signal to NK cells to prevent cytolysis of edited T cells. For this theory to be tested using a canine SCT (peptide-b2M-DLA-79 SCT), a pure canine NK cell population must be obtained and a reliable cytotoxicity assay is needed to evaluate function. We hypothesized that a culture depleted of CD5 expressing T cells would yield the purest and most robust NK cell popula- tion. NK cells were identified by the NKp46+/CD3- phenotype and function demonstrated by lysis of MHC class I null K562 cells. Experiments using MACS separation and flow cytometry over a 21 day period comparing CD5 selected, CD5 depleted, and unmanipulated apheresis product showed that CD5 depleted cultures produce the purest cell population (> 65%) that demonstrate the phenotypic and cytolytic characteristics of NK cells. A cytotoxicity assay using CellTraceTM Violet and 7-AAD to detect lysis of K562 cells at 3 different effector to target ratios confirmed the population’s cytotox- icity against K562 cells. This generation of relatively pure and viable NK cells marks a vital step in enabling the study of missing self in canine UCAR-Ts. Following these experiments, SCTs will be transduced into K562 cells and b2M knock- out canine T-cells to evaluate the SCT’s protective properties against cultured NK cells.

Research Grant: U54: Engineering the Next Generation of T-Cells Student Support: NIH T35 OD010919 Training Grant

342 • 2021 National Veterinary Scholars Symposium Detection of tick-borne pathogens by immunohistochemical (IHC) staining

Sabrina Valdes, Mariano Carossino, and Udeni B. R. Balasuriya

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Tick-borne pathogens are a significant concern for human and animal health. As tick-borne diseases become more prev- alent, it is crucial to identify the viral, bacterial, and parasitic pathogens transmitted by ticks in Louisiana. Thus, it is important to develop new tests to diagnose various pathogens in ticks rapidly and accurately. Identifying pathogens’ tissue and cellular location in ticks is vital to better understand the disease transmission mechanisms. Ticks lack a circulatory system, so pathogens must migrate through the tissues and cells via another method. Hemocytes, cells analogous to white blood cells, are believed to be responsible for translocation of pathogens from the midgut to other tick tissues, such as reproductive organs and salivary glands. In this study, we standardized immunohistochemistry (IHC) staining procedures for formalin-fixed, paraffin-embedded (FFPE) tick sections based on various antigen-retrieval (AR) techniques to identify the tissue and cellular localization of Rickettsia. First, we stained whole tick sections with H&E to better understand tick anatomy. We found that internal structures are well preserved in Davidson’s Fixative and are easily visualized with H&E fixation. Subsequently, we stained experimentally infectedAmblyomma maculatum (The Gulf Coast Tick) whole tick sections with anti-Rickettsia antibody (RcPFA) using Leica Bond-Max fully automated system. The development of these techniques sets the groundwork for future studies to investigate the tissue and cellular distribution of tick pathogens. It also allows us to better understand the cellular mechanisms of disease transmission within the tick.

Research Grant: LSU SVM Startup Funds to Dr. Udeni Balasuriya (PG2165) Student Support: Kenneth F. Burns Trust and LSU SVM

Evaluating accuracy of two 3D surface scanners when compared to anatomical measurements of canine pelvic limbs

Max Veenema, Alberto Gines

Laboratory for 3D Printing and Regenerative Medicine (Veenema, Gines), Department of Veterinary Clinical Sciences (Gines), School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana

Conventional bandage immobilization of the distal aspect of the canine pelvic limb requires the use of a cast or splint, which can lead to common complications such as skin irritation, ulcers, and in some cases poor immobilization. Custom casts can be created by obtaining a mold of the limb, however this usually takes several weeks to be perfectly fitted to the patients due to the inaccuracy and inefficiency of this method. The emerging technology of 3D scanning and 3D printing should be able to quickly produce a more accurate mold for custom cast fabrication which could lead to fewer compli- cations associated with conventional immobilization practices. The objective of this study was to evaluate the precision of two different scanners: a low-cost 3D scanner (Sense 3D scanner from 3D Systems) and a high precision wireless 3D scanner (Leo wireless scanner by Artec 3D) when compared with manual measurements in the distal aspect of canine pelvic limbs. The limbs of 7 cadaver dogs were scanned by each scanner and time for scanning was recorded. Manual measurements of the perimeter and computer-generated measurements were performed at descending intervals of 25 mm. Initial results demonstrate that the high precision 3D scanner has an overall accuracy of 1.92% versus 7.38% for the low- cost scanner. Pending statistical analysis, the high precision wireless 3D scanner appears to create an accurate representa- tion of the limb that can be used for custom cast creation. The inaccuracy seen with the low-cost scanner warrants further analysis before this type of scanner can be used for creating accurate models of the distal pelvic limb in dogs.

Research Grant: National Institutes of Health (NIH) Student Support: None

2021 National Veterinary Scholars Symposium • 343 T cell activation induced by dendritic cell infection with Cryptosporidium parvum in serum-free media

Brayden J Veith, Jack Guinan, and Brina S. Lopez

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Several studies have investigated and reported on T cell activation in serum-supplemented media; however, T cell activa- tion after exposure to Cryptosporidium parvum (CP) by dendritic cells in a serum-free model has yet to be investigated. Benefits of using serum-free media include more consistent performance, better control over physiological responsiveness, and reduced risk of contamination by serum-borne adventitious agents in cell culture. Adult barren cattle were used for this experiment. Monocyte-derived dendritic cells (MoDC) were generated from whole blood using recombinant bovine GM-CSF and IL-4. Following exposure to CP, MoDC were co-cultured with fluorophore labeled T cells from unrelated cattle and cultured in a 50:50 mixture of conditioned medium (CP-exposed MoDC medium) and a commercially available serum-free medium for 3-4 days. T cell proliferation, cellular metabolism, cytokine secretion, and cellular phenotype were subsequently evaluated. T cell activation (CD25 expression) and proliferation were measured via flow cytometry (Guava easyCyte). Changes in cellular metabolism were evaluated via Seahorse XFe96. Cytokines were quantified via a commer- cially available bovine multiplex cytokine kit. The MoDC exposed to CP effectively stimulated T cells, as indicated by increased T cell proliferation and CD25 surface marker expression, and reprograming of cellular metabolism. Collectively, these findings suggest that MoDC stimulation of T cells contributes to cattle immunity during ex vivo CP infection. The use of serum-free medium to culture CP-exposed MoDC with T cells provides an ideal model for drug discovery, disease modeling, and CP therapy moving forward.

Research Grant: Midwestern University Faculty Fund Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Characterizing infectivity of Spondweni virus and Zika virus isolate in human placenta and neuronal cell lines

Jossette Velazquez and Matthew Aliota

Department of Veterinary and Biomedical Sciences (Velazquez, Aliota), College of Veterinary Medicine, University of Minnesota, Twin Cities.

Spondweni virus (SPONV) is a flavivirus and the closest relative to Zika virus (ZIKV).After ZIKV was introduced into South America, it was discovered that gestational infection with ZIKV caused debilitating congenital and neurological anomalies such as microcephaly and retinal defects. Together, these adverse pregnancy outcomes are termed congenital Zika syndrome (CZS). Similarly, recent findings indicate SPONV can cause fetal harm and also fetal demise, analogous to ZIKV. Despite SPONV and ZIKV displaying similar pathogenic potential, the molecular mechanisms used by these two viruses to harm the fetus remain poorly understood, but both must likely traverse the placental barrier in order to reach the fetus. The placenta is comprised of three different trophoblast cell types, which are defined by their functional and ana- tomical differences, and these differences may lead to altered susceptibility to viral infection among trophoblast cell types. This raises the question of whether or not there is phenotypic heterogeneity between SPONV and ZIKV including in their overall ability to infect and replicate in the placenta. Our study determined the capacity for SPONV and ZIKV to repli- cate in human placenta and neuronal cell lines. To measure viral burden and infectivity, plaque assays and growth curve experiments were performed. Since, SPONV and ZIKV are so closely related, our study may contribute to understanding its potential emergence risk to prevent another epidemic to pregnant women along with other mosquito-borne viruses.

Research Grant: NIH/NIAID: R01AI132563 Student Support: Supported by the NIH training grant T35OD011118

344 • 2021 National Veterinary Scholars Symposium In vivo radiation dose-response relationships for a syngeneic orthotopic murine model of oral carcinoma

Raisa Velez-Contreras, Karen L. Marcus, Michael W. Nolan

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University

Growth of Mouse Oral Cancer 2 (MOC2) cells in mice provides a powerful tool for studying oral squamous cell carci- noma (OSCC). Because OSCC is commonly treated with radiotherapy (RT), the objective of this study was to charac- terize the radiation dose-responsiveness of orthotopically grown MOC2 tumors in their syngeneic host. Female C57Bl/6 mice underwent intralingual injection of 4x105 MOC2 cells. Four days later, the resultant tongue tumors were irradiated (5 daily fractions of 0, 4.5, 5.5, 6.5, or 7.5 Gy; N = 6 mice per dose level) using an experimental irradiator. Mice were monitored daily, and euthanized when they had lost more than 20% body weight and presented inability to prehend food or water, dyspnea, facial wiping indicating pain, and if tumors reached measurement endpoint (6 mm or wider). Necropsy was performed to evaluate for metastasis. Tumors developed in 29/30 mice (97%). Subjectively, tumors were difficult to measure, and often associated with necrosis of adjacent tongue tissue. In sham-irradiated mice, tumor growth necessitated euthanasia by day 9. At 22.5 Gy, there was minimal initial tumor regression followed by progression by day 11 post-IR. Higher radiation doses did not cure tumors but did further delay tumor progression and prolong survival. One mouse had equivocal regional lymphadenomegaly, but necropsy identified no other evidence of metastasis in any mouse. Despite the aforementioned limitations, these results indicate a clear radiation dose-response relationship, and this work lays the groundwork for future experiments that are designed to test novel combinatorial therapies to improve the care of HNSCC patients.

Research Grant: This work was supported by a grant from the American College of Veterinary Radiology, philanthropic donations in support of the Nolan lab, and funds from NC State College of Veterinary Medicine Student Support: NIH-T-35 Interdisciplinary Biomedical Research Training Program (IBRTP)

Skeletal muscle fiber type changes contribute to muscle weakness in sheep hypophosphatasia

Constance Vernham, Joshua Bertels, Alyssa Falck, Shannon Huggins, Larry J. Suva, Dana Gaddy

Departments of Veterinary Integrative Biosciences (Vernham, Bertels, Gaddy), and Veterinary Physiology and Pharmacology (Huggins, Suva), College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Hypophosphatasia (HPP) is a rare inherited disorder that results from mutations in the tissue-nonspecific alkaline phos- phatase (TNSALP) gene (ALPL) that manifests with decreased mineralization and muscle weakness. Using CRISPR/ Cas9, a c.1077 C>G point mutation engineered in the Rambouillet sheep ALPL sequence recapitulated human HPP. Observations of 6 mo HPP mutant founder sheep revealed variable gluteal muscle fiber size and altered fiber types. We hypothesized that low TNSALP activity resulted in decreased oxidative fiber area to mediate muscle weakness in HPP. The purpose of this study was to determine if fiber type changes were consistent across muscle groups, and whether the diminished fiber size was unique to a fiber type. Gluteal, gastrocnemius, and soleus muscle biopsies were collected from 1 year old Wild type (n = 4) and Homozygous (n = 4) sheep and frozen sectioned. Myosin heavy chain Type 1 oxidative, Type 2a, and Type 2x glycolytic fiber types were detected by immunofluorescence and quantified. No consistent differ- ences were observed between genotypes in fiber size of any type in any muscle group; nor were significant differences in Type 2a fiber area observed. However, HPP sheep Type 1 fiber (slow twitch) area was diminished in gluteal muscle but increased in soleus muscle, and Type 2x fiber (fast twitch glycolytic) area was increased in soleus muscle. These fiber type changes across specific muscle groups are entirely consistent with a diminished oxidative capacity leading to generalized muscle weakness in HPP sheep. These different fiber type changes across muscle groups lead us to investigate the energy production in specific muscle groups to provide a more mechanistic basis for muscle weakness in HPP.

Research Grant: SoftBones Foundation, NIH R21-DE028076-01 Student Support: NIH T35OD010991-16, Texas A&M College of Veterinary Medicine & Biomedical Sciences

2021 National Veterinary Scholars Symposium • 345 Canine osteosarcoma cells induce pro-inflammatory gene expression in macrophages

Adriana V. Villasenor, Mike Povelaitis, Erika J. Gruber

Department of Population Health and Pathobiology, College of Veterinary Medicine, NC State University, Raleigh, NC

Macrophages are innate immune cells that adopt a wide variety of phenotypes, the extremes of which are pro-inflammato- ry (M1) or anti-inflammatory (M2). Many tumors reprogram macrophages to an immunosuppressive phenotype to evade immune destruction, thus we hypothesized that canine osteosarcoma induces an anti-inflammatory M2 phenotype. Aims of the study were to determine whether canine osteosarcoma cells 1) secrete signals that promote pro- or anti-inflammatory gene expression in canine macrophages, and 2) differ in their effect when stimulated with pro- or anti-inflammatory stim- uli. Abrams osteosarcoma cells were treated with or without pro-inflammatory (lipopolysaccharide [LPS], interferon-gam- ma [IFNg], or LPS/IFNg) or anti-inflammatory (IL-4) stimuli. After 24 hours, Abrams cell-conditioned media (Abrams- CM) was removed, filtered, and added to canine macrophage-like DH82 cells for 24 hours. Relative gene expression of macrophage M1 markers (TNFa, CXCL10) and M2 markers (TGM2, CD23) was measured by quantitative PCR (qPCR). We found that Abrams-CM significantly upregulated expression of TNFa and CXCL10 genes, but had no effect on TGM2 or CD23. Interestingly, conditioned media from IFNg-treated Abrams cells had no effect on TNFa or CD23, but induced a 1000-fold increase in CXCL10 and 5-fold increase in TGM2. Together, these data suggest that canine osteosarcoma cells induce a pro-inflammatory, rather than an anti-inflammatory phenotype in macrophages. Future studies will investigate the signals mediating this effect. These studies begin to tease apart the complex signaling between osteosarcoma cells and macrophages, with potential implications for the treatment of canine osteosarcoma.

Research Grant: NC State College of Veterinary Medicine Faculty Start Up Funds Student Support: NC State Veterinary Scholars Program; Boehringer-Ingelheim Veterinary Scholars Program

Canine otitis externa diagnosis and treatment

Kristopher Vine, Dr. Pamela Guy

Department of Small Animal Clinical Sciences. College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama

The tubular portion of the outer ear is called the ear canal, which carries sound to the eardrum. The most common disor- der of the canine ear canal is called otitis externa. Otitis externa occurs when the layer of cells that line the external ear canal become inflamed. Signs include headshaking, redness of the skin, swelling, scratching, scaly skin, and increased wax production. The dog may also be painful or itchy depending on the cause or duration of the condition. Otitis externa is caused by many different factors, such as parasites, foreign objects, and allergies that cause inflammation. Other etiolo- gies include bacteria, yeast or middle ear infection that perpetuate the condition. In order to properly treat otitis externa, a detailed history and thorough physical examination will need to be completed. Using an MDS-Vet endoscope will help to properly examine the ear canal and identify important structures of the ear such as the malleus, the eardrum, and the epi- thelium; which are key structures in diagnosing otitis externa. By examining the external ear canal of dogs and collecting ear swabs for cytology, the otic microflora can be evaluated. The aim of this study is to determine normal and abnormal microflora to aid in properly diagnosing and treating otitis externa.

Research Grant: TUCVM Summer Scholarship Student Support: HRSA COE Grant #D34HP00001

346 • 2021 National Veterinary Scholars Symposium Prescribing practices on the prevalence of methicillin-resistant S. pseudintermedius infections in dogs

Elizabeth Walsh, William Love, Cristina Lanzas

Department of Population Health and Pathobiology, College of Veterinary Medicine, NC State University, Raleigh, NC

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a pathogen of concern for dogs as it is commonly associated with urinary tract infections, pyoderma, and other skin diseases. Antimicrobial resistance is one of the biggest challenges in both human and animal health. The mecA gene confers resistance to methicillin and other commonly used b-lactam antibiotics in S. pseudintermedius, and co-occurrence of other resistance genes may allow for further selection of multi-drug resistant MRSP. The objective of the current study was to understand how previous antimicrobial prescriptions contribute to the development of MRSP infections. A retrospective cohort study of 153 records of patients with S. pseud- intermedius cultured between 2014-2016 were reviewed for antimicrobial prescriptions, date of prescription, and presence of mecA. Analysis was accomplished by logistic regression, and AIC was used for model selection and to compare fit of models with prescription time windows. Covariates not included for fit were assessed for confounding.The final model included patient age, sex, and weight in addition to a variable for any antimicrobial prescribed in the 30, 60, or 90 days before culture. Time frame did not appear to meaningfully change the model (AIC = 155.98, 156.13, 156.28 respectively). When comparing antimicrobial exposure at days 30, 60, and 90, no significant change was seen (p = 0.53 or higher) which suggests that recent treatment with an antimicrobial did not increase the risk of being colonized by MRSP as compared to mecA negative S. pseudintermedius. Further analysis will look at the relationship between specific drug classes and mecA positive Staphylococcus pseudintermedius.

Research Grant: FDA U01FD007057 Student Support: FDA U01FD007057 NC State Veterinary Scholars Program (VSP)

Behavioral impact and pharmacokinetics of transdermal fentanyl in healthy corn snakes

Benjamin Walter, Stephen Johnson, Kurt Sladky, Mary Thurber

Comparative Biosciences (Johnson), Surgical Sciences (Sladky, Thurber), School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin

Snakes are common household pets and are prominently featured in zoo collections. Geriatric snakes often develop os- teoarthritis, leading to low quality of life that leads to humane euthanasia. Snakes feel pain, but it has been difficult to find an analgesic drug that shows clinical efficacy using laboratory models.Anecdotally, transdermal fentanyl (TF) appears to cause vast clinical improvement (increased activity) in osteoarthritic snakes who are presumed to be in pain. Our research goals are: 1. to evaluate the impact of TF on normal snake behavior using constant video-monitoring to determine if TF-dependent changes are non-specific or possibly related to pain relief; and 2. to measure serum fentanyl concentrations in corn snakes during a 4-week TF application period to correlate fentanyl levels with behavioral changes. An ethogram was developed to compare behavior, such as time spent active or in the hide box, before and after TF application in the control snakes. Pharmacokinetics were determined by measuring serum fentanyl concentrations at 0, 1, 2, and 4 weeks after patch application. Preliminary behavioral data indicate that healthy control corn snakes have a 94% reduction in activity level for 48 hours; as time after patch application increases, behavior varies by snake. This suggests that TF does not non-specifically increase activity and may be relieving pain in osteoarthritic snakes. Preliminary pharmacokinetic data show that serum fentanyl concentrations are much greater on average (10.4 ng/mL) than those considered therapeutic in mammals (1 ng/mL) at two weeks post-TF application. Overall, preliminary results suggest that TF may provide safe, clinically effective analgesia in snakes for at least 2 weeks.

Research Grant: UW SVM Companion Animal Fund Grant, University of Wisconsin-Madison School of Veterinary Medicine Student Support: Summer Scholars Program, University of Wisconsin-Madison School of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 347 Risk Factors for Antimicrobial Resistance among Campylobacter coli from Agricultural Swine Populations

C. Annie Wang, Will J. Love, Manuel A. Jara, Siddhartha Thakur, Cristina Lanzas

Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Wang, Love, Jara, Thakur, Lanzas)

Antimicrobial resistance (AMR) is a major threat to human and animal health today, rendering once treatable infections untreatable. Although antimicrobial use is known to directly select bacterial populations that are resistant to those drugs, other important risk factors influencing the epidemiology of AMR are incompletely understood. Several complex mech- anisms enable the exchange of genetic material among populations of unrelated bacteria (e.g. mobile genetic elements), thereby influencing the rise of multidrug resistant bacterial populations.Thus, resistance to an antibiotic may still arise even without using that specific antibiotic. Traditional analytical methods used in epidemiological research rely solely on generalized linear models to identify risk factors for health outcomes; however, these methods are less suitable for eval- uating AMR risk factors while simultaneously accounting for the complex, non-linear dynamics of AMR selection and persistence. In this study, we utilize a multi-layered chain graph model to identify risk factors for phenotypic resistance while also accounting for potential complex genetic mechanisms underlying AMR selection and persistence. We applied this model to populations of Campylobacter coli isolated from agricultural swine herds experiencing varying degrees of antimicrobial exposure and other management practices. In addition to antimicrobial usage, we found that risk for fluoro- quinolone- and macrolide-resistance differed based on biosecurity practices employed at each farm and whether animals were reared entirely indoors or outdoors. Results and computational methods from this study are applicable to human public health surveillance data.

Research Grant: George H Hitchings New Investigator Award in Health Research, NIH R35 NIGMS(R35GM134934), NIH F30 ORIP(F30OD030022) Student Support: George H Hitchings New Investigator Award inHealth Research, NIH F30 Training Grant F30OD030022

Quantifying Disease Transmission Risk in Infants Using Video Data

Wenjun Wang, Jeremiah Cox, Abisola Osinuga, Hugo Gonzalez Villasanti, Rebecca Garabed, Kelly Baker

College of Veterinary Medicine (Wang, Garabed), College of Public Health (Cox), and Crane Center for Early Childhood Research and Policy (Gonzalez Villasanti), The Ohio State University, Columbus, OH; College of Public Health (Osinuga, Baker), University of Iowa, Iowa City, IA

Nowadays, more and more people rely on nonparental care while they’re working outside the home. However, children in daycare centers are more likely to get enteric diseases than those who are in home care. Children’s unhygienic habits, such as mouthing and touching objects and other people, facilitate the spread of infectious agents. The objective of this study is to quantify enteric disease transmission risks in infants using video data, and apply this observation method to the animal population for diseases transmitted through the same route in the future. 18 infants from 8 different rooms at 3 daycare centers were selected to participate. Diarrheal illnesses of infants were reported from parents, facility records, and patho- gen detection from fecal samples. However, only 13 infants from 6 rooms were documented on the videos. The caretak- ers from other two rooms refuse to be filmed on the camera. 3 to 4 on-site cameras were placed in each room to provide structured observations. One-minute videos from the same room with different views were then complied into a ten-min- ute video for enumerators to view at once. A specialized software, LiveTrack, was used to translate the video into a digital record. After thorough observation of infants’ behavior and facility infrastructure, categories of locations, object mouthed, object touched, human interaction and diapering were customized to annotate the videos. Specific protocols were estab- lished to reduce inter-observer errors. Annotation testing was used to train enumerators to decrease intra-observer varia- tion, and the output data was then evaluated using Krippendorff’s Alpha to measure inter-observer reliability.

Research Grant: The Ohio State University Infectious Diseases Institute Student Support: NIH T35 Training Grant

348 • 2021 National Veterinary Scholars Symposium Differentiation of H3N2 IAV-S antigenic sites by RT-qPCR

Courtney A. Wangler, Suzanna M. Storms

Applied Livestock Epidemiology Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL

IAV-S is a negative-strand RNA virus that is an endemic disease in swine production. It rapidly mutates, which can cause continuous reinfection and has zoonotic implications. PCR is used to diagnose and differentiate between IAV-S strains. To our knowledge, PCR has not been used to differentiate antigenic sites specifically. The objective of this study is to devel- op PCR primers for antigenic sites of the hemagglutinin (HA) gene of two H3N2 strains. The HA antigenic sites of two H3N2 strains were evaluated at amino acid sites previously recognized in human IAV. These amino acid sites have been identified as the main antigenic mutation points and the reason for the loss of cross-protection between strains. HA-spe- cific primers were designed for two antigenically discrete IAV-S clade IV H3N2 viruses: NY/A01104005/2011 and MN/ A01668936/ 2016 following IDT and ThermoFisher primer design guidelines. The virus was grown and passaged on MDCK cells. RNA was extracted with Qiagen Viral RNA mini kit and Cytiva Sera-Xtracta nucleic acid extraction kit using the manufacturers’ instructions. RT-qPCR was completed using a QuantStudio 3 according to WHO influenza PCR detection procedures. We expect to be able to distinguish between antigenically distinct clades of H3N2. Viruses with different antigenic sites will have negative PCR reactions, indicating a lack of cross-protection between strains.This rapid approach will allow veterinarians to determine what H3N2 clade is present in a herd, and better equip them to make pro- duction decisions for disease management.

Research Grant: Foundation for Food and Agriculture Research Vet Fellows Program Student Support: Foundation for Food and Agriculture Research Vet Fellows Program

Microenvironmental immune effects of stereotactic body radiotherapy and immunotherapy in canine solid tumors

Remy Watts, Lauren Harrison, Leone Hopkins, Erin Trageser, Daniel Regan, Steven Dow, Mary-Keara Boss

Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI (Watts), and Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, CO (Harrison, Hopkins, Trageser, Regan, Dow, Boss)

Purpose: Combining radiotherapy with immunotherapy is an emerging approach to treating cancer. We characterize the microenvironmental immune effects of canine tumors treated with stereotactic body radiotherapy (SBRT) and OX40 an- tibody/TLR9 immunotherapy (SBRTi) compared to SBRT + control (SBRTc). Methods: A randomized prospective study was performed in 12 dogs with solid tumors. Tumor samples were obtained pre- and 2 weeks post-treatment. Immuno- histochemistry (IHC) analysis of immune cell density was evaluated using markers for CD3 (T cells), FoxP3 (Tregs), and CD204 (macrophages). Nanostring gene expression analysis was performed using a customized 48 gene immune panel. Results: Intratumoral Tregs increased post-SBRTc (mean = 130.2%; range = 67.4 to 201.5%) and decreased following SBRTi (mean = -51.0%; range = -20.4 to -86.0%). The ratio of post-/pre-treatment %FoxP3+ cells was decreased follow- ing SBRTi compared to SBRTc (p = 0.0042). There is a trend where Treg-related genes (CTLA-4, FoxP3, GATA3, IL-10) are increased post-SBRTc and decreased post-SBRTi, and effector T cell-related genes are decreased post-SBRTc and increased post-SBRTi (GZMB, IFNg, OX40). FoxP3 and IFNg expression is inversely related: SBRTc increased FoxP3 and decreased IFNg while SBRTi decreased FoxP3 and increased IFNg post-treatment. Differences in expression also included decreased IL-8 post-SBRTi compared to post-SBRTc (P = 0.043) and increased ICOS post-SBRTc relative to pre-treatment (P = 0.036). Conclusion: These results suggest that SBRTi suppresses the density and influence of immuno- suppressive Tregs in the tumor microenvironment and causes a shift towards upregulated effector T cell cytotoxic activity and cytokine signaling.

Research Grant: Eldred Foundation Student Support: AVMA/AVMF 2nd Opportunity Summer Research Scholarship

2021 National Veterinary Scholars Symposium • 349 Pathophysiology of tau hyperphosphorylation and glial inflammation in aged cats

Danielle F Weaver, Analeis Cofield, McKenzie Richards, Amelia Hines, Breonna R Kusick, Stephanie McGrath, Julie A Moreno

Clinical Sci (Weaver, Kusick, McGrath), Environ and Radio Health Sci (Richards, Moreno), Microbio, Immuno, and Path (Hines), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft Collins, CO; College of Agriculture, Environmental, and Nutrition Science (Cofield), Tuskegee University, Tuskegee, AL

Medical advances in human and veterinary medicine are resulting in longer lifespans for people and their pets alike. Therefore, age-related medical concerns like Alzheimer’s disease in humans and cognitive dysfunction syndrome in dogs and cats have become increasingly common and concerning. In this study, we seek to address the pathophysiologic pro- cesses behind feline cognitive dysfunction syndrome (FCD). Currently, little is understood about FCD, a syndrome that af- flicts 28% of cats over the age of 11 and up to 50% of cats over the age of 15. The pathophysiology includes amyloid-beta accumulation, tau protein hyperphosphorylation, gliosis, and neuronal loss. We hypothesize that the brain pathophysiolo- gy, specifically the presence of hyperphosphorylation of the tau protein and glial inflammation, will be present in aged cats compared to cats under the age of four. Immunohistochemistry is performed on sections of feline brain tissue from mul- tiple cats of a wide variety of ages to look for hyperphosphorylation of tau, a protein found to aggregate and form fibrils, and glial inflammation, marked by GFAP for astrocytes and IBA1 for microglia; increases in these markers are associated with cognitive dysfunction in other species. Our data show a significant increase in all protein markers when compared between different age groups of cats. This study furthers the understanding of the pathophysiology of FCD, which can be used as a physiologic model of Alzheimer’s disease. Better understanding of the disease process could lead to improved early diagnosis and treatment of both FCD and human Alzheimer’s disease.

Research Grant: None / Unknown Student Support: Boehringer Ingelheim

Active cell fractions in bone marrow aspirate concentrate (BMAC) for improved treatment of osteoarthritis

Meghan Webster, Katie Sikes, Kelly Santangelo, Ariel Timkovich, Lyndah Chow, Dean Hendrickson, Steven Dow, Lynn Pezzanite

1Department of Clinical Sciences (Webster, Sikes, Chow, Hendrickson, Pezzanite), College of Veterinary Medicine, Colorado State University, Fort Collins, CO and 2Department of Microbiology, Immunology, Pathology (Santangelo, Timkovich, Dow), College of Veterinary Medicine, Colorado State University, Fort Collins, CO

Osteoarthritis (OA) is a common malady affecting both horses, dogs, and humans, but there remains a lack of effective treatment options to reduce pain and improve quality of life without significant adverse effects. Increased FDA regulation of cellular therapies in the U.S. has prompted investigation of other cell therapy options, including the use of bone mar- row aspirate concentrate (BMAC). While poorly defined, the benefits of BMAC injections have been attributed to rare mesenchymal stem cells, and BMAC injections are often extremely inflammatory and painful.We hypothesized that the efficacy of BMAC therapy could be improved and inflammation reduced by identifying most active cell fractions within the otherwise very complex cell mixture. To address the question, purified BMAC cell subpopulations were evaluated for their relative ability to reduce joint inflammation and cartilage degradation in a mouse OA model. The following BMAC fractions were evaluated: unseparated BMAC, BMAC with RBC removed, BMAC CD45+ cells, BMAC CD45- cells. Operated mice were injected with cells once (5x105 cells/injection intra-articularly following destabilization medial me- niscus (DMM), and the impact of cell therapy on gait/activity and joint histology were assessed over 4 weeks. The relative anti-inflammatory properties of purified equine BMAC subpopulations were further compared using in vitro co-culture assays with chondrocytes and synoviocytes. These studies are important for optimizing and improving cellular therapy using BMAC cells for OA in both animals and humans.

Research Grant: Animal Health and Disease Grant No. 19HMFPXXXXG039150001 USDA National Institute of Food and Agriculture, Carolyn Quan and Porter Bennett, NIH/NCATS CTSA 5TL1TR002533-02, and NIH 5T32OD010437-19 Student Support: Veterinary Summer Scholars Program NIH Fellowship Grant Trainee

350 • 2021 National Veterinary Scholars Symposium Interrogating the role of hydrogel electrical and physical properties on neural cell behavior

Nicole Wegrzyniak, Alena Casella, and Kent Leach

School of Veterinary Medicine (Wegrzyniak), Department of Biomedical Engineering (Casella, Leach), Department of Orthopaedic Surgery (Leach), University of California, Davis, Davis, CA

Cellular bioelectricity, which is crucial for proper tissue function and healing, has received limited attention for its role in tissue regeneration. Conductive biomaterials direct electroactive cell behavior and differentiation. Graphene is an electron conductor used in tissue engineering to support nerve activity while alginate is used as a cell transplantation vehicle due to its tunability and biocompatibility. While electrical and mechanical properties are independently known to influence cell behavior, the relationship between material electrical and physical properties is poorly understood. We hypothesized that the interplay of hydrogel electrical and biophysical properties, including conductivity, storage modulus, and degradation, influences cell adhesion and viability and promotes neural cell morphology and behavior. Graphene was added in concen- trations of 0, 10, 25, and 50 v/v% to medium viscosity sodium alginate hydrogels modified with arginine-glycine-aspar- tate. We observed a positive correlation between graphene concentration and conductivity by electrical characterization, and mechanical testing indicated that storage modulus was unchanged. Optimally, hydrogel conductivity would be notably increased while maintaining storage modulus. Because the conductivities are not statistically different, future studies may include groups of higher graphene concentrations (75 v/v%). Future studies will subsequently investigate hydrogel degra- dation, lysozyme and bovine serum albumin adsorption, and PC12 cell adhesion to model neural behavior. These studies aim to elucidate advantages of conductive materials for biological applications while optimizing the level of graphene for cytocompatibility and biological efficacy.

Research Grant: National Institutes of Health R01 DE025475 Student Support: NIH

Physiological synchronization between horse and human in EFP for veterans with PTSD

Chuyan Wei, Zoe Fournier, Laurie McDuffee, and William Montelpare

Departments of Health Management (Chuyan Wei, Zoe Fournier, Laurie McDuffee) and , Applied Human Sciences (William Montelpare), Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island

Evidence has shown that traditional treatment programs have limited efficacy for veterans with severe post-traumatic stress disorder (PTSD). To support veterans’ mental health challenges, equine facilitated psychotherapy (EFP) has been studied as one of innovative therapeutic models in recent years. Human-horse bonding formed during EFP has often been hypothesized as a contributing factor to improvements. The objective of this study is to investigate human-horse bonding using physiological measures and examine if the physiological effects of human-horse interactions on horses and humans are synchronized. We hypothesized that certain degrees of physiological synchronization in human-horse dyads could be captured using heart rate variability (HRV) data and hormone response. Four cohorts of veterans participated in the 8-week EFP program. Each cohort consisted of 4 human-horse dyads, with cohort 1 to 3 having 4 dyads, and cohort 4 having 2 dyads (n = 14). The saliva was collected from horse and human passive drool before and after each psychother- apy session, which was later analyzed for the cortisol levels in the lab. The HRV data was collected using the polar heart rate monitors during each session, which was then processed by the Kubios HRV software. The Bland Altman approach was used to evaluate the level of agreement within the human-horse dyad. Preliminary findings showed strong agreement in mean heart rates between humans and horses of cohort 1 (t = 0.13; df = 1;n.s.). Further measures will include time-do- main, frequency-domain and non-linear measures of HRV, as well as pre- to post-measures of cortisol of cohort 1 and cohort 4. These results are encouraging as they show interspecies physiological synchronization.

Research Grant: Horses & humans research foundation Student Support: AVC Veterinary Summer Research Award & Boehringer Ingelheim Veterinary Scholar

2021 National Veterinary Scholars Symposium • 351 Evaluating the confidence and competency of team communication skills gained via BLS andALS training course

Ilana Weisberg, Claire Tucker, Kelly Hall

Urgent Care and Critical Care Services, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

In human emergency medicine, teamwork and communication amongst the medical team have been shown to improve patient outcomes. Current research in emergency nursing highlights that employees trained in effective communication have improved work environments and patient outcomes, and feel more empowered. Comparatively, research in veter- inary medicine related to teamwork is lacking and there is a need for further education and research. The RECOVER Basic Life Support (BLS) and Advanced Life Support (ALS) online courses use cardiopulmonary resuscitation (CPR) to highlight important teamwork aspects in triage. The objective of this project is to evaluate confidence and competence in team communication of in person training participants (primarily veterinary students and technicians) during their Emer- gency and Critical Care rotation. We hypothesize that these characteristics will improve following their exposure to the RECOVER online modules, the in-person component of the RECOVER BLS course, and a series of author-developed ALS scenarios. A further intervention to expose learners to teamwork communication topics- such as the group pause and closed-loop communication- is placed between the first and second simulations. Participants’ confidence in communicat- ing is measured on a self-reported Likert scale (1-5) before and after the in-person simulations. Competence is evaluated via a trained observer with 1-5 rating of communication skills. This study will inform design of veterinary school curricu- la leveraging best practices in veterinary teamwork training to improve patient outcomes and increase confidence of new graduates to lead teams and communicate effectively, especially during emergencies.

Research Grant: Internal Funding Student Support: Veterinary Summer Scholars Program

The role of calmodulin-dependent protein kinase II nitrosylation in cardiac ischemia-reperfusion injury

Brandon M. Weiss, Kenneth S. Ginsburg, Adam Wilder, Juliana Mira Hernandez, Donald M. Bers, Julie Bossuyt

School of Veterinary Medicine (Weiss) and Department of Pharmacology, School of Medicine (Ginsburg, Wilder, Hernandez, Bers, Bossuyt), University of California, Davis, Davis, CA

Ischemia-reperfusion (IR) injury is an important cause of morbidity and mortality in both humans and animals. As such, interest has grown in discovering novel methods of mitigating damage caused by acute IR injury. Preclinical studies have shown a critical role for nitric oxide synthase and S-nitrosylation in providing protection against IR damage, with better calcium handling and smaller infarcts. Critical NO targets are unknown but inhibition of Calmodulin-dependent protein kinase II (CaMKII), a master regulator of calcium fluxes in cardiomyocytes, improves post-IR recovery. Interestingly our group has recently shown dual regulation of CaMKII by nitrosylation: inhibition by nitrosylation at C273 and activation via C290 nitrosylation. However, the functional importance of CaMKII S-nitrosylation in IR injury has yet to be exam- ined. We hypothesize that nitrosylation of the C273 site on CaMKII will inhibit enzyme activity and improve recovery from IR injury, while nitrosylation of the C290 site will be stimulatory and worsen damage. Using established IR proto- cols for Langendorff perfused hearts we will examine the effect of triggering nitrosylation at C273 by pretreatment with GSNO and BAPTA prior to CaMKII activation with isoproterenol. We will also utilize novel C290A mice that are resis- tant to nitrosylation at C290. We will examine infarct development and CaMKII activation by western blotting of CaM- KII and key targets such as phospholamban. These experiments provide key insight into the functional role of CaMKII nitrosylation and the potential benefit of therapeutically targeting CaMKII in IR injury

Research Grant: NIH R01 HL142282 Student Support: NIH T35 Training Grant

352 • 2021 National Veterinary Scholars Symposium Validating behavioral indications of stress in red-tailed hawks

Paige L. Weiss, Annette Ahlmann, Julia B. Ponder, and Jamie K. Clarke

The Raptor Center (Weiss, Ahlmann, Ponder, Clarke) and Department of Veterinary Population Medicine (Ponder), College of Veterinary Medicine, University of Minnesota, St. Paul, MN

Maintaining a wild bird in captivity for rehabilitation causes stress which can be detrimental to critical body systems (Blas 2015). In red-tailed hawks, stress has anecdotally been identified through visualization of certain behaviors. Little scien- tific research, however, has gone into validating these behaviors as indicators of stress making these assumptions sus- ceptible to flaws like anthropomorphism. We looked to validate stress-behaviors in red-tailed hawks by correlating them with increases in heart rate and plasma corticosterone levels. These physiological parameters were first measured during a control event where no stress was induced and a stressed event where stress was induced via a person opening and closing a door repeatedly over a 7 minute time interval. Video recordings of the events were then analyzed for specific behaviors. These behaviors were further categorized into 1 of 6 behavioral categories: posture, locomotion, maintenance, appetitive, reactionary, and elimination. Behavior was then correlated with fluctuations in the measured physiological parameters. Preliminary results indicate increased frequency and duration of locomotion, elimination and reactionary behaviors in the stressed event when compared to the control event. Increased frequency and duration of maintenance behaviors and in- creased duration of perching in the control event compared to the stressed event was also observed. Heart rate was higher in the stressed event when compared to the control event further suggesting that behaviors observed during the stressed event were true stress-behaviors. Correlating plasma corticosterone levels with behaviors occurring during the two events is our next step.

Research Grant: University of Minnesota College of Veterinary Medicine Resident & Graduate Student Research Grant Partners for Wildlife Student Support: University of Minnesota College of Veterinary Medicine Summer Scholars Program

A novel surgical approach to laryngeal collapse in brachycephalic canines

Jamie E. Wells, Elizabeth A. Rozanski

Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts.

Brachycephalic obstructive airway syndrome (BOAS) is a debilitating pathologic condition whereby soft tissue narrows the airways, increasing resistance and limiting respiration. The primary components of BOAS, including stenotic nares, elongated soft palate, and hypoplastic trachea, result in negative pressure within the airway due to increased respiratory effort. Over time, negative pressure can then lead to laryngeal collapse, with late-stage cases having a guarded prognosis for survival. Children with subglottic stenosis face similar challenges to dogs with laryngeal collapse, mainly, increased respiratory effort and decreased laryngeal lumen width, with collapse on inspiration. Laryngotracheal reconstruction, either with intercostal cartilage or plastic grafts, is effective in treating pediatric cases of subglottic stenosis.This study will modify the technique for dogs and test its efficacy in expanding laryngeal lumen and decreasing airway resistance in a cadaver model. Six cadavers from donated dogs weighing less than 10 kg will be enrolled in this study. Upper airway resistance will be measured using a pressure transducer with a known flow rate before and after the insertion of a homolo- gous intercostal cartilage graft between the arytenoid cartilages, and measurements will be compared using a paired t-test. If successful, this surgical technique could permanently correct laryngeal collapse in dogs and revolutionize treatment of animals with BOAS.

Research Grant: None Student Support: Cummings School of Veterinary Medicine at Tufts University

2021 National Veterinary Scholars Symposium • 353 CRAPP: A microCT-based method for P2 fracture scoring in a murine Down Syndrome model

Casey A. Welsh, Kirby M. Sherman, Alyssa Falck, Shannon Huggins, Lindsay A. Dawson, Dana Gaddy, and Larry J. Suva

Departments of Veterinary Physiology and Pharmacology (Welsh, Sherman, Huggins, Dawson, Suva), Veterinary Integrative Biosciences (Falck, Gaddy), College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21) occurs in ~1 in 700 live births. Patients with DS have a variety of well-characterized congenital deficits and low bone mass, putting them at significantly increased risk of long bone and vertebral fractures. DS patients, as well as the murine DS model DP16C57/Blk (Dp16), have significantly de- creased bone mineral density associated with decreased bone turnover, and, delayed accrual of peak adult bone mass. To test the hypothesis that fracture healing is impaired in DS, we used Dp16 DS mice and generated fractures in the middle phalanx (P2) digits since they do not require post-fracture fixation and the mice ambulate normally post-fracture. P2 was fractured at 15 weeks of age and in vivo microCT was used to analyze fracture healing over 93 days post fracture (DPF). To further evaluate fracture healing in DS and WT mice, we developed the Computed Radiographic Assessment of P2 (CRAPP) method. Modeled after the RUST fracture scoring system, CRAPP assigns quantitative measurements to mi- croCT analysis of P2 fractures to assess callus bridging. At 93DPF, 0% of DS male fractures (n = 12) exhibited bridging, compared to 50% of the male WT littermates (n = 28). Dp16 DS females had 0% fractures healed (n = 8) compared to 50% of female WT littermates (n = 16) at 93DPF. While microCT demonstrated that Dp16 DS fractures had robust peri- osteal callus formation, histologic analysis revealed significant unbridged callus formation across the fracture gap.These studies provide the first direct evidence of impaired bone bridging and conversion of cartilage to woven bone resulting in impaired P2 fracture healing in DS.

Research Grant: R01 HD102909-01A1 (LJS) and Texas A&M University College of Veterinary Medicine & Biomedical Sciences Student Support: NIH T35OD010991-16, Texas A&M University College of Veterinary Medicine & Biomedical Sciences

Experimental infection of ferrets with different SARS-CoV-2 lineages

William T. Weng, David A. Meekins, Konner Cool, Chester D. McDowell, and Juergen A. Richt

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

The emergence of SARS-CoV-2 has resulted in a global pandemic with significant negative impacts on global health and socioeconomics. Recently, the disease ecology of SARS-CoV-2 has been complicated by the emergence of variants of concern (VOCs), i.e. mutant isolates with increased transmissibility that have supplanted the original circulating viral lineage. Mustelid species, including mink (Neovison vison) and domestic ferrets (Mustela putorius), are highly susceptible to experimental SARS-CoV-2 infection, making them species of concern for becoming potential reservoirs. Moreover, fer- rets have been successfully used as a model species to investigate SARS-CoV-2 virulence, transmission, and host adapta- tion. In this study, we used the ferret model to investigate the infectivity and transmission of a lineage A virus and an alpha VOC, also known as lineage B.1.1.7. Ferrets were inoculated intranasally with equal amounts of the original SARS-CoV-2 lineage A isolate (USA-WA1/2020) and an Alpha VOC lineage B.1.1.7 isolate (USA/CA_CDC_5574/2020). Uninoculated ferrets were co-housed with the infected ferrets to determine the transmissibility of the respective isolates. Clinical sam- ples (nasal washes/oropharyngeal swabs) were collected and subjected to next generation sequencing and bioinformatic analysis to determine the relative percentage of lineage A and lineage B.1.1.7 genetic material in each sample. Cumula- tively, the results of this study provide essential information regarding the relative fitness of the SARS-CoV-2 alpha VOC, including its transmissibility, in an established model species, and provides information regarding the potential of ferrets to act as a secondary reservoir for recently emerged SARS-CoV-2 VOCs.

Research Grant: 1) NIH - NIGMS P20GM130448 2) DHS - HSHQDC 16-A-B0006 Student Support: T35OD029981

354 • 2021 National Veterinary Scholars Symposium Chronic pen outcomes based on diagnosis and the economic loss due to bovine respiratory disease

Kaitlyn M. Wesley, Renee D. Dewell, Suzanne T. Millman, Grant A. Dewell

Veterinary Diagnostic & Production Animal Medicine (Wesley, G. Dewell, R. Dewell, Millman), Center for Food Security & Public Health (R. Dewell), Biomedical Sciences (Millman), College of Veterinary Medicine, Iowa State University, Ames, IA

Outcomes from feedlot chronic pens based on diagnosis and economic loss due to bovine respiratory disease (BRD) with- in the chronic pen are uninvestigated areas of study within the beef industry. The goal of this project was to investigate outcomes of feedlot cattle that are in chronic pens. The objectives were to provide chronic pen statistics evaluating health and well-being of cattle, economic losses, and develop strategies to improve chronic pen outcomes. Three assessments of cattle housed in chronic pens were conducted over a nine-month period at five Iowa beef feedlots. Data collected included diagnosis, animal welfare assessment (e.g body condition score, candidate for euthanasia), date of entry and exit, and eco- nomic data such as purchase and treatment costs. Primary diagnosis was BRD (57%), followed by lameness (33%), and 10% for other issues (bullers, poor doers or digestive problems). Of the 9% of cattle that died or were euthanized, 82% were diagnosed with BRD, followed by lameness (12%) and other issues (6%). BRD cattle that died or were euthanized spent an average of 83 days in the chronic pen, with an average treatment cost of $85 and a net profit of -$946.50.These results suggest refinements to chronic pen management, perhaps separating cattle in chronic pens by diagnosis or severity to provide individualized treatment. This could potentially increase efficiency of returning cattle back to normal health with welfare and economic benefits. More research is needed to further assess the effect of separating cattle in chronic pens based on diagnosis as well as determining economic costs associated with managing BRD cattle in a chronic pen.

Research Grant: Iowa Veterinary Medical Association Student Support: USDA NIFA project # 2021-67015-34080

Evaluation of prednisolone pharmacokinetics and toxicity in dogs with lymphoma or immune-mediated disease

Madeleine Westbrook, Sarah Shropshire, Beck Ringdahl-Mayland, Steve Dow, Daniel Gustafson, Susan Lana

Flint Animal Cancer Center (Westbrook, Ringdahl-Mayland, Dow, Gustafson, Lana) and the Department of Clinical Sciences, (Shropshire) College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado

Despite being one of the most widely used drugs in veterinary medicine, little is known regarding the actual plasma con- centrations achieved by current dosing recommendations of prednisolone in dogs with spontaneous disease. Our objective is to assess pharmacokinetic parameters as well as toxicity in dogs with lymphoma or immune mediated disease before and after oral dosing of prednisolone using two commonly recommended doses (1mg/kg and 2mg/kg). Ten patients in each disease group (n = 20) will be randomly placed in two dosing groups. Plasma will be collected pre and at 1, 4, 6, 8, 24 hours and 7 days post dosing. Free prednisolone levels will be measured by (ESI) mass spectrometry to evaluate pharmacokinetic parameters. All adverse events (AE) will be graded according to the VCOG CTCAE v2.0. CBC will be evaluated pre, 24hrs, and 7 days post dosing. Chemistry panel, UA, and UPC will be assessed pre and 7 days post dosing. To date 6 dogs with lymphoma and 5 dogs with immune disease have been enrolled. The total number of drug attributable AEs was 47, with the average for the 1mg/kg group and the 2mg/kg group being 2.8 and 5.5 AEs respectively. The 2mg/ kg group had more gr 2-4 AEs (7) than the 1mg/kg group (4). All cases had increases in ALP, ALT, and BUN. PU/PD occurred in 4 cases. GI toxicity occurred in 2 cases. USG decreased in only 7 cases, proteinuria was present pre dosing in 4 dogs and post in 1 dog. 2 patients developed evidence of infection (UTI, pneumonia) prior to day 7 assessment. Weight loss occurred in 9 patients with 6 loosing > 5 and < 10% BW. These findings with be correlated with PK parameters (AUC, Cmax, CL, t1/2) to be assessed in batch at the end of the study.

Research Grant: Young Investigator Award, Center for Companion Animal Studies and the Quinn Foundation Student Support: Boehringer Ingelheim Veterinary Scholar

2021 National Veterinary Scholars Symposium • 355 Inhibition of fibrotic mediators in partially obstructed mouse bladders by administration of microRNA-29a

Hannah Wildermuth, Samantha Hassin, Robert Spitz, Zunyi Wang, and Dale Bjorling

Department of Surgical Sciences, University of Wisconsin-School of Veterinary Medicine, Madison, Wisconsin

Many bladder disorders (e.g., outlet obstruction stemming from benign prostatic hyperplasia, spinal injury, chronic inflam- mation) can result in increased bladder wall stiffness, reduced compliance, and decreased emptying.These outcomes are typically the result of increased extracellular matrix (ECM), predominantly collagen, in the bladder wall. micoRNA-29a (miR-29a) inhibits signaling pathways associated with fibrosis and may be an effective target to suppress deposition of ECM in the bladder wall. microRNAs are small (~20 nucleotides), noncoding RNAs that regulate protein-encoding genes via inhibition of mRNA translation and other processes. miR-29a, specifically, has been shown to suppress expression of mRNAs encoding ECM components including collagen isoforms, fibronectin, and elastin.This study was conducted to investigate the efficacy of miR-29a to suppress fibrosis of the bladder. We compared the relative miR-29a abundance and mRNA concentrations of fibrogenic-associated molecules in the bladders of male mice with surgically induced partial bladder outlet obstruction (pBOO) or sham operation. Our preliminary data show that 10 days of pBOO significantly de- creased abundance of miR-29a and increased expression of message for components of the ECM in the detrusor. Systemic treatment of mice with a miR-29a mimic prevented the observed increase in message for components of the ECM. We are currently evaluating histological samples specifically for collagen deposition, as well as analyzing results of studies of bladder function. Results to date appear to confirm the capacity of miR-29a to inhibit increased ECM in damaged bladders and suggest that this may be a viable treatment option for patients with bladder fibrosis.

Research Grant: Research Grant: NIH R01 DK118145 Student Support: Student Support: NIH T35 OD011078

Stereotactic radiation therapy (SRT) outcomes on the treatment of canine nasal tumors

Emily Willis, Isabelle Vanhaezebrouck

Department of Radiation Oncology, College of Veterinary Medicine, Purdue University, West Lafayette, IN

Stereotactic Radiation Therapy (SRT) has risen in prevalence for the treatment with curative intent of canine nasal tumors because it can deliver higher radiation doses to more concentrated areas in less fractions compared to previous forms of radiation. Preliminary studies utilizing SRT technique have reported moderate toxicities. Rare severe cases associated, were reported with single dose fraction or tumor involvement to the hard palate or the skin. Treatment margins surround- ing tumor volumes (TV) are variable between studies. The purpose of this study was to analyze Purdue’s experience utiliz- ing tight TV margins and taking 1 day of rest between fractions. The medical records for 11 dogs receiving SRT for nasal tumors between 2014-2019 were reviewed. Any dogs that received prior radiation therapy (RT) were excluded. Follow-up information was collected, if available, from hospital records, primary veterinarians, and owners. The median survival time (MST) was 627 days. Nine of 11 (81%) dogs presented mild acute toxicities. Two dogs were euthanized before data was collected for late toxicities. Of the 9 dogs remaining, 5 (55%) had moderate late toxicities. Toxicities may have been under reported. A linear accelerator was used to deliver 3 fractions with intensity modulated radiation therapy (IMRT) at a dose of 8 Gy every other day, for a cumulative dose of 24 Gy. Modified Adam’s staging for canine nasal tumors was used to classify tumor progression. PTV margins averaged 3 mm, except in 2 cases. Organs at risk (OAR) that received higher radiation doses appeared to have a higher risk for side effects. Conclusions are yet to be determined.

Research Grant: None Student Support: Boehringer Ingelheim, Purdue College of Veterinary Medicine

356 • 2021 National Veterinary Scholars Symposium Novel biomarkers of canine gastrointestinal health

Erin L Willis, Lara Sypniewski, Kristina M Hiney, Melinda L Upton, Lindsay L Donnelly, Adel Pezeshki, Pratyaydipta Rudra, Dianne McFarlane

College of Veterinary Medicine (Willis, Sypniewski, McFarlane), Dept of Animal & Food Science (Hiney, Pezeshki) and Dept of Statistics (Rudra), Oklahoma State University, Stillwater, OK; Oncology/Internal Medicine Referral Service, Tulsa, OK (Upton); College of Veterinary Medicine, University of Missouri, Columbia, MO (Donnelly)

The gut microbiota is known to play an important role in immunity. Altered bacterial populations can led to changes in intestinal homeostasis, and can be impacted by factors such as diet, disease states and drug therapy. Gut dysbiosis has been documented in canine GI diseases and a previous study in dogs with chronic enteropathy found decreased levels of the protective anti-inflammatory enzyme intestinal alkaline phosphatase (IAP). However, knowledge of the canine micro- biome and intestinal health is limited, particularly in conditions that may lead to mild intestinal alterations. Therefore, the initial goal of this study was to further investigate potential biomarkers of intestinal health by examining groups of dogs with differing microbiota. Fecal samples obtained from dogs consuming either a processed diet (n = 27) or unprocessed diet (n = 28) were assessed for microbiome differences and candidate biomarkers.The microbiome was characterized by 16S amplicon sequencing. ELISAs for the candidate biomarkers IgA and IgG were validated for use in fecal samples, while fecal IAP was measured by an enzymatic activity assay. As expected, diet substantially impacted the microbiome. In addition, 3.2- to 2.5-fold differences were found in fecal IAP, IgA and IgG between dietary groups (P < 0.05). IAP was positively associated with fecal IgA and IgG (r = 0.32-0.39; P < 0.05) and multiple correlations were observed between these protective factors and specific bacterial genera. Currently, the impact of chemotherapy treatment on gut microbiota and these candidate biomarkers is being assessed. Results of this study may also provide insight into potential therapies to protect against GI dysfunction and drug complications in dogs.

Research Grant: American Holistic Veterinary Medicine Foundation, Oklahoma State University College of Veterinary Medicine, Office of the Associate Dean of Research Student Support: Oklahoma State University College of Veterinary Med, Veterinary Clinical Sci. Henthorne Chair

Hepatocyte ploidy in dogs with hepatocellular carcinoma and hepatitis

Kaitlynn Willis, Ingeborg Langohr, Chin-Chi Liu, Andrea Johnston

1 Veterinary Clinical Sciences (Willis, Liu, Johnston), School of Veterinary Medicine, Louisiana State University, Baton Rouge LA and Pathobiological Sciences (Langohr), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Polyploidy is commonly exhibited in mammalian liver cells. Though there is evidence to suggest that polyploidy may play both a role in tumorigenesis and suppression, the function of polyploidy is still not clearly understood. Furthermore, canine liver ploidy has not been documented. The objective of this study is to 1) quantify hepatocellular ploidy in normal dogs; 2) quantify hepatocellular ploidy in dogs with hepatitis and HCC; 3) and compare these results. We hypothesize that hepatocyte ploidy is not statistically different in dogs with HCC or hepatitis versus normal dogs.To test this hypothesis, fluorescence image-based analysis will be used to quantify nuclear size and ploidy. The role that hepatocellular ploidy plays in canine hepatitis and HCC pathogenesis is not well understood. Therefore, exploring the characteristics of canine hepatitis and HCC and their relationship to hepatocellular polyploidy may provide a clearer understanding of disease pro- gression and treatment.

Research Grant: Kenneth F. Burns Student Support: Kenneth F. Burns

2021 National Veterinary Scholars Symposium • 357 Predominance of fungal colonies in the feces of canines with diabetes mellitus versus nondiabetic control dogs

Alec T. Wilson, Ross Monasky, Jared Jaffey, Ogi Okwumabua, and Shankar Thangamani

College of Veterinary Medicine, Midwestern University, Glendale, AZ

Candida spp. is normally present in the microbiome of both humans and canines. However, they usually only make up a very small percentage of the total microbiome. One of the many diseases that could cause a patient to have increased fungal colony isolation is diabetes mellitus. Diabetic canines have immune dysregulation, increased comorbidities, and greater metabolic disorders when compared to healthy canines. Many of these downstream effects can lead to an increased incidence of fungal colonies. We hypothesized that dogs with type-1 diabetes mellitus (T1DM) would have Candida spp. colonies isolated more commonly from feces and urine than age, sex, and breed matched healthy control dogs. Feces and urine samples were spread on agar plates to allow the fungal species to proliferate, and individual fungal colonies were isolated. The fungal burden of each sample was calculated and MALDI-TOF was used to confirm the identity of the fun- gal species. Twenty-eight total dogs were enrolled (T1DM, n = 14, controls, n = 14). Dogs with T1DM (36%, 5/14) were significantly more likely to haveCandida spp. colonies isolated from fecal cultures than controls (0%, 0/14, P = 0.04). Fungi colonies were not isolated from urine cultures in any dog. Larger studies are needed in the future to confirm the results of our pilot study. If fungi are consistently identified more frequently in diabetic dogs than studies can be designed to investigate its potential clinical consequences. Moreover, these findings could support use of the dog as a viable animal model for humans with T1DM to explore gastrointestinal fungi colonization.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and Federal Work Study

Bacterial burden, moisture content, and mouse preference of DietGel 76A over time

Rachel Wilson, Glenn Jackson

College of Veterinary Medicine and Biomedical Sciences (Wilson), Texas A&M University, College Station, TX; Center for Animal Resources and Education (Jackson) at Cornell University, Ithaca, NY

DietGel 76A is a commonly used commercial gel diet for rodents needing oral nutritional and hydration support during ill- ness, injury, or post procedure. Typically, the entire container is not used in one treatment, requiring storage of remaining product for later use. Inconsistency in storage practices has been noticed in mouse facilities and among animal handlers. The manufacturer recommends storing opened DietGel 76A up to 2 weeks in a sealed bag either at room temperature or refrigerated. We hypothesize that after opening, DietGel can be used past the manufacturer recommendation of two weeks. The aim of this study is to validate manufacturer recommendations regarding storage of opened containers of DietGel, and obtain data supporting extended storage. Each of the DietGel 76A containers were opened, then the foil lids set back in place. Half the containers were then sealed in recloseable plastic bags. All samples were refrigerated or stored at room temperature for 6 days, 14 days, or 31 days. Samples were evaluated for bacterial load, water content, and mouse prefer- ence, compared against freshly opened, sterile DietGel. Preliminary impression of the data suggests that DietGel stored in bags retains higher moisture content, but there is no obvious difference in bacterial load or mouse preference among samples. These preliminary findings suggest that opened DietGel can be kept up to 31 days in a sealed bag at room tem- perature or refrigerated to prevent excess waste of opened containers.

Research Grant: Department funding from Center for Animal Resources and Education at Cornell University Student Support: ASLAP Summer Fellowship

358 • 2021 National Veterinary Scholars Symposium Racehorse Safety in Louisiana (2009-2021)

J. Windham, A. Chapman, M. Mirza, A. Briscoe, A. Bertrand, A. Distefano, J. Miller, C. Mauberret, F. Andrews

Equine Health Studies Program, Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA. Louisiana Racing Commission, New Orleans, LA

Since 2009, the Louisiana Animal Disease Diagnostic Laboratory (LADDL) has performed necropsies on the horses that died/were euthanized at the Fair Grounds Race Course (FGRC) as a part of the National Thoroughbred Racing Associa- tion Safety and Integrity Alliance created to improve racehorse safety. The results of those necropsies have been reported to the Alliance, but a comprehensive analysis of the data has not been reported. Records of the number of races, starters, ages of the horses, and the number of fatalities will be collected from the Louisiana Racing Commission data base for the FRGC, Evangeline Downs, Louisiana Downs, and Delta Downs in the State of Louisiana from 2009-2021. Data will be entered into a spread sheet and compared to data taken from other years. The purpose of this study is to collect the current and archived necropsy data at the FGRC to determine if there were more catastrophic injuries in racehorses in years 2019- 2021 when compared to years 2009-2018, and if there were more injuries in 2-year-old horses compared to > 2 years-of- age horses. Data on the number of horses that died and the number of horses racing will be evaluated to determine number of deaths/starts and races. In a previous study similar information was collected from the year 2009-2018 with the purpose of comparing the number of deaths and catastrophic injuries. This study will use this information as a foundation to build upon and will continue the analysis into the years 2019-2021 in order to provide a complete and inclusive data collection and analysis.

Research Grant: Boehringer Ingelheim Animal Health Student Support: Boehringer Ingelheim Animal Health, School of Veterinary Medicine Louisiana State University

Borrelia burgdorferi colonization of gastrointestinal tract mucosa in Mus musculus

Caroline G. Winiarski, Christa D. Jackson, Charles R. Brown

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri

Expanding geographical ranges for ticks has resulted in an analogous increase in the spread of tick-borne diseases in humans and companion animals alike. Lyme Disease, a tick-borne illness, is caused by Borrelia burgdorferi, a spirochete bacterium that initiates an erythema migrans rash, inflammation, and generalized pain in the infected individual.Though B. burgdorferi is susceptible to antibiotics, persistence of the bacterium in an unknown location within the host may cause chronic infection. The objective of this study is to determine whether B. burgdorferi is present in the gastrointestinal tract (GIT) mucosa of mice. We hypothesize that the bacterium will be present in the GIT mucosa due to the specialized immune system and corresponding tolerance to various bacterial species within the GIT. Wildtype mice will be euthanized at least 29 days post-inoculation with B. burgdorferi, after which the acute infection is cleared. The duodenum, jejunum, ileum, cecum, and colon will be harvested and rinsed with phosphate-buffered saline (PBS) to remove fecal contamina- tion and the bladder will also be harvested as a positive control for B. burgdorferi. DNA will be isolated from the samples using a TRIzol reagent and qPCR will be run to determine relative expression of borrelial flagellin flaB( ) versus murine nidogen (nid-1). We expect that B. burgdorferi will be present in the GIT mucosa. Such results would provide a target for the treatment of chronic Lyme Disease for doctors in the medical and veterinary fields.

Research Grant: NIH R21 AR075160 Student Support: University of Missouri College of Veterinary Medicine Office of Research

2021 National Veterinary Scholars Symposium • 359 The molecular clock gene, CLOCK, in the human placenta

Emily Winn, Soo Ahn, Thu Duong, Margaret G. Petroff, Hanne Hoffmann

Departments of Pathobiology & Diagnostic Investigation (Winn, Ahn, Petroff) and Microbiology & Molecular Genetics (Duong), College of Veterinary Medicine, Michigan State University, East Lansing, Michigan Department of Animal Science, College of Agriculture & Natural Sciences, Michigan State University, East Lansing, Michigan

Preeclampsia is a maternal vascular disease of pregnancy caused by malperfusion of the placenta, and manifests as life-threatening hypertension. Increasing evidence suggests that the bodies internal time-keeping system, represented by circadian clock genes, are involved in placental function as well as the timing and duration of labor. The important role of molecular clock genes and circadian rhythms in a healthy pregnancy is supported by human studies showing that disrup- tion of these rhythms can lead to reproductive pathologies and are associated with increased abortion and miscarriage rates. Despite this knowledge the role of circadian rhythms in the placenta during normal and pathologic pregnancies is poorly understood. The objective of this study is to explore the expression of the molecular clock gene CLOCK in the placenta across human gestation and in placentas affected by preeclampsia. Using publicly available microarray data, we identified a reduced expression of CLOCK in the placenta from women with preeclampsia, independent of gestational stage (term vs preterm). To validate this data, we analyzed placental protein from the first, second, and third trimesters of pregnancy, as well as from preeclamptic and matched control pregnancies, by SDS PAGE and western blot. We expect that quantities of CLOCK will be regulated in a gestation stage-specific manner and will be reduced in placentas from preeclamptic pregnancies. This study aims to provide a basic understanding of the mechanisms by which CLOCK is regu- lated in the placenta, and how circadian rhythms influence placental function and pathology.

Research Grant: NICHD R00 HD084759 NIH R01 HD100832 Student Support: Student Funding provided by NIH Grant 5T35OD016477-19 to Michigan State University

Evaluation of histopathologic sectioning of canine soft tissue sarcomas

Hannah E. Wittorff, Ryan N. Jennings

Department of Veterinary Biosciences (Jennings), College of Veterinary Medicine, The Ohio State University, Columbus, OH

Soft tissue sarcomas (STS) are a group of mesenchymal neoplasms with the potential for metastasis and recurrence. The current method for determining excision status of soft tissue sarcomas is histological assessment of radial sections. However, recent research argues tangential, or en face, sectioning provides a complete margin evaluation and therefore, is a more sensitive method in some tumors. A more sensitive margin assessment may better inform prognosis. The goal of this study was to compare standard radial sectioning to en face sectioning in a retrospective study of 21 canine soft tissue sarcomas. For this study, cases of canine STS were evaluated by radial and en face sectioning. The total percent “dirty” surface areas from en face margins were measured and calculated using image analysis. The measured histologic tu- mor-free margins (HTFM) of the radial sections were compared against the “gold standard” en face results using statistical analysis and a receiver operating characteristic (ROC) curve. Out of a total of 14 negative radial margins, 8 (57.1%) were positive on en face margin analysis. Histopathologic evaluation of radial sectioning had a low sensitivity (46.7%; 7/15) when compared to en face sectioning when positive margins were defined as HTFM = 0 mm. The mean HTFM of nega- tive en face margins (n = 6) was 6.79 6 4.58 mm while the mean HTFM of positive en face margins (n = 15) was 0.367 6 1.03 mm. These results suggest that radial sections with HTFMs > 0 mm should not always be considered completely excised. Future studies should use methods to increase the accuracy of image analysis and should consider case follow-up to determine the prognostic significance of positive margins detected by en face sectioning.

Research Grant: None Student Support: The Dr. William M. Busey Endowed Summer Research Fund

360 • 2021 National Veterinary Scholars Symposium Mosquito diversity, abundance, and virus associations of Binder Park Zoo in Battle Creek, Michigan

Hunter E. Wojtas, Morgan E. Ramsdell, John B. Keven, and Edward D. Walker

College of Veterinary Medicine (Wojtas), and Department of Entomology (Ramsdell, Keven), and Department of Microbiology and Molecular Genetics (Walker), Michigan State University, East Lansing, MI

Mosquitoes are vectors of pathogens to both people and animals. In Michigan, three mosquito-borne diseases of concern are Eastern Equine Encephalitis virus (EEEV), West Nile virus (WNV), and Jamestown Canyon virus (JCV). One site of interest for virus presence is Binder Park Zoo in Battle Creek, Michigan, where, in 2019, two Mexican gray wolf pups contracted EEEV. The goal of this project is to initiate virus surveillance for the zoo and quantify mosquito abundance, diversity, and virus associations of the mosquito community there. Further, zoos present a unique environment to study the nexus of people, animals, and mosquito-borne pathogens. Mosquitoes have the potential to spread diseases between zoo employees, patrons, wild and exotic animals, and pets and people of neighboring communities. By analyzing blood meals, we will illuminate the relationships between mosquito-borne viruses, people, and animals. We are sampling mosquito pop- ulations at eight sites on zoo property weekly from June through July, using three methods (light traps, gravid traps, and resting boxes). After collections, we identify species, pool samples, and test them for virus infection in collaboration with the Michigan Department of Health and Human Services. We will compare abundance and diversity of mosquito species by site in the zoo and over time by week in the summer. We also expect to determine patterns of positive virus results associated with specific species, locations, and weeks. Blood meal analysis will reveal mosquito biting patterns on animals and people in the zoo, and resultant risk of virus transmission. Ultimately, information from this study will help prevent the spread of viruses effectively by targeting specific mosquito species.

Research Grant: CDC U50 723K866 “CDC Midwest Center of Excellence in Vector-Borne Disease” and CDC BAA 75D301-20-R-67837 “CDC Innovations in vector control” Student Support: Support provided by Boehringer Ingelheim and the Graduate School at Michigan State University

Multispecies proteomics reveals potential biomarkers for osteoarthritis

Sydney J. Womack, Camila Carballo, Scott A. Rodeo, Heidi L. Reesink

Department of Clinical Sciences (Womack, Reesink), College of Veterinary Medicine, Cornell University, Ithaca, New York; Hospital for Special Surgery (Carballo, Rodeo), New York City, New York

Post-traumatic osteoarthritis (PTOA) following osteochondral fragmentation in horses, cranial cruciate ligament rupture (RCCL) in dogs, and anterior cruciate ligament (ACL) injury in humans is a leading cause of disability across all three species. RCCL in dogs is an attractive target as a comparative animal model for ACL injury in humans, while PTOA in horses has comparative relevance to human intra-articular fracture. The goal of this study was to reveal protein biomarkers and potential therapeutic targets for osteoarthritis (OA) in synovial fluid across the three species. Synovial fluid sam- ples were collected from 16 equine carpal joints, 16 canine stifle joints, and 10 human knee joints. Human samples were paired, while dog and horse samples were from independent animals. Proteomics data was collected using nano-scale reverse phase chromatography and tandem MS. Proteins were identified using Proteome Discoverer 2.3 and analyzed in MetaboAnalyst 5.0. In all species, Principal Component Analysis demonstrated distinct groupings between healthy and OA samples. Several proteins including immunoregulatory (a-2-macroglobulin) and macromolecule transport proteins (apolipoprotein B-100) were significantly up- or down-regulated in OA as compared to healthy synovial fluid samples across two or more species. Periostin, an extracellular matrix protein involved in wound healing, was the most upregulat- ed protein by fold change among ACL-injured limbs in dogs and humans. A multi-species approach enables more robust identification of proteins of interest and may offer insight into naturally occurring animal models for human OA. Future work may use the results of this study to focus therapeutic or OA screening protocol development.

Research Grant: Weill Cornell Medical College/CTSC NIH Pilot Award 1 UL1 TR002384 Student Support: Boehringer Ingelheim, Cornell University College of Veterinary Medicine

2021 National Veterinary Scholars Symposium • 361 Serine protease inhibitor supplementation to enhance host response to Intranasal and intraperitoneal vaccines

Rachel M. Woodfint, Logan E. Mazik, Prosper N. Boyaka

Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus, OH

Zoonotic respiratory viruses remain a major public health concern world-wide. These viruses include influenza virus and SARS-COV-2, which is responsible for the COVID-19 pandemic, and infect hosts via the airways. Vaccination strategies that promote mucosal IgA immunity at the pathogen portal of entry are believed to provide better protection than tradition- al injected vaccines, which only stimulate immunity in the bloodstream. Previous work from our lab demonstrated that the presence of neutrophils is inversely related to IgA production. Sublingual vaccination, supplemented with a pharmacolog- ical serine protease inhibitor (SPI) that specifically targets neutrophil elastase increases serum and fecal IgA, improving the mucosal immune response. Therefore, we hypothesize that serine protease inhibitors represent a new class of adjuvant that can improve the efficacy of intranasal vaccines and afford protection in mucosal tissues. Using inan vivo model of either intranasal or intraperitoneal primary vaccination with intranasal vaccine boosts, containing SerpinB1 or Neutrophil elastase inhibitor, we examined the role of these serine proteases in secretory IgA response, using the SARS-COV-2 spike protein as the antigen.

Research Grant: Unknown Student Support: NIH T35 Training Grant T35OD010977

Social group and behavioral changes of white-tailed deer treated with immunocontraceptive PZP

Emma K. Wunderlich, Kali Pereira, and Allen T. Rutberg

Center for Animals and Public Policy, Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts

Deer-human conflicts in suburban areas have led to the investigation of potential solutions like the immunocontraceptive porcine zona pellucida (PZP), a non-lethal and non-hormonal method of reducing deer populations. While many studies show the efficacy and benefits of treating wildlife with PZP, very few have evaluated possible changes in social behav- ior of vaccinated white-tailed deer (Odocoileus virginianus). The aim of this field study is to investigate these potential behavior changes, specifically comparing group composition and interactive behaviors between vaccinated and non-vac- cinated does at Avalon Nature Preserve and the surrounding area in Head-of-the-Harbor Village in New York, where a multi-year study on PZP cost-effectiveness is currently underway. From June to August 2021, data will be collected through approximately 100 hours of direct observation and a grid of 10 motion-sensitive infrared trail cameras. Group compositions (# of does/bucks, adults/yearlings/fawns) will be compared between treated and non-treated does using a t-test. In addition, treated and non-treated does will be compared using a Mann-Whitney U test for frequency (# per unit observation time) of aggressive and affiliative behaviors between individuals, such as striking, nursing, and grooming.We expect to see a change in group composition between individuals who have been treated with PZP but we do not expect to see a difference in the frequency of aggressive and affiliative behaviors between the two groups.There is currently very little known about PZP effects on white-tailed deer natural behavior, which may be critical when considering the viability of administering PZP to control deer populations on a long-term and wider scale.

Research Grant: Burkett-Plack Foundation Student Support: Tufts Center for Animals and Public Policy, Cummings School of Veterinary Medicine

362 • 2021 National Veterinary Scholars Symposium Developing 3D-printed osteotomy guides to improve accuracy of surgical correction of angular limb deformities

Amelie L. YaDeau, Anna M. Massie, Adriana Barba, and Thomas P. Schaer

University of Pennsylvania School of Veterinary Medicine (YaDeau), Department of Clinical Science & Advanced Medicine (Massie), Department of Clinical Studies New Bolton Center (Barba, Schaer), University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania

Angular limb deformities (ALDs), a common cause of canine lameness, often have both multiplanar angular and rotation- al malformations. ALDs can be corrected by closing wedge osteotomy (CWO) surgery (excision of a wedge-shaped piece of bone). Current freehand CWO techniques are imprecise and can cause lameness and osteoarthritis. Patient-specific 3D-printed saw guides for CWOs exist, but they do not correct the rotational element of the deformity. We hypothesized that 3D-printed patient-specific combined saw and pin osteotomy guides would improve distal femoral CWOs. First, a guide that creates both the angular and rotational components of an ALD was designed. Ovine femora from previously sacrificed animals were scanned by a clinical CT scanner (Neurologica) to develop distal femoral osteotomy jigs. Scan data were imported into a computer-aided design program Meshmixer. Sheep-specific osteotomy guides were designed using varus and torsion angles consistent with clinically relevant angles from canine ALD patients. These guides allowed manipulation of femoral alignment to create prespecified rotational and angular deformitiesin silico. Then a guide that induces 25° varus and 20° anteversion deformities was 3D-printed by the University of Pennsylvania Biomedical Library. Using the guide, a CWO was performed on a cadaveric ovine femur. Post-operative CT scans showed that the guide cre- ated an ALD with 16.7° varus and undefined anteversion angles. Thus, we successfully developed a workflow for creating patient-specific osteotomy guides, validated their utilityin silico, and begun investigating their ex vivo performance. In vivo implementation of guides to correct ALDs is under investigation, pending further validation studies.

Research Grant: Unknown Student Support: NIH T35 Training Grant T35 OD010919

The Pharmacokinetics of Meloxicam and its effects on COX in Ball Pythons Python( regius)

Sean S Yamada, Curtis Eng, Manish Issar, Sebastien Fuchs

Western University of Health Sciences - College of Veterinary Medicine (Yamada, Eng), College of Osteopathic Medicine (Issar, Fuchs)

Ball pythons are among the most popular reptiles being sold in the pet market currently. Meloxicam (Mel) has been used for many years as a pain management and anti-inflammatory drug in snakes for a variety of medical conditions. However, dosing recommendations for snakes have been principally based on other non-serpentine reptiles and bird species. The study of pharmacokinetic and pharmacodynamic properties of Mel, or any other non-steroidal anti-inflammatory drug (NSAID), in snakes is lacking. Our study will determine, for the first time in snakes, the half-life of Mel and peak concen- trations in plasma after a single dose administration. The measurement of plasma Mel concentration will be determined by HPLC approach. We have determined that the minimum detectable concentration is 20 ng/ml of Mel in snake plasma. In addition, tissue samples of the kidneys and liver will also be used to analyze toxicity and to measure the inhibition of snake cyclooxygenase (COX) by Mel. These results will allow us to evaluate the pharmacokinetic properties based on the bioavailability of an NSAID, and the ability of the NSAID to inhibit snake COX. Mel has selectivity against COX-2 enzymes in other species. We will also attempt to measure its enzymatic properties in snakes. We hypothesize that a single 0.3mg/kg of Mel, administered IM will achieve an effective inhibitory concentration for at least 24 hours. Dosing is based on textbook recommendations and is also supported by an observational behavior study. We intend to investigate further to support those recommendations. At the completion of this project, we hope to provide pharmacological evidence to support a safe and effective use of Mel as pain management and anti-inflammatory drug.

Research Grant: None Student Support: Western University of Health Sciences

2021 National Veterinary Scholars Symposium • 363 Reverse Trendelenburg and the incidence of gastroesophageal reflux (GER) in Isoflurane anesthetized dogs

Erin Wendt Hornickle, Kirby Yamamoto

University of Minnesota Department of Veterinary Clinical Sciences (Wendt Hornickle), University of Minnesota College of Veterinary Medicine (Yamamoto)

The regurgitation of gastric contents, or gastroesophageal reflux (GER), is a common surgical complication. This study analyzed if the reverse Trendelenburg position (158 elevation of the head relative to the horizon) reduced the incidence of GER in dogs receiving elective stifle procedures. Based on a block randomization scheme, half of the study dogs were assigned to the experimental condition while the rest served as a standard horizontal control position. Dogs were excluded if they required general anesthesia for pre-operative radiographs, or had received gastric antacids/prokinetics within the last month. They were also excluded if they had a prior history of GER, regurgitation, vomiting, esophagitis, or cough- ing. Dogs receiving nonsteroidal anti-inflammatory drugs were not excluded, although this usage was noted.To monitor for GER, an esophageal probe was placed at induction. and removed at extubation following post-operative radiographs. Episodes of GER were defined as pH drops below 4 (gastric acid) or above 7.5 (bile) exceeding 30 seconds.The timing of movement to and from the OR table was noted in the data set. Hydromorphone or atropine boluses were given if the mean arterial pressure or heart rate fell or rose by more than 20% of baseline (this number was determined pre-incision). The number and time of these boluses was noted. At the time of this abstract’s writing, the data for this experiment was still being collected. However, the findings in this data will be discussed within the body of this poster.

Research Grant: None Student Support: Boehringer Ingelheim Veterinary Scholars Program and the U of M College of Veterinary medicine

Recoverability of metagenome assembled genomes within the fecal microbiome of commercial swine

Jared Young, Noelle Noyes

Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota

Metagenomic sequencing can be a powerful tool for investigating microbiome dynamics within commercial swine pro- duction systems, but is often limited by inconsistent strain-level identification of microbial taxa. Recent advances in metagenomic binning have allowed for the construction of metagenome-assembled genomes (MAGs) and greater charac- terization of individual microbes within a given microbiome. Utilizing these recent advances, the aim of this project was to characterize MAGs that may be representative of commensal intestinal microbes within commercial swine. Given the computationally intensive nature of the MAG workflow and the short project timeframe, 12 composite fecal samples from a recent National Pork Board funded study were selected for analysis. These samples were selected because of availability of deep sequencing data and a well-defined source population with distinct group-level structure. MAGs were assembled following the methods described in the PIGC pipeline, excluding sample co-assembly. Samples underwent adapter and host DNA removal prior to contig assembly with MEGAHIT. Assembled contigs next underwent metagenomic binning and downstream processing using metaWRAP and genome dereplication using dRep. Preliminary results demonstrate that MAGs have been successfully assembled for this dataset. Further analysis is being performed to assess sample-level vari- ation in draft genome quality, taxonomic classification, phylogenetic analysis, genome annotation, and relative abundance. While current MAG workflows still have many limitations for metagenomic datasets, refinement of these approaches has the potential to further our understanding of complex microbiome dynamics, particularly at the strain level.

Research Grant: Funding, wholly or in part, was provided by both the National Pork Checkoff and theAgricultural Research, Extension and Education Transfer Technology (AGREET) Program through the State of Minnesota Student Support: Boehringer Ingelheim VSS program and the College of Veterinary Medicine, University of MN

364 • 2021 National Veterinary Scholars Symposium Can Influenza A Virus infect sialic acid knock out mammalian cells through artificial antibody receptor?

Kirsten Young, Simon Frueh, Wendy Weichert, Oluwafemi Adu, Colin Parrish

Royal Veterinary College, University of London, London, England, and Leadership Program for Veterinary Students at Cornell University (Young), Baker Institute for Animal Health, College of Veterinary Medicine (Frueh, Weichert, Adu, Parrish) Cornell University, Ithaca, NY

The biological process of Influenza A virus (IAV) hemagglutinin (HA) binding toa 2,6 or a2,3-linked sialic acid (SIA) and initiating viral endocytosis has been explored intensively over several decades. However, a lot less is known about the molecules that the SIA are attached to, or how those mediate endocytic entry and infection. More recently IAVs (HA17 and 18) from bats have been shown to bind to glycoprotein receptors, most likely MHC Class II. While some studies have analysed IAV internalization through cellular receptors binding to the glycan on the viral glycoprotein, such as C-type lectins, little is known of any IAV infecting through receptors other than SIA. This project investigates IAV infections using an artificial chimeric antibody receptor made from portions of an antibody fused to a transferrin receptor (fTfR) and expressed in suppressed SIA HEK 293 cells. The chimeric antibody-based receptor contained the H and L variable domains of a broadly neutralizing antibody (5J8, that has known interaction with HA) and residues 1-122 of the TfR stalk. H1N1 pdm09 infections were tested in HEK 293 WT cells, HEK 293 knocked out for SIA (DSLC35A1) cells, HEK 293 DSLC35A1 cells transfected with the chimeric antibody, and HEK 293 DSLC35A1 cells transfected with the TfR. Infec- tions were quantified through immunofluorescence and flow cytometry. The WT cells were susceptible to infection, and initial results show increased infection in DSLC35A1 cells expressing the artificial chimeric antibody compared to the control cells.

Research Grant: None. Student Support: NIH T35 A1007227

Ex vivo heme-induced NETs in canine neutrophils as a model for NETs in canine immune-mediated hemolytic anemia

Austin D. Yungmeyer, Nghi Nguyen, Ronald H. L. Li

Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California

Despite being a common immune-mediated disease, immune-mediated hemolytic anemia (IMHA) continues to be a lethal disease in dogs with a high mortality rate and limited treatment options. Cell-free hemoglobin (Hb), released by hemoly- sis, rapidly undergoes oxidation to release heme, which may exacerbate systemic inflammation and thrombosis by activat- ing neutrophils to release neutrophil extracellular traps (NETs). The primary aim of this study was to elucidate the mech- anisms by which cell-free heme leads to NETs formation. Specifically, we wished to determine if inhibition of reactive oxygen species (ROS) generation could modulate heme-induced NET formation in dogs. We hypothesized that iron-bound heme or free heme could induce NETs formation in canine neutrophils via ROS generation. Neutrophils, isolated from 6 healthy dogs, were treated with hemin or iron-bound heme) in the presence or absence of LPS or diphenyleneiodonium chloride (DPI). Phorbal 12-myrisitate 13-acetate (PMA)-activated neutrophils served as positive control. Cell-free and cellular DNA of live cells were stained with SytoGreen and SytoxOrange and NETs were quantified by fluorescent -mi croscopy. We found a dose-dependent increase in NETs formation in hemin- and LPS- treated neutrophils (P < 0.010) with 20 mM hemin causing the most significant elevation in NETs compared to vehicle control (P = 0.037). Iron-bound heme, however, did not induce significant increase in NETs. Preliminary data suggested that inhibition of ROS generation by DPI modulated hemin- and PMA-induced NETosis. Cell-free heme released during hemolysis in IMHA dogs may further exacerbate inflammation and systemic coagulation by inducing NET formation via ROS generation.

Research Grant: Center for Companion Animal Health (School of Veterinary Medicine intramural grant) Student Support: Morris Animal Foundation

2021 National Veterinary Scholars Symposium • 365 An exploratory study of stress behavior in dogs

Gianna Zavota, Seana Dowling-Guyer, Emily McCobb

Center for Shelter Dogs and the Department of Clinical Sciences, Tufts University Cummings School of Veterinary Medicine, Grafton, MA (Zavota, Dowling, McCobb)

The stressors that dogs are exposed to can impact their behavior and welfare. Results from studies examining dogs’ reac- tions to stressful stimuli tend to show that stress adversely affects welfare (Beerda et al.,1998;Tuber et al.,1999). Because of this, it is important for owners and animal caretakers to be able to identify signs of stress in dogs in an easy and non-in- vasive way. Since many methods for stress assessment rely on physiological measures such as cortisol collection which is itself stressful, a non-invasive method to assess stress would be useful to help monitor dogs in a variety of situations. Be- havioral measurements show promise to be utilized as a convenient stress assessment tool (Kerswell et al.,2009; Rooney et al.,2009), however, additional investigation is needed to fully understand which behaviors across varying contexts are useful indicators of stress in different dogs. Therefore, this study examines patterns of stress-related behavior in videos of dogs in various confined settings. To do this,100 videos were coded using Noldus Observer XT, version 15. Last summer, I coded 40 videos of owned dogs isolated in a lab room, and this summer I completed the final 60 videos of kenneled dogs in shelters, which will be the focus of this poster. In each video, I coded 33 behaviors documented in an ethogram for either occurrence (count) or duration (number of seconds). Descriptive statistics and principal component analysis will be used to analyze the behavioral data.This study’s results will contribute to the development of a behavioral tool that can be used in a variety of settings, which could be utilized by the animal care industry to improve welfare of shelter animals.

Research Grant: NIH student scholar program Student Support: Unknown

Manipulation of DNp63 and Fosl1 modulate the regenerative capacity of pulmonary progenitors

Hanna M. Zayas, Andrew E. Vaughan

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Distal basal-like bronchiolar progenitors (DNp63+ progenitors) are activated by pulmonary injury, acquire expression of Krt5, proliferate, and migrate extensively from the distal airways into the damaged alveolar niche. Post-influenza injury, these Krt5+ cells migrate into the pulmonary parenchyma, forming an ectopic bronchiolar-like epithelium which is thought to not perform gas exchange. DNp63, a well-characterized transcription factor in the epidermal epithelium, is also locally expressed in pulmonary dysplasia and is thought to play a role in regeneration. Prior to injury, DNp63 deletion resulted in the failure of DNp63+ progenitors to both proliferate and migrate from the airways. In contrast, DNp63 deletion post-in- jury allowed for Krt5+ dysplastic cells to convert into AT2 cells, a functionally beneficial cell type typically present in the uninjured alveolar epithelium. As an alternative to dysplastic repair, we simultaneously investigated functionally benefi- cial regeneration of AT2s by targeting Fosl1, a subunit of the AP1 complex that is upregulated in injured AT2s. We utilized an AAV highly specific to AT2 cells to transmit an engineered plasmid encoding the Fosl1 gene driven by doxycycline-re- sponsive elements. Administration of doxycycline in vivo thus allowed us to upregulate Fosl1 expression, which in turn activates a pro-regenerative state. Conversely, we suppressed AP1 in vitro by treating AT2 organoids with an AP1 inhibi- tor, preventing AT2 entrance into this state, evidenced by reduced colony size. Overall, these experiments seek to investi- gate maladaptive Krt5+ ectopic bronchiolization and euplastic AT2 regeneration in hopes to therapeutically eliminate the former and/or promote the latter.

Research Grant: R01HL153539 Student Support: NIH T35OD010919, Boehringer-Ingelheim, and Department of Biomedical Sciences

366 • 2021 National Veterinary Scholars Symposium The nose knows: Validation of an olfactory test in dogs with and without nosework training

Gabriella Zayas Santiago, Micheal Khan, Margaret Gruen

Department of Behavior Medicine, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Olfaction is a cognitive process used for communication and discrimination. Assessment is difficult in dogs as untrained pet dogs rely on other cognitive and searching strategies when completing olfactory trials, and will primarily choose visual cues over olfactory ones. The ability to assess olfaction in dogs would be valuable as a way to monitor sensory decline associated with age and change in cognition. The study aims to validate an olfactory test using adult dogs with and without nosework training. Trials using a hidden food reward are conducted first in light and then in dark to eliminate visual cues, thus requiring dogs to rely on olfaction. The number of correct trials in each setting and latency to find the treat are recorded. It is hypothesized that both control and trained groups will perform better in the dark than in the light, and that the trained dogs will perform better overall than the controls. 20 control and 15 trained dogs completed the trials. On average, control and trained dogs performed similarly in the light and the dark with no significant difference between the groups in both settings (Light P = 0.08; Dark P = 0.27). Across both groups, the dogs performed significantly better in the dark than in the light (P < 0.001), with an average improvement of 18.9%. Trained dogs were slightly faster at the dark trials than control dogs (mean: 13.16s vs 16.63s). The results prove that olfactory trials conducted in the dark drives dogs to engage their noses more frequently than in the light. The similarity between the groups may indicate how alike their capabilities are. These findings establish a standard behavior of healthy adult dogs and allow for a more accurate investi- gation of sensory decline in dogs

Research Grant: None Student Support: Merial Veterinary Research Scholars Program

Efficacy of alfaxalone vs tricaine methanesulfonate as immersive anesthetics in rainbow outtr

Shoshana Zenilman, Daniel Savson, Carmen Smith, Erin Daugherity, Bhupinder Singh, Rodman Getchell

Center for Animal Resources and Education at Cornell University (Zenilman, Savson, Daugherity, Singh), Department of Biomedical Sciences, Section of Anatomic Pathology (Smith), Department of Microbiology and Immunology (Getchell), College of Veterinary Medicine, Cornell University, Ithaca, NY

Tricaine methanesulfonate (MS-222) is currently the only FDA-approved anesthetic for sport fish used for research pur- poses. However, the prolonged withdrawal time of MS-222 impedes the release of wild-caught fish back into the field. Al- faxalone has previously been shown to be a reliable immersive anesthetic in ornamental koi, however, its effects in sport fish are unknown. The purpose of this study was to evaluate the immersive anesthetic profile of alfaxalone versus MS-222 in rainbow trout (Oncorhynchus mykiss). In a complete crossover design, fish (n = 48) were anesthetized, held for 10 min- utes in a recirculating maintenance system, and then recovered. Treatment groups included low-dose alfaxalone (5mg/L induction, 1mg/L maintenance), high-dose alfaxalone (5mg/L induction, 2mg/L maintenance), or MS-222 (150mg/L induction, 100mg/L maintenance). Response to noxious stimuli, opercular rate, time-to-induction (TTI) and time-to-re- covery (TTR) were recorded. We hypothesized that both doses of alfaxalone immersion would provide a stable plane of anesthesia equivalent to that of MS-222, with comparable TTI and TTR. Results indicate both alfaxalone treatments had a significantly longer TTI than MS-222 (P < 0.001). The mean TTI for low-dose alfaxalone, high dose alfaxalone, and MS-222 were measured at 87.2 6 4.24, 91.0 6 4.19, and 58.9 6 4.19 seconds, respectively. Both low and high-dose al- faxalone had a longer mean TTR than MS-222 (10.26 6 0.452, 14.06 6 0.448, 4.72 6 0.452 minutes, respectively), with high-dose alfaxalone significantly longer than low-dose and MS-222 (P < 0.0001), and low-dose significantly longer than MS-222 (P < 0.0001). We conclude that alfaxalone is a clinically viable anesthetic for rainbow trout.

Research Grant: Departmental funding from Center for Animal Resources and Education at Cornell University Student Support: The American Society of Laboratory Animal Practitioners

2021 National Veterinary Scholars Symposium • 367 Efficacy of chitosan and polycaprolactone biomaterials as antibiotic delivery vehicles in osteomyelitis

Haley K. Zetterholm, Luke J. Tucker, Malley A. Gautreaux, Xavier J. Person, and Lauren B. Priddy

Department of Agricultural and Biological Engineering, Bagley College of Engineering, Mississippi State University, Mississippi State, Mississippi

Osteomyelitis, an infection of bone, is often caused by Staphylococcus aureus and poses a significant threat following traumatic fractures or orthopedic hardware implantation. Current treatment modalities include systemic antibiotic ther- apy and debridement surgery; there are no local antibiotic therapies that persist long enough to clear infection. Relapses frequently occur, sometimes leading to limb amputation. The aim of this pilot study was to evaluate the ability of chi- tosan-based local delivery vehicles to reduce bacterial load in a rat osteomyelitis model. Twelve female CD rats were assigned to three groups (n = 4): chitosan gel (CH), chitosan-fosfomycin gel (CH-FOS), or chitosan-fosfomycin gel surrounded by a polycaprolactone scaffold (CH-FOS-PCL). FOS dose was 3 mg. Osteomyelitis was induced via place- ment of an ATCC 6538-GFP S. aureus-soaked screw into a bicortical defect in the mid-diaphysis of the femur. Blood and urine samples were taken periodically starting at Day -1. Bone and soft tissue samples were collected at Day 35 to eval- uate bacterial load. Four rats (1 CH-FOS, 3 CH-FOS-PCL) experienced spontaneous femur fractures and were eutha- nized early. Bone CFU/mL was highest in the CH group and lowest in the CH-FOS group, while soft tissue CFU/mL was highest in the CH-FOS-PCL group and lowest in the CH-FOS group, though differences were not statistically significant. ATCC 6538-GFP was not detected in blood at any time point. The plasma was frozen and will be used to measure infec- tion markers. Urine will be evaluated for fosfomycin to determine elution over time. Future work will further examine chitosan-fosfomycin gel as a local antimicrobial therapy.

Research Grant: National Institutes of Health 5T35OD010432 Student Support: Summer Research Experience, College of Veterinary Medicine, Mississippi State University

Examination of canine peripheral blood NK cells

Cong Zhu, Kristin Snyder, Robert Hullsiek, Kate Dixon, Jianming Wu, Bruce Walcheck

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN

Natural killer (NK) cells are lymphocytes of innate immune system that respond to infected cells and malignant cells with- out prior sensitization. NK cells recognize various ligands and attached antibodies on tumor cells, resulting in natural cy- totoxicity and antibody-dependent cell-mediate cytotoxicity (ADCC), respectively. During these events, NK cells release the cytolytic components perforin and granzyme that result in target cell killing, and they also release various cytokines and chemokines that augment the immune response. CD16A is an IgG Fc receptor (FcgR) expressed by human NK cells that recognizes the Fc portion of IgG antibodies and exclusively mediates ADCC. Much less is currently known about dog NK cells. The focus of my studies is on further characterizing these cells and their ability to kill tumor cells by ADCC. Dog NK cells are identified by their expression of low levels of CD5, lack of CD20 (B cell marker), lack of CD3 (T cell marker), and lack of CD11b (neutrophil and monocyte marker). My studies focused on the enrichment and expansion of dog NK cells. This involved the separation of lymphocytes from canine peripheral blood samples and the enrichment of NK cells by bead-based cell sorting. The enriched cells were subjected to various cell culture conditions using different ratios of artificial antigen presenting cells and various concentrations of IL-2 and IL-15.These conditions were examined to determine if this stimuli induced NK cell expansion. In other assays we examined expression of FcgRs by dog NK cells and their ability to mediate ADCC. The goal of our studies is to further examine phenotypic markers that identify dog NK cells, and to examine their function and expansion for therapeutic use.

Research Grant: UMN CVM, Signature Programs in Comparative Medicine Student Support: Boehringer Ingelheim Veterinary Scholars Program

368 • 2021 National Veterinary Scholars Symposium Evaluation of neuropathic pain mediators in acute and chronic laminitis

Jordyn Zoul, Britta Leise

School of Veterinary Medicine, University of Wisconsin (Zoul), Madison, WI; Department of Veterinary Clinical Sciences (Leise), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA

Laminitis is a disease in horses wherein the lamellar tissues binding the hoof wall fail to adhere to P3. In addition to being extremely painful, it is also common with 13% of horse owners reporting at least one affected horse and a resulting $13 million loss in 2019. Despite the understanding that the disease is painful, little is known regarding the mechanisms of this pain. Because anti-inflammatory therapy is often unsuccessful, there are indications that neuropathic pain may play a role. It is critical to determine which pain mediators, such as neuropeptides, are present in the tissue to successfully study the signaling mechanisms involved. Thus, the objectives of this study are: 1) to determine the gene expression of nerve growth factor (NGF), calcitonin-related gene peptide (CGRP), activating transcription factor 3 (ATF3), and substance P (SP) in the lamellar tissue from horses with laminitis (acute and chronic) and without laminitis (unaffected/control), and 2) to localize the expression of SP in the lamellar tissue. We hypothesize that horses with laminitis will see increased expres- sion of NGF, CGRP, ATF3, and SP when compared to unaffected horses with further differences seen in chronic versus acute. Additionally, we hypothesize that SP will be seen perivascularly within the dermis. To meet Objective 1, gene ex- pression will be evaluated via qRT-PCR. Objective 2 will be met using immunohistochemistry to determine the location of SP expression. Archived tissue samples of laminitic and unaffected horses will be used. It is our hope that this study will lead to conclusions that can provide insight on managing pain in laminitis thereby increasing quality of life and prognoses of laminitic horses.

Research Grant: United States Department of Agriculture (USDA) Student Support: National Institutes of Health (NIH)

2021 National Veterinary Scholars Symposium • 369