CVM Research Forum Jan

January 20, 2012

CVM Annual Research Forum and Litwack Lecture

Friday, January 20, 2012

The Litwack Lectures are supported by the Martin Litwack Fund, an endowment created by family and friends as a living memorial to Dr. Litwack, who was an acknowledged leader in establishing the College of Veterinary Medicine at North Carolina State University. Dr. Litwack was a charter member of the North Carolina Veterinary Medical Foundation and was active in many professional organizations including the North Carolina Veterinary Medical Association, the American Animal Hospital Association and the American Veterinary Medical Association. He served as president of the Triangle, Eastern North Carolina and North Carolina Veterinary Medical Associations. Dr. Litwack was also very active in community affairs and received many awards in recognition of his service and leadership, including the North Carolina Association of the Professions Special Recognition Award. Dr. Litwack is perhaps best remembered for his love of his family and profession, his deep commitment to education and to North Carolina State University, and his strong sense of obligation to the North Carolina livestock industry and to the general public.

2012 Litwack Lecturer : Dr. Oliver Smithies

Dr. Oliver Smithies, Excellence Professor of Pathology and Laboratory Medicine at the University of North Carolina School of Medicine, Weatherspoon Eminent Distinguished Professor, and co-recipient of the 2007 Nobel Prize in physiology or medicine

Schedule of Events

9:00am – 11:30am Oral Presentations

11:30am-1:00pm Posters Judged (Lunch)

1:00pm – 3:00pm Oral Presentations

3:00pm-4:00pm Informal session with Dr. Smithies for Grad

Students

4:00pm-5:00pm Litwack Lecture – Dr. Oliver Smithies

5:00pm – 5:30pm Awards Ceremony (presented by Dr. Smithies) Poster Presentations

Author Poster Abstract (Classification- Title # on Page Mentor) Cassandra Bare 1 (VS-Bailey) THE USE OF CARBETOCIN FOR 1 LONG-TERM ESTRUS SUPPRESSION LAMP: A NOVEL DETECTION 2 Adam Beard METHOD FOR RICKETTSIAL 2 (VS-Breitschwerdt) DISEASES NOVEL COMPOUNDS THAT ALTER Leslie Beck TGFΒ-INDUCED EPITHELIAL- 3 3 (VS-Sannes) MESENCHYMAL TRANSITION IN A549 CELLS AN INVESTIGATION OF CELLULAR Kathleen Bedard PROLIFERATION IN DOGS WITH 4 4 (VS-Mariani) MENINGOENCEPHALITIS OF UNKNOWN ETIOLOGY (MUE) Javier Benito EVALUATION OF MELOXICAM AND 5 5 (GS-Lascelles) TEPOXALIN IN CATS

Javier Benito OWNER-ASSESSED INDICES OF 6 (GS-Lascelles) QUALITY OF LIFE IN CATS AND THE 6 RELATIONSHIP TO THE PRESENCE OF DEGENERATIVE JOINT DISEASE. PRACTICAL MORPHOMETRICS IN VARIOUS STRANDED MARINE Heather Beveridge MAMMAL SPECIES IN THE NORTH 7 7 (VS-Correa) ATLANTIC: REFERENCE VALUES FOR NECROPSY AND DRUG DOSAGE APPLICATIONS. MICROBUBBLE DELIVERY OF DII TO Sydney Cartiff THE OCULAR POSTERIOR SEGMENT: 8 (VS) COMPARISON OF INTRAVITREAL 8 AND SUPRACHOROIDAL INJECTION ROUTES PRACTICES AND PERSPECTIVES OF Caitlin Cavanaugh 9 NORTH CAROLINA VETERINARIANS 9 (VS-Cowen) TOWARD WILDLIFE CARE

LACK OF GENOMIC Jaewook Chung IMPRINTING OF DNA PRIMASE, 10 10 (GS-Piedrahita) POLYPEPTIDE 2 (PRIM2) IN HUMAN TERM PLACENTA AND WHITE BLOOD CELLS.

Carolyn Collier NEUTROPHIL RESPONSE AND MMP9 11 11 (VS-Yoder) EXPRESSION IN ZEBRAFISH (DANIO RERIO) EMBRYOS EXPOSED TO IMMUNE AGONISTS

Katelyn Cordle INHIBITION OF CYCLOOXYGENASE-1 12 (VS-Fogle) AND -2 IN EQUINE BLOOD BY 12 PHENYLBUTAZONE, FLUNIXIN, MELOXICAM AND FIROCOXIB: AN EX VIVO ANALYSIS IS THE INHIBITORY ROLE OF MHCI IN 13 Hayley Dirscherl NATURAL CYTOTOXICITY 13 (GS-Yoder) EVOLUTIONARILY CONSERVED? CYTAUXZOON FELIS CYTOCHROME Megan Downey B GENOTYPE IS ASSOCIATED WITH 14 14 (GS-Birkenheuer) SURVIVAL IN DOMESTIC CATS WITH CYTAUXZOONOSIS EXPLORATION OF WILD CANID GENOMES USING CHROMOSOME- Shannon Duke 15 SPECIFIC PROBES SHOW THEY 15 Becker (GS-Breen) SHARE EVOLUTIONARY BREAKPOINTS

Brett Gulledge COMPARISON OF SELECTED STRETCH POSITIONS OF THE 16 (VS-Marcellin- 16 PIRIFORMIS MUSCLE USING Little) COMPUTED TOMOGRAPHY AND BIOMODELING T REGULATORY CELLS HAVE A Jennifer Hendricks HIGHER PRODUCTIVE VIRAL LOAD 17 (VS-Fogle, THAN T HELPER CELLS DURING 17 Tompkins) BOTH A CHRONIC AND ACUTE FIV INFECTION IN CATS. Amanda Jeffries OLFACTORY THRESHOLD OF 18 (VS- Dorman) C4/TOLUENE BY EXPLOSIVE 18 DETECTION DOGS MOUSE KERATINOCYTE SIDE- POPULATION PLAYS A UNIQUE ROLE Sun Hye Kim (GS- 19 DURING MALIGNANT PROGRESSION 19 Rodriguez-Puebla) TO SKIN SQUAMOUS CELL CARCINOMAS EFFECT OF ASYMPTOMATIC FELINE David Kleisch HYPERTROPHIC CARDIOMYOPATHY 20 20 (VS-DeFrancesco) ON ACTIVITY LEVELS AND QUALITY OF LIFE PHYLOGENETIC ANALYSIS OF CAMPYLOBACTER FROM Leanne Magestro COMMERCIAL AND 21 (VS-Thakur) ANTIMICROBIAL-FREE (ABF) 21 PRODUCTION SYSTEMS REVEALS COMMON ANCESTRY AND A NON- CLONAL POPULATION RELEASE RATE OF CRYSTALLINE Megan Mathias RAPAMYCIN IN VITRO AND 22 22 (VS-Gilger) INTRAVITREALLY IN EX VIVO HORSE EYES. TORIN1 IS A POTENT INHIBITOR OF Kathleen McGinnis 23 MTOR-MEDIATED CELL SIGNALING 23 (VS-Sannes) AND LIPOGENESIS IN H292 CELLS

GARP-BOUND TGFB COMPLEXES ON Michelle Miller FELINE TREGS ARE UPREGULATED 24 (GS- Tompkins) DURING ACUTE FIV INFECTION AND 24 CONTRIBUTE TO IMMUNE SUPPRESSION OF CD4 TH VIA LIGATION OF TH TGFBRII.

OPTIMIZATION OF REAL TIME Momo Tanaka MICROSCOPY FOR ASSESSMENT OF 25 (VS-Piedrahita) RATE OF HISTONE EXCHANGE 25 AFTER SOMATIC CELL NUCLEAR TRANSFER (SCNT) USING GFP- LABELED HISTONE 2B Christina Park BACTERIAL COUNTS IN BEDDING 26 (VS-Anderson) MAY PREDICT BULK TANK 26 BACTERIAL POPULATION CANINE ORAL MELANOMA - CYTOGENETIC CHARACTERIZATION 27 Kelsey Poorman OF FRESH TISSUE, CELL LINES AND 27 (GS-Breen) ARCHIVAL TISSUES

DEVELOPMENT OF A NOVEL Jessica Tarter COGNITIVE/MOTOR PARADIGM TO 28 28 (VS-Sherman) DOCUMENT FIV DISEASE IN LABORATORY CATS Bradley Waffa CLINICAL ANATOMY OF THE BALL 29 29 (VS-Stoskopf) PYTHON (PYTHON REGIUS)

Jessica Wofford SEQUENCING ANTIDIURETIC 30 (VS-Hauck) HORMONE IN CATS WITH CENTRAL 30 DIABETES INSIPIDUS Guanxi Xiao THE ROLE OF EPIDERMAL GROWTH 31 (GS-Ghashghaei) FACTOR RECEPTOR IN 31 NEUROGENESIS AND GLIOGENESIS

Oral Presentations

Author Abstract Time (Classification- Title on Page Mentor) Joanne

Fernandez- ANGIOPOIETIN-2 SERUM LEVELS IN 9:00-9:10 Lopez 32 CANINE MALIGNANT MELANOMA (VS-Hauck) PATIENTS

INCIDENCE OF ONYCHECTOMY IN Laura Lockhart CATS PRESENTED FOR VETERINARY 9:10-9:20 (VS-Posner) CARE NEAR RALEIGH, NC AND 33 EDUCATIONAL ATTITUDES TOWARDS THE PROCEDURE Ryan Bray STARGAZERS AND STEM CELLS: A (VS- POTENTIAL THERAPEUTIC 9:20-9:30 34 Ghashghaei) APPROACH TO TREATING IDIOPATHIC SEIZURES Sehwon Koh GENERATION OF PUTATIVE (GS- INDUCED PLURIPOTENT STEM CELLS 9:30-9:40 35 Piedrahita) (IPS) FROM ADULT CANINE FIBROBLAST THE TRANSCRIPTION FACTOR Huixuan Liang SPECIFICITY PROTEIN 2 (SP2) IS (GS- 9:40-9:50 REQUIRED FOR PROGRESSION 36 Ghashghaeis) THROUGH THE CELL CYCLE OF

NEURAL PROGENITORS Laura MECHANISMS OF VESICULAR Sommerville 9:50-10:00 TRAFFICKING IN THE NEURAL STEM 37 (GS- CELL NICHE Ghashghaei) Nagendran A PHARMACOGENETIC TOOL TO Muthasamy STUDY PHYSIOLOGICAL FUNCTIONS 10:00-10:10 (GS- 38 OF POSTNATAL NEUROGENESIS IN Ghashghaei) THE OLFACTORY BULBS.

MITOCHONDRIAL GENOME SEQUENCES RESULT IN IMPROVED Megan Downey UNDERSTANDING OF THE 10:10-10:20 (GS- 39 PHYLOGENETIC RELATIONSHIPS OF Birkenheuer) PIROPLASMA THAT INFECT COMPANION ANIMALS Rachael PROTECTING THE HEART: HSP25 AS Stebbing 10:20-10:30 AN INNATE RESPONSE TO VIRAL 40 (GS-Sherry) INFECTION

Kristin THE SEDATIVE EFFECTS OF Messenger ORAL-TRANSMUCOSAL 10:30-10:40 41 (GS-Papich) DETOMIDINE GEL IN COMPARISON WITH INTRAVENOUS DEXMEDETOMIDINE IN DOGS.

Kate Medl EFFECTS OF MS 222 ON Bailey 10:40-10:50 ELECTRORETINOGRAPHY (ERG) IN 42 (HO-Posner) KOI FISH

A.J. Van Wettere ACTIVATION OF THE TGF-Β 10:50-11:00 (GS-Kullman, SIGNALING PATHWAY IN A DMN- 43 Law) INDUCED FISH MODEL OF HEPATIC FIBROSIS MAST CELL CORTICOTROPIN- Amelia Gibson RELEASING FACTOR RECEPTORS 1:00-1:10 (GS-Moeser) PLAY OPPOSING ROLES IN 44 MEDIATING STRESS-INDUCED BARRIER DYSFUNCTION THE DEVELOPMENT OF A LARGE Liara Gonzalez ANIMAL MODEL FOR 1:10-1:20 (GS-Blikslager) TRANSLATIONAL STUDIES OF 45 INTESTINAL DISEASE FOCUSING ON INTESTINAL STEM CELLS Brittney EARLY WEANING IMPAIRS GUT McLamb MUCOSAL DEFENSES AND 1:20-1:30 46 (VS-Moeser) EXACERBATES CLINICAL DISEASE IN F18 E. COLI INFECTION CRF-MEDIATED MAST CELL Laura Riggs ACTIVATION AND INTESTINAL 1:30-1:40 47 (VS-Moeser) BARRIER INJURY IS MEDIATED BY THE ENTERIC NERVOUS SYSTEM EVALUATION OF DOPPLER AS A Jason Heitzman TECHNIQUE TO DETECT CHANGES 1:40-1:50 (VS-Bailey) ASSOCIATED WITH ASCENDING 48 BACTERIAL PLACENTITIS IN THE MARE Meghann ELASTOGRAPHIC EVALUATION OF 1:50-2:00 Lustgarten 49 THE EQUINE DISTAL FORELIMB (HO-Seiler) THE CANINE MHC CLASS I ALLELE, Peter Ross DLA-88*50801, PRESENTS PEPTIDES 2:00-2:10 50 (GS-Hess) OF VARYING LENGTHS WITH FOUR KEY ANCHOR RESIDUES CLINICAL ANATOMY OF THE Emily Wayne 2:10-2:20 BEARDED DRAGON (POGONA 51 (VS-Stoskopf) VITTICEPS) DEVELOPMENT OF A QUESTIONNAIRE TO ASSESS Javier Benito 2:20-2:30 CHRONIC PAIN ASSOCIATED WITH 52 (GS-Lascelles) FELINE DEGENERATIVE JOINT DISEASE. CHANGES IN VON FREY QUANTITATIVE SENSORY Alexander THRESHOLDS AS A RESULT OF 2:30-2:40 Bennett 53 TOTAL HIP REPLACEMENT IN THE (HO-Lascelles) TREATMENT OF COXOFEMORAL JOINT OSTEOARTHRITIS Morika 54 COMPARISON OF THERMAL Williams & THRESHOLD LATENCIES BETWEEN Amy 2:40-2:50 NORMAL DOGS AND DOGS WITH Kirkpatrick HINDLIMB OSTEOARTHRITIC (VS-Lascelles) ASSOCIATED PAIN

THE USE OF CARBETOCIN FOR LONG-TERM ESTRUS SUPPRESSION

Cassandra A. Bare, Anne R. Schramme, C. Scott Bailey, Jason M. Heitzman, Renan D. Sper, Kate Archibald, Michael Whitacre North Carolina State University, College of Veterinary Medicine North Carolina Horse Council, internal NCSU CVM, and Veyx Pharma GmbH (Germany)

Estrus behavior can negatively impact performance mares. Prolongation of the interovulatory interval through oxytocin injections represents a safe, reversible means of controlling estrus behavior. The purpose of this study was to determine whether an oxytocin analog, carbetocin, could increase the interovulatory interval similarly to oxytocin. We hypothesized that mares would have a longer interovulatory interval after treatment with oxytocin or carbetocin than during untreated cycles. Twelve cycling mares were randomly assigned to one of two groups (CARB and OXY). One normal cycle was documented in all mares during April/May. From day 7 to 14 post ovulation, mares were administered either 1.19mg of carbetocin (CARB) or 60 IU of oxytocin (OXY) once daily via IM injection. Blood was drawn from all mares once weekly for progesterone assay. Mares were teased every other day to a fertile stallion to detect estrus behavior and subsequently examined by ultrasonography to detect ovulation between May and August. Administration of carbetocin decreased the interovulatory interval in all mares (18±1.1 vs. 21.8±2.2; P=0.003). Administration of oxytocin increased the interovulatory interval in 4/6 mares (67%) (48±29.9 vs. 21.8±1.5; P=0.03) and delayed estrous behavior in 3/6 mares (50%). Two mares failed to return to estrus before the end of the study; the diestral length at that time was used for calculations. Luteal regression and subsequent ovulation was confirmed with serum progesterone concentration. This work suggests that carbetocin, an analog of oxytocin, cannot be used for long-term estrus suppression in the mare.

1 LAMP: A NOVEL DETECTION METHOD FOR RICKETTSIAL DISEASES

Adam W. Beard1,2 and William L. Nicholson1, Dr. Ed Breitschwerdt 1Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Atlanta, GA 2North Carolina State University College of Veterinary Medicine, Raleigh, NC

Simple and effective amplification methods of detection of rickettsial species are not readily available in many parts of the world, particularly those with limited resources for laboratory support. LAMP (Loop Mediated Isothermal Amplification) offers a specific and sensitive method of detection for nucleic acids in various specimens at low cost. Rickettsia species are important pathogens globally and are often not recognized due to their non-specific symptomology and lack of diagnostic laboratory capacity. Development of robust assays for the detection of these pathogens would be useful, and LAMP assays provide a number of advantages over PCR assays. We have designed multiple LAMP assays for the major spotted fever group rickettsiae (Rickettsia conorii and R. africae) and for the typhus group rickettsiae (R. prowazekii and R. typhi) that cause human illness in many parts of the world. The primer sets were designed using PrimerExplorer software. Reaction conditions were optimized through systematic testing of multiple magnesium concentrations, primer concentrations, reaction temperatures, and reaction times. This study reports the ongoing results from the use of these assays to detect rickettsiae in a variety of specimen types, describes their parameters, and compares the detection limits to those obtained using our conventional PCR assays.

Funding: CDC Rickettsial Zoonoses Branch funded the research Student was not funded for this project

2 NOVEL COMPOUNDS THAT ALTER TGFβ-INDUCED EPITHELIAL- MESENCHYMAL TRANSITION IN A549 CELLS

Leslie L. Beck, Donna R. Newman, Philip L. Sannes Molecular and Biomedical Sciences Department North Carolina State University College of Veterinary Medicine Raleigh, NC

Idiopathic pulmonary fibrosis (IPF) is a terminal condition characterized by excessive accumulation of connective tissue with the complete loss of normal lung morphology and function. A potential target for treatment is the fibrogenic cytokine, transforming growth factor beta (TGF-β). We hypothesized that heterotaxin, a novel inhibitor of TGF-β-signaling, might alter the latter’s involvement in the epithelial-mesenchymal transition (EMT) process characteristic of IPF. A549 cells, a pulmonary adenoma cell line, were induced to undergo EMT with TGF-β with or without treatment with heterotaxin or one of its analogs, and monitored for morphologic changes indicative of EMT. Western blots were performed to analyze the expression of epithelial or mesenchymal protein markers. Neither heterotaxin nor an inactive analog completely inhibited the EMT phenotype triggered by TGF-β. A more-potent analog caused A549s to assume a dendritic morphology uncharacteristic of either epithelial or mesenchymal cells. Western analysis revealed that after 48 hours of treatment with both the potent analog and TGF-β, A549 cells retained the epithelial marker E-cadherin while some mesenchymal markers, such as collagen 1A1 and connective tissue growth factor, were clearly evident at 48 hours. These results suggest a novel mechanism behind this disruption of TGF-β-induced EMT and lay the groundwork for further research. These findings suggest that heterotaxin and its potent analog may be of potential value in treatment of conditions involving EMT. If successful in IPF lungs, these or similar compounds could prove beneficial in the treatment of fibrosis in other organs such as the liver, eye, heart, and kidney.

3 AN INVESTIGATION OF CELLULAR PROLIFERATION IN DOGS WITH MENINGOENCEPHALITIS OF UNKNOWN ETIOLOGY (MUE)

Kathleen M. Bedard, Stan Dunston, Chris L. Mariani NCSU CVM

Meningoencephalitis of unknown etiology (MUE) is a common, debilitating disorder in dogs. Despite its frequency, little is known about the pathogenesis of MUE. Believed an aberrant immune response directed against cells of the CNS, current treatment centers on the use of immunosuppressive medications. Despite suggestions of the effectiveness of this method of treating MUE, the exact role of these drugs in MUE therapy remains unknown. We hypothesized that MUE involves excessive proliferation of immune cells around brain vasculature and that immunosuppressive drugs target this cellular proliferation. The first goal of this study was to identify abnormal cellular proliferation in archival MUE brain sections by utilizing immunohistochemistry for Ki-67. The second goal was to investigate the effects of immunosuppressants on proliferating immune cells. Peripheral blood mononuclear cells (PBMCs) were labeled with CFSE, stimulated with mitogens and cultured in vitro with or without the immunosuppressive drug Cytarabine, and cellular proliferation was evaluated using flow cytometry. Many MUE brain sections showed significant proliferation (2.9- 13% Ki-67+ cells), particularly those from untreated patients. However, in vitro proliferation studies showed no appreciable difference with the addition of Cytarabine. Our IHC results indicate that cellular proliferation occurs in the brain of dogs with MUE. The failure of Cytarabine to show an anti-proliferative effect may indicate that this drug acts via a different mechanism to alleviate the symptoms of MUE, or may be an artifact of this in vitro system. Double labeling immunohistochemistry studies are underway to characterize the nature of the proliferating cells in the archival sections.

Funded by Merck-Merial

4 EVALUATION OF MELOXICAM AND TEPOXALIN IN CATS

Benito J 1, Charlton AN1, Simpson W 2, Lascelles BD 1 1. Comparative Pain Research Laboratory (CPRL) & Center for Comparative Medicine and Translational Research. Dept. of Clinical Sciences. NCSU CVM. 2. Morrisville Cat Hospital. Morrisville, NC.

There have been recent concerns surrounding the use of NSAIDs to treat pain in cats. The purpose of this study was to review retrospectively the prescriptions of meloxicam (Metacam®) and tepoxalin (Zubrin®) in cats, and furthermore to evaluate the effect of tepoxalin on different blood and urine diagnostic values. Medical records (n=262) were obtained from the Morrisville Cat Hospital. The criteria for this review were to include clinical pathology panels before and after courses of NSAIDs. Age, diagnosis, NSAIDs prescriptions, dose, duration, pre-existing diseases, concurrent medications, adverse events were recorded. Two-tailed paired tests were performed for exploring significant relevance (p<0.05) between initial versus final laboratory tests (chemistry and CBCs panels, urinalyses and T4 tests) in cats prescribed with tepoxalin prescription cases. A total of 78 medical records fit the inclusion criteria for this study (n=32 and n=46, meloxicam and tepoxalin respectively). The average doses administered were 0.024 and 12.09 mg/kg/day (meloxicam and tepoxalin, respectively). The median prescription durations were 149 (4-1604) and 10.5 (2-807) days for meloxicam and tepoxalin respectively. Suspected adverse events were reported for meloxicam (18.75%, 6/32 cats) and tepoxalin (8.69%, 4/46 cats) after prescription started (66 days and 0.0085mg/kg/day for meloxicam and 3.5 days and 10.79mg/kg/day for tepoxalin). For cats prescribed with tepoxalin there were no statistically significant changes for chemistry (n=9) and CBCs (n=6) panels, and urinalyses (n=40) and T4 (n=6) tests Tepoxalin and meloxicam can be used to treat pain in cats. Further investigation is warranted for tepoxalin.

Funding Source(s): Comparative Pain Research Laboratory-NCSU.

5

OWNER-ASSESSED INDICES OF QUALITY OF LIFE IN CATS AND THE RELATIONSHIP TO THE PRESENCE OF DEGENERATIVE JOINT DISEASE.

Benito J 1, Gruen ME 2,1, Thomson A 1, Simpson W 3, Lascelles BDX 1 1.Comparative Pain Research Laboratory (CPRL) & Center for Comparative Medicine and Translational Research. Dept. of Clinical Sciences. NCSU CVM. 2.Animal Behavior Service. Dept. of Clinical Sciences. NCSU CVM. 3.Morrisville Cat Hospital. Morrisville, NC.

Little is known about what items owners consider important for their pet’s quality of life (QoL). Mobility alterations are thought to be associated with degenerative joint disease (DJD) in cats. The objectives of the study were to describe the types of items considered important by owners for their cats’ quality of life; to describe the proportion of these items that involve mobility; to evaluate what patient factors, including the severity of DJD, affect this distribution. This prospective, observational study included 166 client-owned cats. A weighted QoL questionnaire was used to collect data. QoL items were categorized into six behavioral domains and further designated by the degree of mobility involved. Items were divided into active (AA), inactive (IN) and items with implied activity (IA). Distributions of AA, IA and IN items were described. Backwards stepping regression analysis was used to assess the effect of variables on QoL item distributions. 840 client-generated items were evaluated. Regardless of DJD status, 40% of items listed involved mobility, while 60% were “inactive” items. Increasing age was associated with decreased AA frequency (P<0.0001) and increased IN frequency (P<0.0001). Increased bodyweight was associated with decreased IA frequency (p=0.0012). No other variables affected score distribution. Overall QoL score was significantly lower in cats with worse temperament (p=0.0029) and higher DJD scores (p=0.01); age was not significant in this model. These results highlight the need to assess non-active items that owners consider constitute QoL to fully assess the impact of diseases like DJD.

Funding Source(s): Morris Animal Foundation; Novartis Animal Health Global Fellowship Program.

6 PRACTICAL MORPHOMETRICS IN VARIOUS STRANDED MARINE MAMMAL SPECIES IN THE NORTH ATLANTIC: REFERENCE VALUES FOR NECROPSY AND DRUG DOSAGE APPLICATIONS.

Heather Beveridge, Dr. Craig Harms, Dr. Maria Correa NCSU-CVM, Center for Marine Sciences and Technology, NCSU-CVM

The aim of the study was to develop a reference interval based on field data collected between 1991 to present. We examined the correlation of total body length and weight, as well as, organ volume to organ weight in marine mammal species stranded along the North Atlantic coast. Necropsy data on stranded marine mammals were provided by the Center for Marine Sciences and Technology, University of North Carolina at Wilmington and the Virginia Aquarium Stranding Program. The data were analyzed using univariate and multivariate regression models (organ weights). We used polynomials models for length and weight. Correlation coefficients were obtained for bottlenose dolphins’ organ volumes and weights. A strong positive association (R-sq > 90% except for the harbour porpoise) was found in the bottlenose dolphin and other marine mammal species including the common dolphin, Risso’s dolphin, pygmy sperm whale, dwarf sperm whale, and striped dolphin. Strong linear correlations were found between organ volumes and organ weights in bottlenose dolphins for the adrenals, spleen, kidneys, testes, and ovaries (Pearson correlation > 90%). The regression equations generated in this study may be used as a tool for scientists and veterinarians to estimate the body weight of a marine mammal if a body length is available or vice versa. One of the most important uses for these equations is for necropsy or drug dosage under field conditions. The data collected for the study could also be used as a reference to determine whether an organ is reduced or enlarged.

Funding source(s): Various university funding (NCSU-CVM, CMAST, UNCW, Virginia)

7 MICROBUBBLE DELIVERY OF DII TO THE OCULAR POSTERIOR SEGMENT: COMPARISON OF INTRAVITREAL AND SUPRACHOROIDAL INJECTION ROUTES

Sydney Cartiff, Gabriela S. Seiler2, Paul Dayton3, Jacklyn H. Salmon2, Brian C. Gilger2 1Clinical Sciences, 2Molecular Biosciences, North Carolina State University, Raleigh, NC; 3Biomedical Engineering, North Carolina State University and The University of North Carolina, Chapel Hill, NC.

Dye distribution to the ocular posterior segment via an intravitreal (IV) or anterior suprachoroidal space (SCS) injection of free or microbubble-contained 1,1’-Dioctadecyl- 3,3,3’,3’-tetramethylindocarbocyanine iodide (DiI) was evaluated. Canine cadaver eyes received an IV or SCS injection (250 uL) of free or microbubble DiI. Eyes injected with microbubbles were simultaneously imaged with ultrasound and received 0, 1, or 3 pulses to destroy microbubbles once in the posterior segment. Eyes were frozen in liquid nitrogen, stored at -80°C until sectioned at 5-10 um at -30°C. Dye observed using fluorescent microscopy was semi- quantified using scores of 0 (none) to 4 (saturated) in areas of the superior, central, and inferior retina. Mean cumulative scores were analyzed using wilcoxon rank sum test and differences significant at P<0.05. Ultrasound illustrated that microbubble contrast was present in the ocular posterior segment within 10 seconds of injection. Mean cumulative scores of retinal dye were similar between IV (2.0 +/- 1.7) and SCS (1.8 +/- 0.88) for free DiI, and higher for 1 pulse microbubble delivery of DiI (IV: 2.9 +/- 1.4 ; SCS: 2.8 +/- 2.3). However, 3 pulse microbubble destruction resulted in significantly higher delivery of DiI to SCS (P=0.043) (4.1 +/- 0.8) compared to IV (2.7 +/- 1.1). Microbubbles as a delivery vehicle and ultrasound may improve drug delivery to the ocular posterior segment. Furthermore, the SCS may allow targeted drug delivery to the ocular posterior segment and use of these modalities together (microbubbles and SCS injections) may result in more effective drug delivery to the retina.

8 PRACTICES AND PERSPECTIVES OF NORTH CAROLINA VETERINARIANS TOWARD WILDLIFE CARE

Caitlin Cavanaugh, Dr. Peter Cowen The Piedmont Wildlife Center, NCSU CVM

Little data is available on veterinary practice trends in wildlife care and treatment. Information such as common species seen, amount of care given, and average costs to the veterinarian and client are essentially unknown. In 2010, a survey was created to assess the current practices of veterinarians in North Carolina. The survey also served to gauge interest, previous educational background, and preferences in regards to wildlife education opportunities for veterinarians. Furthermore, it also sought reasoning for lack of interest, which incentives encourage treatment of wildlife, and to identify whether a network between veterinarians, wildlife centers, and the public is both warranted and feasible. The questionnaire was conducted at the North Carolina Veterinary Conference in November 2010. Of approximately 800 veterinarians in attendance, 123 completed surveys were returned. The survey consisted of 17 questions and a section for comments. Data frequency analysis was conducted by hand and by the program Epiinfo. Survey responses showed variability in amount of care given, as well as reasoning for not providing wildlife care. Many respondents showed interest in wildlife medicine education, but reported being unaware of wildlife related training opportunities. As to financial responsibility for wildlife, there was not a consensus for a single entity financially responsible. Time, cost, and transportation were considered relevant factors impacting veterinarian decisions to engage in wildlife practice. Contingincies for interest in participation in a wildlife care network included lack of time or training, and practice goals. Overall, availability of transportation was valued to participate in a wildlife care network.

Funding: Piedmont Wildlife Center

9 LACK OF GENOMIC IMPRINTING OF DNA PRIMASE, POLYPEPTIDE 2 (PRIM2) IN HUMAN TERM PLACENTA AND WHITE BLOOD CELLS.

Jaewook Chung1, Shengdar Tsai1, Andra H. James2, Betty H. Thames2, Stephanie Shytle2 and Jorge A. Piedrahita1 1Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC and 2Department of Obstetrics and Gynecology, Duke University School of Medicine, Durham , NC

Genomic imprinting is an epigenetic process that leads to allelic differential gene expression in a parent-of-origin specific way, influencing development, growth and behavior in mammals. Over 100 genes have been conclusively demonstrated to be imprinted in mammals, however, both in silico prediction studies and massive parallel sequencing suggest that there may be as many as 1,000 imprinted genes. PRIM2, encoding a large subunit of primase involved in DNA replication and transcription, is highly expressed in the placenta, and is crucial for mammalian development and growth. Its role in placenta function is not well understood. Recently, PRIM2 has been reported as an imprinted, maternally- expressed gene in human white blood cells, whereas we identified PRIM2 as a gene up-regulated in small for gestational age (SGA) placentas. In order to further understand the role of PRIM2 in the human placenta we examined whether the PRIM2 gene was imprinted in the placenta, as reported for WBC. Here, we report our failure to confirm imprinting of the PRIM2 locus in human placenta or white blood cells. Our specific PCR assay, followed by utilization of a commercial sequencing, proves that the discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database, which may lead to miscalculations of PRIM2 allelic frequencies in a population and lead to misinterpretation of results.

Funding Sources: NIH Grant HD048510 to JP and AJ, and ATPM/CDC SUBAWARD #TS-1051

10 NEUTROPHIL RESPONSE AND MMP9 EXPRESSION IN ZEBRAFISH (DANIO RERIO) EMBRYOS EXPOSED TO IMMUNE AGONISTS

Carolyn M. Collier, Iván Rodriguez, Shila K. Nordone, Jeffrey A. Yoder North Carolina State University College of Veterinary Medicine

Matrix metalloproteinase 9 (mmp9) is involved in extracellular-matrix remodeling during several cell-migration events, such as neutrophil chemotaxis, in vertebrate species. Results from previous microarray studies using zebrafish embryos exposed to two immune agonists, PAM3CSK4 and poly(I:C), showed increased levels of mmp9 mRNA (Heffelfinger and Yoder unpublished). However, it is uncertain whether increased mmp9 expression was specific to neutrophil activity (i.e. neutrophil number and/or migration) or was a generalized response of embryonic cells to immune stimuli. This study determined if PAM3CSK4- or poly(I:C)-induced mmp9 upregulation reflected increases in neutrophil number and migrations in zebrafish embryos. Neutrophil numbers and distribution were examined using a transgenic zebrafish line in which GFP is under the control of the myeloperoxidase promoter resulting in GFP-expressing neutrophils. As embryos are transparent, neutrophil number and location may be observed with fluorescent microscopy. Studies were conducted using three-day old embryos exposed to PAM3CSK4 or poly(I:C) in water. At various time points after exposure, embryos were sedated with a tricaine solution and either photographed under fluorescent microscopy or homogenized and subjected to flow cytometry. Although microscopy results revealed biological variability in neutrophil number among individual embryos in the same treatment group, preliminary results suggested a possible increased neutrophil number 24 hrs after PAM3CSK4 exposure. Preliminary flow cytometry analyses suggested a possible increased neutrophil number 4 and 24 hrs after PAM3CSK4 exposure and 24 hrs after poly(I:C) exposure. These studies will help elucidate the role of mmp9 and neutrophils in the innate immune response in a whole organism that may provide insights into immune function in other vertebrate species.

This project was supported by Merial and NIH R21 AI076829.

11 INHIBITION OF CYCLOOXYGENASE-1 AND -2 IN EQUINE BLOOD BY PHENYLBUTAZONE, FLUNIXIN, MELOXICAM AND FIROCOXIB: AN EX VIVO ANALYSIS

Katelyn Cordle, Callie A. Fogle, John F. Marshall, Anthony T. Blikslager North Carolina State University College of Veterinary Medicine

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in equine clinical practice for relief of pain and inflammation in cases of colic, lameness, and endotoxemia. They produce anti-inflammatory and analgesic effects by inhibiting cyclooxygenase enzymes (COX-1 and COX-2) in the arachidonic acid cascade, thereby blocking the production of prostaglandins (PGE2) and thromboxane (TXB2). While COX-2 is inducible and pro-inflammatory, COX-1 is expressed constitutively and serves “housekeeping” functions, such as maintenance of renal blood flow and protection of the gastrointestinal mucosa. The use of nonselective NSAIDs in particular has therefore been associated with adverse effects such as renal toxicity and gastrointestinal injury. Our aim in this study was to measure the ex vivo effects of phenylbutazone, flunixin, meloxicam and firocoxib on cyclooxygenase activity in the horse. Three clinical doses of each drug were administered to healthy horses (n=3). Blood samples were collected at 13 timepoints throughout each dosing period (0 to 120 hours). TXB2 and PGE2 concentrations were determined using commercially available EIA kits to serve as indicators of COX-1 and COX-2 activity, respectively. Administration of each drug was associated with suppression of TXB2 and PGE2, suggesting inhibition of COX-1 and COX-2 activity. The duration of suppression of PGE2 and TXB2 was at least 24 hours after final administration of q 12 and q 24 drugs. By 92 hours, however, PGE2 levels had risen above baseline levels for horses receiving phenylbutazone, firocoxib or meloxicam. Statistical analysis of comparative COX-1/COX-2 activity for each drug is ongoing.

This project was made possible by my mentor’s startup funding.

12 IS THE INHIBITORY ROLE OF MHCI IN NATURAL CYTOTOXICITY EVOLUTIONARILY CONSERVED?

Hayley Dirscherl1,2 and Jeffrey A. Yoder1 1. Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC 27606 2. Joint Department of Biomedical Engineering, University of North Carolina – Chapel Hill and North Carolina State University, Raleigh, NC 27695

Mammalian natural killer (NK) cells recognize and directly lyse transformed or infected cells through a process termed cytotoxicity. Cytotoxicity in mammals is mediated by activating and inhibitory NK receptors (NKRs) which regulate whether or not an NK cell will kill another cell. While there is data to suggest that many mammalian NKRs recognize major histocompatability complex I (MHCI) as ligands on target cells, little is known about the molecular mechanisms of cytotoxicity in non-mammalian vertebrate species such as zebrafish. Herein we demonstrate that zebrafish splenocytes can recognize and directly kill YAC1 murine tumor cells, K562 human tumor cells, ZF4 zebrafish fibroblast cells, and transgene derived zebrafish tumor cells. Additionally, a novel PCR-based MHCI genotyping strategy has been used to develop three different MHCI matched zebrafish lines. Together these tools will be used to test the hypothesis that the role of MHCI as a marker of “self” is evolutionarily conserved in zebrafish, providing a new model for understanding how immune cells recognize transformed and infected cells.

13 MITOCHONDRIAL GENOME SEQUENCES RESULT IN IMPROVED UNDERSTANDING OF THE PHYLOGENETIC RELATIONSHIPS OF PIROPLASMA THAT INFECT COMPANION ANIMALS

Megan Downey1, Henry Marr1, Jaime Tarigo1, Leah Cohn2, David Bird3, Betsy Scholl3, Mike Levy1, Adam Birkenheuer1. 1. North Carolina State University College of Veterinary Medicine, Raleigh, NC. 2. University of Missouri College of Veterinary Medicine, Columbia, MO. 3. North Carolina State University College of Agriculture and Life Sciences, Raleigh, NC

Based on phylogenetic analyses using 18S ribosomal ribonucleic acid (rRNA) gene sequences, parasites in the order Piroplasmida have been sub-categorized into at least five distinct groups1. Piroplasma from these groups infect common companion animal species—dogs and cats. In this study, we characterized the mitochondrial genome sequence and structure from these piroplasma. We hypothesized that use of mitochondrial gene sequences would improve upon previous phylogenetic analyses that used 18S rRNA genes alone. Blood was collected from dogs or cats infected with species representing the proposed sub-groups: Babesia microti-like sp. (AKA, Theileria annae), B. conradae, B. canis canis, B. canis rossi, B. canis vogeli, an un-named Babesia sp. (“Coco”), and Cytauxzoon felis. DNA was isolated and the entire mitochondrial genome (5.6-5.9 kb) was PCR-amplified in overlapping fragments. Amplicons were sequenced bi-directionally, and contigs were assembled using a commercially available software package2. Mitochondrial genomes were annotated for cox1, cox3, cytb and ribosomal subunit genes. Phylogenetic trees were constructed using both mitochondrial genes and 18S rRNA gene (Neighbor-joining, 1000 bootstrap replicates3). Utilization of mitochondrial genes improved bootstrap support and resolved uncertainties concerning the relationship between Cytauxzoon species and Theileria species compared to trees based on 18S gene sequence alone. Additionally, one sub-group characterized by its tropism for ungulid hosts was shown to include a species (Babesia sp. “Coco”) that parasitizes dogs. In conclusion, we demonstrate the utility of the mitochondrial genome structure and sequence for phylogenetic analyses, and propose that future studies include mitochondrial genes to further define evolutionary relationships between Piroplasmida species.

Funding provided by: An anonymous Donor

14 EXPLORATION OF WILD CANID GENOMES USING CHROMOSOME-SPECIFIC PROBES SHOW THEY SHARE EVOLUTIONARY BREAKPOINTS

Shannon E Duke Becker1, Rachael Thomas1,2, Vladimir A Trifonov3, Robert K Wayne4, Alexander S Graphodatsky3, Matthew Breen1,2,5

1 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, 1060 William Moore Drive, Raleigh, NC 2 Center for Comparative Medicine and Translational Research, NC State University, Raleigh, NC 3 Department of Molecular and Cellular Biology, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia 4 Department of Ecology and Evolutionary Biology, University of California Los Angeles, 621 Charles E. Young Drive South, Los Angeles, CA 5 Cancer Genetics Program, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC

Evolutionary breakpoint regions (EBRs) may reflect naturally occurring fragile regions that have been reused as part of evolutionary related translocation events. In human cancers, common translocations often span EBRs and it has been theorized that the chromosomal reorganization events leading to speciation may also be associated with cancers. In this context, we study genome organization in the Canidae, a group with chromosome numbers ranging from 2n=34+Bs (red fox) to 2n=78 (domestic dog). This karyotypic range developed via breakage- fusion events involving whole-arm segments during speciation, reflecting a high rate of karyotypic evolution since their divergence from a common ancestor 10MYA. We explored canine EBRs using multicolor fluorescence in-situ hybridization analysis to physically map groups of dog-derived bacterial artificial chromosome (BAC) clones to the karyotypes of eleven species of wild canid. As the panels were hybridized to test species, the order of hybridization signals revealed the orientation of the dog-syntenic regions. Shared EBRs were narrowed to ~1- 3Mb regions and compared across each species. Our findings suggest that the EBRs associated with speciation in the Canidae are compatible with recent phylogenetic groupings and provide evidence that these breakpoints are also recurrently associated with spontaneous canine cancers. We identified several regions of domestic dog sequence that share homology with canid B chromosomes, including additional cancer associated genes, suggesting that these supernumerary elements may represent more than inert passengers within the cell. We propose that the complex karyotype rearrangements associated with speciation of the Canidae reflect unstable chromosome regions described by the Fragile Breakage Model.

Funding Sources: This study was supported by a grant from the Morris Animal Foundation awarded to MB (D08ZO-022). SEDB was funded in part by the Comparative Biomedical Sciences Graduate Program at NCSU. RW was supported by funds from the National Science Foundation (DEB 0614585) and ASG and VAT were supported by funds from the Program on Molecular and Cellular Biology (MCB) and Russian Foundation of Basic Research (RFBR).

15 COMPARISON OF SELECTED STRETCH POSITIONS OF THE PIRIFORMIS MUSCLE USING COMPUTED TOMOGRAPHY AND BIOMODELING

Brett Gulledge*; David Levine, Ph.D.**; Larry J. Tillman, Ph.D.**; Ola Harrysson, Ph.D.‡; Jason Osborne, Ph.D.†; Denis Marcellin-Little, DEDV* *Department of Clinical Sciences, College of Veterinary Medicine, NCSU **Department of Physical Therapy, College of Health, Education and Professional Studies, University of Tennessee at Chattanooga †Department of Statistics, College of Agriculture and Life Sciences, NCSU ‡Edward P. Fitts Dept of Industrial and Systems Engineering, College of Engineering, NCSU

In humans, piriformis syndrome is a non-discogenic form of sciatica characterized by radiating pain in the buttock and leg as a result of an inflamed or hypertrophic piriformis muscle (PM) compressing the sciatic nerve. The conventional treatment of piriformis syndrome includes stretching. Little is known, however, about optimal stretching protocols. OBJECTIVES: To quantify the femoral position and PM elongation in two stretches commonly used to conservatively treat piriformis syndrome and to identify stretch positions that would optimize PM elongation. Seven subjects measuring less than 165 cm were enrolled in a clinical study. The subjects underwent CT scans of the sacrum, pelvis, and proximal femur in supine and in two stretch positions. The CT scans were retro-reconstructed. A numerical computing program was designed to use point coordinates to measure the femoral position and PM length and to compute the femoral position that would maximize PM elongation for each subject. The subjects were female. Piriformis muscle elongation for the two stretch positions were 11.2 ± 3.9% and 12.5 ± 5.0% (p = 0.103). The optimal stretch position was determined to be hip flexion of 117°, external rotation of 40°, and adduction of 27°, resulting in PM elongation of 15.6 ± 5.0%. The PM stretches analyzed in this study were similarly effective. Hip flexion and adduction may play more important roles in PM stretching than previously thought. Stretches that focus on maximizing flexion and adduction may offer greater improvement in clinical outcome in patients conservatively managing their piriformis syndrome.

Funding Sources: This project was funded by the University of Tennessee at Chattanooga and by the Biomodeling Laboratory, Fitts Department of Industrial and Systems Engineering, College of Engineering, NCSU.

16 T REGULATORY CELLS HAVE A HIGHER PRODUCTIVE VIRAL LOAD THAN T HELPER CELLS DURING BOTH A CHRONIC AND ACUTE FIV INFECTION IN CATS.

Jennifer A. Hendricks, Jonathan E. Fogle, Mary B. Tompkins North Carolina State University College of Veterinary Medicine

Feline Immunodeficiency Virus (FIV) infects CD4+CD25+ T regulatory (Treg) cells and CD4+CD25- T helper (Th) cells. Virus load in Treg cells and Th cells has not been examined in FIV chronic or acute infections in vivo. We tested the hypothesis that Treg cells have a higher virus load than Th cells in acute and chronic FIV+ cats. For acute infection, cats were inoculated with FIV NCSU1, and blood was collected at week 1, 2, 3 and 4. Lymph nodes (LNs) were biopsied at 6 weeks post-infection (p.i.) from each cat. For chronic infection, blood was collected from cats infected with FIV for at least 2 years. Lymphocytes from blood and LNs were sorted by FACS into CD4+CD25+ T cells and CD4+CD25- T cells. RNA was isolated from the sorted cells and the plasma collected from each cat. FIV-gag-mRNA levels were measured by real-time RT-PCR. Plasma viremia peaked at 2 weeks p.i., while the highest virus load in T cell was at 3 weeks p.i. In both plasma and T cells, the virus became barely detectable by 4 weeks p.i. In both acute and chronic infections, Treg cells had a higher virus load than Th cells. As Treg cells are infected early in FIV infection and have a higher virus load than Th cells, Treg cells need to be considered in the research of anti-viral drugs.

This work was funded by NIH grant R01-AI080288 and 1KO8AI074445. .

17 OLFACTORY THRESHOLD OF C4/TOLUENE BY EXPLOSIVE DETECTION DOGS

Amanda L. Jeffries, Melanie L. Foster, and David C. Dorman North Carolina State University College of Veterinary Medicine

The dog’s exceptional sense of smell arises from a large nasal cavity lined with olfactory epithelium. This sense of smell is exploited for explosive and drug detection. While canine olfaction of many substances has been quantified, the ability of dogs to detect a target odor at varied concentrations during different stages of explosive detection training has not been described. Phase I of this study used dogs with either < 45 days or > 100 days of training (n = 8/group). All dogs were trained to detect ~ 100 g of C4. In Phase I, dogs completed a field trial of scent detection on two 90 m courses. 25 g (Course A) or 5 g (Course B) of C4 was placed ~ 60 m from the start of each respective course, with varying false digs throughout. Dogs were given 10 minutes to complete the search. The time from start to odorant detection, number of incorrect responses (e.g., response to a false dig), or failure to identify the target odorant within 10 minutes was recorded for each dog. In Phase II, we will semi-quantitatively measure the olfactory threshold for toluene, an odorant found in C4 explosives. Phase II will use a canine olfactometer that generates a toluene vapor with subsequent dilution to a desired nominal concentration (< 10 ppm toluene in air). We hypothesize that olfactory detection of a target odorant (C4 or toluene) will improve with increased time in training. We expect this to be demonstrated by positive detections at lower concentrations, faster speeds of detection, and fewer incorrect responses.

Funding Source: Office of Naval Research

18 MOUSE KERATINOCYTE SIDE-POPULATION PLAYS A UNIQUE ROLE DURING MALIGNANT PROGRESSION TO SKIN SQUAMOUS CELL CARCINOMAS

Sun Hye Kim, Sung Hyun Lee and Marcelo L Rodriguez-Puebla Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University. Raleigh, North Carolina 27607

Side-population (SP) has been identified in tissues, cell lines and human and experimental tumors as putative adult stem cells (SC) with high efflux capability for antimitotic drugs. The role of SP in tumorigenesis is controversial; though, a role as cancer stem cells has been reported. We have investigated whether the presence of SP is associated with tumor progression. We observed increased percentage of SP in cell lines derived from skin squamous cell carcinomas (SCCs) compared with those arose from epidermis and skin papillomas. Interestingly, SP shows elevated expression of the ABCG2/BCRP1 transporter –responsible for the high efflux capacity of SP- while the expression of the stem cell markers, keratin15, CD34 and Lgr6, were similar between SP and non-SP. Immunofluorescence analysis of skin papillomas and SCCs identified the ABCG2/BCRP1 transporter in a region near/above the hair follicle stem cells (BuSCs). Notably, this region is positive for the thymic epithelial progenitor marker MTS24 which defines a different stem cell population. Therefore, the SP exhibits clear differences compared to BuSCs. The relevant correlation among the SP size and tumor progression suggests that these cells are positively selected during tumor progression. Furthermore, we observed an elevated number of putative stem cells expressing ABCG2/BCRP1 and MTS24 in tumors from K5CDK4 transgenic mouse which showed increased malignant progression to SCCs. Collectively, our results suggest that the putative stem cells SP plays a unique role during the malignant progression to SCCs.

Funding Source: NIH/NCI grant CA116328

19 EFFECT OF APPARENTLY ASYMPTOMATIC FELINE HYPERTROPHIC CARDIOMYOPATHY ON ACTIVITY LEVELS AND OWNER-ASSESSED QUALITY OF LIFE

David J. Kleisch, Amanda M. Erickson, Clarke E. Atkins, Bruce W. Keene, Duncan X. Lacselles, Teresa C. DeFrancesco North Carolina College of Veterinary Medicine, Raleigh, NC

Hypertrophic cardiomyopathy (HCM) is the most commonly diagnosed feline heart disease. With the increased availability of echocardiography and owner willingness to pursue diagnostics, more cats with HCM are being identified prior to the onset of symptoms. We have little objective knowledge of the impact of apparently asymptomatic HCM on either the activity level or quality of life (QOL) of affected cats. The objective of this study was to characterize the impact of apparently asymptomatic feline HCM on activity level and QOL. As a part of a larger ongoing prospective, double-blind, randomized, placebo-controlled clinical trial evaluating the impact of chronic beta blockade in apparently asymptomatic HCM, enrolled cats underwent physical examination, blood pressure measurement and evaluation of serum cardiac biomarkers. Each cat was then fitted with a collar-mounted accelerometer device, which recorded activity data in the home environment for two weeks. A detailed owner QOL questionnaire was completed by the owner at the beginning of the two-week period. To date, we have enrolled thirty cats affected by apparently asymptomatic HCM with left atrial enlargement and fifteen age-, weight-, life-style- and gender - matched control cats free of heart disease as defined by a normal echocardiogram and normal serum cardiac biomarker levels Preliminary results show no significant difference in daily activity levels between groups over a period of seven consecutive days as measured by accelerometry. No significant difference in owners’assessment of activity or quality of life was detected between groups. Cardiac biomarkers (NTproBNP and cTnI) were significantly higher in the HCM-affected cats. These findings suggest that cats with echocardiographic signs of HCM accompanied by left atrial enlargement are indeed asymptomatic for their disease, as assessed by activity levels and owner- percieved QOL.

Morris Animal Foundation

20 PHYLOGENETIC ANALYSIS OF CAMPYLOBACTER FROM COMMERCIAL AND ANTIMICROBIAL-FREE (ABF) PRODUCTION SYSTEMS REVEALS COMMON ANCESTRY AND A NON-CLONAL POPULATION

Leanne M. Magestro1, Macarena P. Quintana-Hayashi1, Jennifer A. Kobylanski, Ashley Whitesell1, Siddhartha Thakur1 1Dept. of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University

Campylobacter is one of the leading pathogens causing foodborne illnesses in the US. Food animals, including pigs, are known reservoirs of Campylobacter strains that infect humans, based on epidemiological evidence. The purpose of this study was to determine the clonality or diversity of Campylobacter coli isolated from conventional and ABF production systems at farm, slaughter and environment using multilocus sequence typing (MLST). A total of 129 C. coli isolates were selected from fecal, environmental and carcass samples of Antimicrobial- Free (ABF) (N= 71) and conventional (N=58) production systems. Seven housekeeping genes (asp, gln, glt, gly, pgm, tkt, unc) were amplified using PCR and the amplified product was sequenced. Identifications of sequence types (STs) were completed as the result of sequence data analysis and allelic profile determination. Based on the analyses, relationships between the genotyped isolated were determined based on the construction of dendrograms and minimum spanning trees. Isolates with similar sequence types were found between the pigs and their environment at

farm and slaughter (ABF: 13, IA = 0.1308; Conventional: 20, IA = 0.1357). Higher genotypic diversity was observed among isolates from the conventional swine production systems (ABF: 0.3455 +/- 0.0901; Conventional: 0.3929 +/- 0.0805). Phylogenetic analysis revealed a genotypically diverse C. coli population with the presence of C. coli isolates sharing a common ancestry in both production systems. Overall, phylogenetic analysis of C. coli isolates from two distinct production systems indicates a diverse genetic makeup and weak clonal population of this species.

21 RELEASE RATE OF CRYSTALLINE RAPAMYCIN IN VITRO AND INTRAVITREALLY IN EX VIVO HORSE EYES.

Megan E. Mathias, Jacklyn H. Salmon, Jennifer L. Davis, Brian C. Gilger North Carolina State University College of Veterinary Medicine

Rapamycin (RAPA) is carbocyclic lactone-lactam macrolide antibiotic with immunosuppressive properties. RAPA has been shown to reduce the amount of ocular inflammation in uveitis as it complexes with FKBP12 to form an immune complex which inhibits the mammalian target of rapamycin (mTOR). Inhibiting mTOR blocks T cell proliferation and cytokine production. The purpose of this study is to evaluate the in vitro release rate of crystalline RAPA to determine if intravitreal injection of RAPA is a potential sustained- release treatment for equine recurrent uveitis (ERU), the most common cause of blindness in horses. RAPA (10 mg) was placed into each of three Spectra/Por® molecularporous membrane tubing which was submerged in 10 mL of PBS and maintained in a water bath at 37° C for 24 hours. Each membrane was transferred to a new vial of PBS every 24 hours for 28 days. The PBS was stored at -80° C until all samples were collected. Samples were analyzed for RAPA content using High Performance Liquid Chromatography (HPLC). At day 28, the mean daily release rate was 20.3 ng/mL (therapeutic levels of RAPA have been reported at 10-20 ng/mL) and there was a mean of 5.97 mg of RAPA remaining in each of the three membranes. Using this data, the duration of in vitro release of RAPA is calculated to be greater than 5 years. RAPA (10 mg) was also injected intravitreally in cadaver equine eyes and aqueous and vitreous humor was collected after 24 hours. The RAPA concentration in the ocular fluids will be compared to the PBS if the ex vivo release rate is similar to the in vitro rate warranting further studies with RAPA as a potential treatment for uveitis. Intravitreal injection of crystalline RAPA may provide sustained release of the drug for a long- term treatment of ERU.

22 TORIN1 IS A POTENT INHIBITOR OF MTOR-MEDIATED CELL SIGNALING AND LIPOGENESIS IN H292 CELLS

Kathleen S. McGinnis1, Helena M. Johansson1 2, Philip L. Sannes1 2 1North Carolina State University College of Veterinary Medicine, Raleigh, NC 2Center for Comparative Molecular and Translational Research, Raleigh, NC

Keratinocyte growth factor (KGF) is a potent inducer of proliferation, lipogenesis, and surfactant production in lung alveolar epithelial type 2 (AT2) cells. This is mediated by the induction of various signaling pathways such as the mammalian target of rapamycin (mTOR) pathway followed by the activation of several lipogenesis-inducing transcription factors, primarily SREBPs and C/EBPα. mTOR exists in 2 distinct protein complexes, mTORC1 and mTORC2. It regulates, besides lipogenesis, cell growth and the cytoskeleton in response to various external stimuli such as nutrients and growth factors. Both complexes can directly be inhibited by the newly identified compound Torin1 through the blocking of the ATP-binding pocket site. Its inhibitory effect on cell growth suggests a potential use as a therapeutic agent, particularly as an anti-cancer drug. We hypothesized Torin1 would alter KGF-induced lipogenesis. To test our hypothesis, a pulmonary epithelial cancer cell line H292, was used as a model for lung AT2 cells. H292 were treated with Torin1, KGF, or a combination of both. Cells were harvested at selected time points to analyze signaling pathways and lipogenesis. Western blot analysis and immunofluorescence revealed that KGF, as previously described, induces the phosphorylation and thus activation of distinct mTOR signaling branches such as p70S6K and Akt. Furthermore, the expression of SREBP1 and its target fatty acid synthase were induced. In contrast, in both Torin1 and Torin1/KGF-treated cells the signal was below basal level (un- stimulated cells). These results indicate that Torin1 is a potent inhibitor of mTOR signaling and blocks KGF-inducible lipogenesis in H292 cells.

23

GARP-BOUND TGFB COMPLEXES ON FELINE TREGS ARE UPREGULATED DURING ACUTE FIV INFECTION AND CONTRIBUTE TO IMMUNE SUPPRESSION OF CD4 Th VIA LIGATION OF Th TGFbRII.

Michelle M. Miller, Jonathan Fogle, Mary B. Tompkins. North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27607 USA

We previously demonstrated up-regulation of TGFb on the membrane (mTGFb) of feline CD4+CD25+ Tregs and TGFbRII on CD4+CD25- Thelpers following FIV infection of cats and proposed a role for mTGFb-mediated signaling in immune dysfunction characterizing FIV. Here we identify a protein marker, Glycoprotein A Repetitions Predominant (GARP) as a Treg specific surface marker regulating expression of mTGFb and contributing to TGFb-mediated anergy. We demonstrated co-precipitation of mature 25 kDa TGFb with GARP in feline CD4+ cells. In support of this, flow cytometry demonstrated TGFb and GARP expressed at similar levels on the CD4+CD25+ Treg population but not CD4+CD25- in both uninfected and chronically FIV-infected cats. Coculture of CD4+CD25+ Tregs or Tregs depleted of GARP+ cells with CD4+CD25- targets revealed that Tregs containing the GARP+ population possess greater suppressor function. These results support the role for GARP as a surface anchor of active TGFb on a discrete population of CD4+CD25+ T cells and as a participant in their suppressor function. To further evaluate the effect of FIV infection on GARP expression and Treg suppressor function, cats were infected with FIV, and GARP:TGFb expression evaluated over a period of 13 weeks. Our results demonstrate an increase in CD25 and GARP:TGFb expression on CD4+ cells during the first week in FIV infection, indicating a Treg activation response. In vitro infection of PBMCs with FIV support the in vivo data. We conclude that GARP-bound TGFb is increased on the surface of Treg cells during FIV and contributes to the CD4+ T cell anergy. Work funded by NIH grant NIAID R01-AI080288

24 BACTERIAL COUNTS IN BEDDING MAY PREDICT BULK TANK BACTERIAL POPULATION

Christina K. Park, Roberta Lyman, Kevin L. Anderson North Carolina State University College of Veterinary Medicine Milk Microbiology and Mastitis Laboratory

Management of the dairy herd environment is a constant area of concern for producers due to the effect on mastitis in a herd. Previous studies have implicated bacterial populations in sawdust bedding as an indicator of clinical mastitis within a herd. This study investigated the association between populations of mastitis pathogens within sawdust bedding and within the bulk tank. Bedding and bulk tank samples were collected from five dairy farms using wood byproducts in free stalls. Bedding samples were collected in triplicate and bacteria were extracted using .85% sterile saline, homogenized and plated on Trypticase soy agar with 5% sheep blood for enumeration at an 8 fold serial dilution. Samples were plated for differential counts of E. coli, Klebsiella spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp. Samples were incubated at 37° C for 24 hours and received additional differential testing to confirm identification. A comparison of coliform differential media was performed also. Preliminary microbial tests showed that similar populations appeared in bedding and bulk tank cultures, particularly coliforms. Bacterial counts in bedding increased with moisture content. Bedding culture enumeration may predict the necessity to change bedding or not, particularly in relation to coliform populations. A previous study suggests 1 million colony forming units of E. coli in bedding increases clinical mastitis. However, in our study many beddings exceeded this level without apparent increase in clinical mastitis. Bedding assessment is part of dairy herd management that can be assessed objectively and help to manage overall mastitis control on the farm.

Funding Sources: Merial Veterinary Scholars Program, North Carolina Dairy Foundation Incorporated

25 CANINE ORAL MELANOMA - CYTOGENETIC CHARACTERIZATION OF FRESH TISSUE, CELL LINES AND ARCHIVAL TISSUES

Kelsey Poorman1, Luke Borst2, Matthew Breen1,3,4 1Department of Molecular Biomedical Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 2Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 3Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA 4Cancer Genetics Program, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA

Canine oral melanoma is a common and aggressive tumor with poor life expectancy. Molecular cytogenetic characterization of these tumors is sparse. There are numerous biological resources available for researchers, including fresh and archival primary tumors, established cell lines, and a series of genomics tools. While it is widely accepted that cell lines may be used to represent the primary disease, they often evolve in vitro beyond what is seen in primary tumors. Additionally, DNA obtained from archival specimens can be degraded, leading to decreased data quality. The purpose of this study was to characterize genome-wide numerical and structural aberrations in canine oral melanomas, using a cohort comprising fresh-frozen tissue, formalin- fixed paraffin embedded (FFPE) tissue and cell lines. We assessed fresh primary tissues (n=5), primary cell lines (n=8), and FFPE archival tissue (n=21) using a combination of high resolution genome-wide array based comparative genomic hybridization (aCGH) and multicolor fluorescence in situ hybridization (FISH). aCGH analysis revealed numerous DNA copy number aberrations in all melanoma samples evaluated. Most notable were characteristic patterns of gain followed by loss on dog chromosomes (CFA) 10, 26, and 30. Various chromosome instabilities were observed and confirmed by FISH. Cell lines recapitulated aberrations evident in primary tumors, suggesting their importance in melanoma pathogenesis. However, cell lines also showed cytogenetic aberrations not evident in primary tumors, suggesting these were secondary events as a result of culture. Among archival FFPE cases, it was evident that the ability to detect copy number aberration declined with the age of the specimen.

26 OPTIMIZATION OF REAL TIME MICROSCOPY FOR ASSESSMENT OF RATE OF HISTONE EXCHANGE AFTER SOMATIC CELL NUCLEAR TRANSFER (SCNT) USING GFP-LABELED HISTONE 2B

Momo Tanaka, Steve Nagar, Xia Zhang, Lauren Jackson, Jorge Piedrahita North Carolina State University College of Veterinary Medicine

Somatic cell nuclear transfer (SCNT) takes advantage of the ability of oocyte histones and mRNA to reprogram the nucleus of a somatic cell, resulting in a cloned embryo with the genetics of the donor. However, after SCNT the rate of development to viable offspring is less than 5% (Maalouf, 2009). One of the key steps affecting SCNT is chromatin remodeling during when somatic histones are replaced by maternal histones (Whitworth and Prather, 2010). Methods that facilitate histone exchange could lead to a more efficient reprogramming. To identify such methods, a system for assessing the rate of histone exchange after SCNT is needed. We propose the development of a real time system based on the use of fluorescently labeled histone and real-time microscopy. The rate of the maternal to somatic histone exchange after SCNT was examined by investigating the affects of reprogramming on the success rate of SCNT. For histone labeling, plasmid vector (pCAG-H2B-sus-N-terminal fusion-eGFP-IFNβ- S/MAR-PGK Puro) was co-transfected with d30-porcine fibroblasts via electroporation. Transfected fibroblasts were able to sustain its viability up to 48hours under microscope with fluorescent exposure of 85ms and intensity of 55%, allowing images to be captured every 15minutes. In addition, conditions required for long-term recording of oocyte development were optimized. Twenty activated oocytes placed in a 50ul in vitro culture medium at 39℃ and 5%CO2, yielded the most efficient rate of cleavage (~80%) and development up to 8-cell stage.Based upon the experiment, H2B-GFP cloned embryos will be observed under microscope for measuring rate of its signal loss. By examining the rates of histone exchange after SCNT, the effects of methods to enhance nuclear reprogramming could be studied and the finding may contribute to improving the development of SCNT embryos to a viable offspring.

Funding: NIH

27

DEVELOPMENT OF A NOVEL COGNITIVE/MOTOR PARADIGM TO DOCUMENT FIV DISEASE IN LABORATORY CATS

Jessica A. Tarter,1 Margaret E. Gruen,1 Lola C. Hudson,1 Andrea E. Thomson,1 Gillian P. Clary,2 Rick Meeker,2 Barbara L. Sherman1 1North Carolina State University College of Veterinary Medicine 2University of North Carolina School of Medicine

Feline Immunodeficiency Virus (FIV) is an important animal model for Human Immunodeficiency Virus (HIV). The goal of this study was to develop an easily-acquired cognitive/motor reaching task that would differentiate infected from uninfected cats and could be used to evaluate disease progression and response to therapeutics. Male laboratory cats intracranially infected with FIV (n=10) or sham-infected controls (n=6) were trained to perform a reaching task by modifying the feline General Test Apparatus (CanCog Technologies). Each test subject was confined in the apparatus and trained to reach with one front paw and draw back a coaster containing a food reward. During testing, the reach-distance from the cat to the coaster varied from 2.5 – 17.8 cm. Cognitive/motor measures included latency (seconds) and number of paw changes to task completion. I was blind to infection status of the subjects. The results confirmed that latency to task completion increased with reach-distance for both FIV and control cats. A 2-way ANOVA revealed more paw changes in the FIV infected cats (p=0.0413), and more paw changes with distance (p=0.0026), with no interaction effect. The results suggest that the reaching task was cognitively more challenging for infected cats. In conclusion, all cats learned the reaching task successfully with minimal training. The increased paw switching at longer distances in FIV infected cats suggests that this motor reaching task can distinguish cats based on FIV status and may be useful to evaluate the effect of novel therapeutics that may have application for HIV treatment.

Grant: NIH R21NS 066843

28

CLINICAL ANATOMY OF THE BALL PYTHON (PYTHON REGIUS)

Bradley J. Waffa, Emily Wayne, Michael K. Stoskopf College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606 USA

Over the past few decades, demand has increased substantially for veterinarians with basic knowledge of reptile medicine. Despite the publication of some excellent reptile medicine textbooks and papers in that time, basic anatomical texts for some of the most commonly kept species such as the ball python (Python regius) are lacking. Euthanized culled adult specimens of both sexes were donated by a commercial breeding facility. Clinically salient anatomical features were documented using ultrasonography (US), digital radiography, computed tomography (CT), T1 and T2 weighted magnetic resonance imaging (MRI), and gross dissection. Weights, dimensions, location, and appearance of internal structures were recorded and photographed. Particular attention was given to anatomic features important for successful routine surgical approaches, vascular access, obstetrical intervention and supplemental feeding. Illustrated anatomical plates detailing internal anatomy and landmarks for tissue sampling and venipuncture were created.

Funding: Teaching Innovation Grant (NCSU CVM 2011); Environmental Medicine Consortium; George H. Hitching’s New Investigator Award in Health Research (Wayne)

29 SEQUENCING ANTIDIURETIC HORMONE IN CATS WITH CENTRAL DIABETES INSIPIDUS

Jessica A. Wofford and Marlene L. Hauck NCSU CVM

Polyuria and polydipsia are common clinical symptoms, especially for cats. Among the many differential diagnoses, central diabetes insipidus (CDI) is generally included yet is a rare diagnosis. CDI occurs when the body fails to produce adequate antidiuretic hormone (ADH) due to pathology of the posterior pituitary or mutation of the ADH gene. Pre-pro-ADH is cleaved into ADH, neurophysin II (NPII, a chaperone protein) and copeptin which has no known physiological role but is released into circulation in equimolar amounts with ADH. The objective of this study was to determine if mutations in ADH are responsible for CDI in three cats. The diagnosis of CDI is often based on response to desmopressin therapy, a synthetic ADH. Over 50 unique mutations have been documented for familial CDI in humans, yet no studies have investigated a genetic etiology in the cat. All three affected cats in this study presented as kittens with polydipsia, polyuria and hyposthenuria. Two have been treated with desmopressin for at least two years and maintain concentrated urine. In normal cats, we determined the mRNA sequence encoding the pre-pro-ADH gene. Of four normal cats sequenced, one was heterozygous for a mutation reported to cause familial CDI in humans. We are now working to amplify the full genomic DNA sequence. We established feline copeptin has high homology to human and are using a human copeptin ELISA to measure serum copeptin in normal and CDI cats. This work will help elucidate the cause and hopefully improve diagnosis of CDI in cats.

Funding sources: Morris Animal Foundation Veterinary Student Scholars Program and George H. Hitchings New Investigator Award

30

THE ROLE OF EPIDERMAL GROWTH FACTOR RECEPTOR IN NEUROGENESIS AND GLIOGENESIS

Guanxi Xiao1, Tang-Cheng Lee2, David Threadgill2, and Troy Ghashghaei1 1 Center for Comparative Medicine and Translational Research, College of Veterinary Medicine and Department of Molecular Biomedical Sciences, NC State University. 2 Department of Genetics, North Carolina State University.

The subependymal zone (SEZ) is the largest germinal region that supports neural stem cells in the postnatal and adult brain. Adult neurogenesis is dependent on distinct populations of transit amplifying progenitors (TAPs) which are derived from neural stem cells and generate large number of migrating neuroblasts. The neuroblasts must emigrate to the olfactory bulb (OB) and terminally differentiate into interneurons. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase, which is currently thought to be ubiquitously expressed by the entire population of TAPs and regulate their proliferation in the SEZ. However, the homeostatic role of EGFR signaling in adult neurogenesis using loss-of-function approaches remains unclear. We used a conditional approach to specifically delete EGFR in the brain, which resulted in profound rostral forebrain and OB defects. Remarkably, these defects subsided after early postnatal development indicating a potent regenerative capacity in the brain during this critical developmental period. Using novel genetic tools we found that loss of EGFR results in a fate-shift in a sub-population of TAPs which surprisingly does not include the entire progenitor pool in the postnatal SEZ. Taken together, our results reveal a novel population of EGFR+ TAPs in the SEZ that is critical for forebrain homeostasis during early postnatal development, but is dispensable for overall survival of mice. The precise identity of the EGFR+ TAPs and potential behavioral deficits in EGFR-null mice remains to be determined.

31 ANGIOPOIETIN-2 SERUM LEVELS IN CANINE MALIGNANT MELANOMA PATIENTS

Joanne Fernandez-Lopez, Jessica Wofford, Julie C. Fisher, Marlene Hauck North Carolina State University College of Veterinary Medicine

New blood vessels are essential for nourishment of growing tumors, the removal of metabolic waste and for tumor progression and metastasis. Angiopoietin-2 (Ang-2) is a pro-angiogenic factor and may have an autocrine or paracrine role in melanoma growth. Serum Ang-2 is prognostic for the development of metastasis and overall survival in human melanoma patients. We hypothesized that serum Ang-2 would be prognostic for disease progression, burden and survival in canine melanoma patients. A secondary hypothesis was that melanoma cell-lines express Ang-2. Levels of Ang-2 mRNA in canine melanoma cell lines were measured using RT- PCR and compared to canine testis. With collected serum from patients (n=20) and supernatant from melanoma cell lines (n=6), Ang-2 levels were measured with a commercial ELISA for human Ang-2 (R&D Systems). Serum levels of Ang-2 from melanoma patients were correlated with disease progression (Student’s t-test), tumor burden and survival (Spearman rank correlation). All melanoma cell lines expressed Ang-2, albeit at a 2- to 6-fold lower expression level than testis. Serum concentration of Ang-2 in canine patients was significantly correlated with the development of metastasis (p=0.008) and marginally correlated with tumor burden (p=0.0541). No correlation with survival was found (p=0.8008). While not statistically significant, the association between volume of disease and serum Ang-2 may be impacted by difficulty in the accurate assessment of disease burden. The correlation between the serum Ang- 2 level and the development of melanoma metastasis encourages evaluation in a larger patient cohort as a possible prognostic factor.

Funding by Merck-Merial and Laboratory Supporters

32 INCIDENCE OF ONYCHECTOMY IN CATS PRESENTED FOR VETERINARY CARE NEAR RALEIGH, NC AND EDUCATIONAL ATTITUDES TOWARDS THE PROCEDURE

Laura E. Lockhart, Alison A. Motsinger-Reif*, Lysa P. Posner NCSU College of Veterinary Medicine; *Bioinformatics Research Center, Department of Statistics

Onychectomy is a controversial procedure. The incidence of onychectomy in cats is currently unknown, and education regarding the procedure appears to vary greatly among veterinary schools. The purpose of this project was to determine the incidence of onychectomy in cats near Raleigh, NC and to document how the procedure is taught in U.S. veterinary schools. Data was collected over a 10-week period from cats seen for appointments from one of five veterinary facilities (two cat-only, two general, and one tertiary). Data collection included signalment and onychectomy status. Simultaneously, 28 U.S. veterinary schools were polled regarding onychectomy education. Data were collected from 1794 cats ranging in age from 8 days to 21 years; 52.3% were female and 95.8% were sterilized. Of those cats, 20.8% had undergone onychectomy. There was a significantly higher rate of declawed cats in general practice compared with the other practice types (p<0.030). Younger cats had a higher rate of onychectomy (p<0.001). Twenty-six veterinary schools responded to the survey (93%). Fifty-four percent of the responding schools did not require a lecture or lab to learn the onychectomy procedure in their curriculum. In the Raleigh, NC area, almost 21% of cats seen in veterinary hospitals are declawed, but less than 50% of veterinary schools included a mandatory lecture or lab to teach the onychectomy procedure in their curriculum. There appears to be a discrepancy between the popularity of the onychectomy procedure and the emphasis placed on its instruction in veterinary schools in the U.S.

Merial Veterinary Scholars Program

33 STARGAZERS AND STEM CELLS: A POTENTIAL THERAPEUTIC APPROACH TO TREATING IDIOPATHIC SEIZURES Ryan N. Bray, Troy Ghashghaei North Carolina State University College of Veterinary Medicine, Department of Molecular Biomedical Sciences; Center for Comparative Medicine and Translational Research

Many idiopathic seizures of the central nervous system (CNS) are due to a breakdown of inhibitory neurotransmission (GABAergic) in distinct CNS foci. Thus, treatment strategies may utilize neural stem cells to increase inhibitory affect in the CNS. Here we utilized a mouse model for idiopathic “absence” seizures, with an underlying mutation of the Stargazer (Stg) gene. Based on the frequency and clearly identifiable characteristics of the behavioral seizure phenotype in Stg mice, we set out to quantify the exhibition frequency of seizures, attempted to molecularly map the foci susceptible to seizure activity, and assess the feasibility of transplanting neural stem cells with the potential to generate GABAergic neurons in susceptible CNS foci. Seizure foci were mapped using the expression of c-Fos, the well-established and neuronal activity-dependent immediate gene, as a molecular marker for behavior-specific neuronal activity. Young adult stg mutants were monitored for seizure frequency and duration. Age- matched mutant and wild-type littermates were sacrificed concurrently 90 minutes after a prolonged seizure (>10 seconds) and their brains were examined for c-Fos protein expression. In parallel pilot experiments we also assessed the feasibility of transplanting neural stem cells harvested from the embryonic medial ganglionic eminence (MGE), which is the source of most inhibitory interneurons in the adult forebrain. MGE transplants were targeted to cortical foci of the adult mouse brain. Based on our preliminary studies we hypothesize that given successful differentiation and integration of MGE transplants into seizure foci identified in stg mutants, seizure duration and frequency may significantly decline after treatment. Funding: Merck-Merial, NIH/AFAR (09-160B)

34 GENERATION OF PUTATIVE INDUCED PLURIPOTENT STEM CELLS (IPS) FROM ADULT CANINE FIBROBLAST

Sehwon Koh2,4, Shengdar Tsai2,4,5, Steve R. Bischoff2,4, Ji-hey Lim3, Rachael Thomas1, Matthew Breen1,2,4, Natasha J. Olby3,4, and Jorge A. Piedrahita1,2,4

1 Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 2 Genomics Program, North Carolina State University, Raleigh, NC 3 Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 4 Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC 5 Department of Pathology, Harvard Medical School, Boston, MA

Pluripotent stem cells such as embryonic stem (ES) and iPS cells can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. Here we report the derivation of putative iPS cells from adult canine fibroblast using retroviral transduction of Oct3/4, Sox2, Klf4 and c-Myc (OSKM). Retroviruses containing OSKM were transduced into adult dog skin fibroblasts and replated onto γ –irradiated mouse embryonic fibroblasts. ES-like colonies were picked at day 21 post-infection and expanded in three different ES culture media containing either FGF (10 ng/mL), LIF(103 units/mL) or both FGF and LIF, and supplemented with two chemical inhibitors (3 uM CHIR99021 and 20 uM PD98059). The isolated cells cultured in FGF and LIF had the strongest alkaline phosphatse activity and those were further analyzed by RT-PCR and immunocytochemistry (ICC), and found to express the pluripotency markers. Karyotyping analysis using array comparative genomic hybridization and fluorescence in situ hybridization showed that genomic aberrations were gained after extended culture periods but not by induction of reprogramming. In vitro differentiation by embryoid body formation and directed differentiation resulted in cells representative of all three germ layers as confirmed by both ICC and RT-PCR. Subcutaneous injection of the putative canine iPS cells into SCID mice created teratomas, and expression of markers for all three germ layers in the tumor were confirmed by both RT-PCR and ICC. This work was funded by a grant to JP and NO from the Canine Health Foundation, grant #01272.

35 THE TRANSCRIPTION FACTOR SPECIFICITY PROTEIN 2 (SP2) IS REQUIRED FOR PROGRESSION THROUGH THE CELL CYCLE OF NEURAL PROGENITORS

Huixuan Liang 1, Nagradan Muthusamy1, Guanxi Xiao1, Laura Sommerville1, Jermaine McGill 1, Haifeng Yin 1, Simon Hippenmeyer 2, Liqun Luo 2, Jonathan M. Horowitz 1, H. Troy Ghashghaei 1 1 Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University 2 Howard Hughes Medical Institute and Dept of Biology, Stanford University, Stanford, CA

Regulation of the cell cycle in tissue specific stem cells is essential for appropriate production of cell types throughout the body. The longevity and developmental potential of stem cells is closely linked to molecular mechanisms that control the rate and fidelity of the cell cycle. Using neural stem cells as a platform, we show that a putative zinc-finger transcription factor Specificity Protein 2 (Sp2) is a key regulator of the cell cycle. A conditional loss-of-function approach was employed to delete Sp2 in neural stem cells, revealing a specific disruption in progression through G2/M phases of the cell cycle. Using transcriptome analysis we confirmed the past findings that Sp2 has little to no transcriptional activity suggesting that although Sp2 belongs to a large family of established transcription factors, it may not function as a transcriptional regulator itself. Cell autonomous function of Sp2 was identified by mosaic deletion of Sp2 using novel genetic tools in combination with time-lapse imaging in culture. We report that Sp2 is localized to the nucleus and is a stable component of the centrosomal complex. Sp2-bound centrosomes are shuffled between perinuclear loci and the intracellular bridge during cytokinesis and in the absence of Sp2 this shuffling becomes excessive. As a result, abscission at the final stage of cytokenesis fails, resulting in multi-centrosomal and multinucleated Sp2-null cells. Our study for the first time reveals the cell biological role of Sp2 and how its absence impacts progression through the mitotic phase of the cell cycle.

Supported by NIH grant R01NS062182 and a grant from the American Federation for Aging Research

36

MECHANISMS OF VESICULAR TRAFFICKING IN THE NEURAL STEM CELL NICHE

Laura Sommerville1, Sue Fang1, Angelito Nepomuceno2, David Muddiman2, Perry Blackshear3, Ken Adler1, and Troy Ghashghaei1

1 Center for Comparative Medicine and Translational Research, College of Veterinary Medicine and Department of Molecular Biomedical Sciences, North Carolina State University. 2 W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, NC State University. 3 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park.

Myristoylated Alanine-rich Protein Kinase C Substrate (Marcks) is a putative scaffolding protein hypothesized to ubiquitously express in eukaryotic cells, with implication in various cell biological processes including cytoskeletal organization, cell migration, and secretion. Marcks is expressed in both the pre- and postnatal mouse brain with its highest concentration in stem cell niches. Although the importance of Marcks has been demonstrated for proper CNS development, its precise cell-autonomous role and cell biological functions remain poorly understood. The postnatal mammalian brain contains two tightly regulated microenvironments that support postnatal neurogenesis: the dentate gyrus in the hippocampus and the subependymal zone (SEZ) of the neostriatum. We show that Marcks is highly expressed in various, but not all, cell types in the SEZ. To focus our study on the ependymal component of the niche, we generated a conditional mouse system which limited Marcks deletion to ependymal cells. This resulted in disruption of intracellular localization of Clca3, a protein specifically expressed in ependymal cells. Using elegant mass-spectrometric approaches, we identified dynamic interactions of Marcks with actin, tubulins, as well as vesicle-associated proteins, and discovered that these associations are governed by the phosphorylation state of Marcks. Real time intracellular imaging in ependymal cells reveals potent regulation of Clca3-bound vesicular trafficking by Marcks and its phosphorylation status. Taken together, these data suggest that Marcks may play a role in microtubule dependent intracellular trafficking in ependymal cells which may underlie the cell biological basis for the various functions attributed to Marcks in different cell types.

Supported by NIH grant RO1NS062182 and a grant from the American Federation for Aging Research

37 A PHARMACOGENETIC TOOL TO STUDY PHYSIOLOGICAL FUNCTIONS OF POSTNATAL NEUROGENESIS IN THE OLFACTORY BULBS.

Nagendran Muthusamy1, Bryan Roth2, and Troy Ghashghaei1.

1Center for Comparative Medicine and Translational Research, College of Veterinary Medicine and Department of Molecular Biomedical Sciences, North Carolina State University. 2Departments of Pharmacology, Medicinal Chemistry, and Psychiatry, University of North Carolina.

Generation of new neurons in the central nervous system (CNS) is prevalent during embryonic development. Postnatal neurogenesis, however, is limited to distinct regions of the mouse CNS including the hippocampus and the olfactory bulbs (OB). Postnatal OB neurogenesis depends on persistence of a distinct neural stem cell niche in the subependymal zone of the lateral ventricles from where neuroblasts migrate via the rostral migratory stream to the OB, where they differentiate into mature interneurons throughout life. Despite enormous progress over the past decade in identification of cellular and molecular mechanisms that regulate postnatal OB neurogenesis, the physiological significance of neurogenesis in the OB remains unknown. This lag is largely due to the absence of tools that allow for understanding the role of a mosaic neuronal population in the OB. Here we report the development of a pharmacogenetic approach to selectively activate and inhibit neuronal activity in newly generated neurons in the OB during postnatal periods. We show that genetically controlled expression of two mutagenized G-coupled acetylcholine receptors, which can only be activated by a designer drug, resulted in activation (by depolarization) and deactivation (by hyperpolarization) of newly generated neurons in the OB. Intraventricular injections of adenoviruses carrying these mutagenized receptors led to their selective expression in postnatally-generated neurons that reached the OB. We provide behavioral data that for the first time illustrate the role of postnatal and adult neurogenesis in physiological olfactory functions in the mouse.

Supported by NIH grant R01NS062182, a grant from the American Federation for Aging Research

38 CYTAUXZOON FELIS CYTOCHROME B GENOTYPE IS ASSOCIATED WITH SURVIVAL IN DOMESTIC CATS WITH CYTAUXZOONOSIS

Megan Downey1, Henry Marr1, Jaime Tarigo1, Leah Cohn2, Mike Levy1, Adam Birkenheuer1. 1. North Carolina State University College of Veterinary Medicine, Raleigh, NC. 2. University of Missouri College of Veterinary Medicine, Columbia, MO.

Cytauxzoon felis is a virulent tick-transmitted protozoan parasite that infects felines. Without treatment, only 3% of infected cats survive. Treatment combining atovaquone and azithromycin (A&A) increased the survival rate to 60%1. In related parasites, resistance to atovaquone has been attributed to mutations in its drug target, cytochrome b (cytb)2. We hypothesized that C. felis cytb genotypes were associated with response (i.e. survival) to A&A treatment. Cytauxzoon felis cytb genes were amplified by PCR from samples collected from 45 cats with cytauxzoonosis that received A&A treatment1. Amplicons were sequenced bi-directionally, chromatograms were inspected for heterozygosity and the sequences were edited accordingly3,4. The majority of samples (30/45) had evidence of a single cytb genotype, while the remaining samples (15/45) had two or more cytb genotypes present. The most common single genotype was arbitrarily assigned as the wild-type (WT). Seven samples had missense mutations compared to the WT, but there was no association with survival or mortality. There was a significant difference (p=0.001) between survival rates of cats infected with C. felis that solely contained the WT cytb (12/12 survived) compared to those infected with C. felis of any other cytb genotype (16/33 survived). However, the mere presence (i.e. alone or concurrent with other genotypes) of the WT cytb was not associated (p=0.35) with survival (15/21 survived) when compared to cats without the WT cytb (13/24 survived). In conclusion, there is a high degree of genetic variability in the C. felis cytb gene, and cytb genotypes may be associated with survival.

Funding provided by: An Anonymous Donor

39 PROTECTING THE HEART: HSP25 AS AN INNATE RESPONSE TO VIRAL INFECTION

Rachael Stebbing, Kim Parks, Barbara Sherry NCSU CVM

Non-replenishable cardiac myocytes are continuously exposed to viruses, yet the heart lacks a “blood-brain barrier”-like structure that protects the similarly vulnerable central nervous system. While the type I interferon response has been well-documented as one of the earliest host defenses against viral infection in many organs including the heart, it is unlikely that the heart would have evolved just one antiviral mechanism. Previously, our laboratory used a proteomic approach to identify additional proteins involved in the cardiac antiviral response. Hsp25 was identified as a protein that is phosphorylated by a virus that does little damage to the heart but that is degraded by a virus that is potently myocarditic. Therefore, we hypothesized that the activation of Hsp25 is an additional innate protective response to viral infection. A cell line was established that over-expresses Hsp25 with GFP as a marker. When treated with H2O2 to mimic ischemic damage and then analyzed by flow cytometry, cells expressing the highest levels of GFP were best protected. Because the cells in the population expressed varying levels of Hsp25, the culture was sorted by FACS into four subpopulations based on their GFP intensities. When the subpopulations were infected with virus and analyzed by flow cytometry, the subpopulation with the highest Hsp25 expression contained the greatest percent of uninfected cells. These preliminary data suggest that Hsp25 protects against viral infection. Future studies will determine if this observed protective effect extends to additional virus families that can infect and damage the heart.

This research was supported by NIH R01 AI083333.

40 THE SEDATIVE EFFECTS OF ORAL-TRANSMUCOSAL DETOMIDINE GEL IN COMPARISON WITH INTRAVENOUS DEXMEDETOMIDINE IN DOGS.

Kristen M. Messenger, Marie J. Hopfensperger, Malte Schwartz, and Mark G. Papich NCSU-CVM

Detomidine gel (Dormosedan Gel®, Pfizer Animal Health) is an FDA-approved equine sedative and is unique because of its oral-transmucosal (OTM) route of administration. Oral- transmucosal detomidine gel may offer an alternative route of administration of a sedative to dogs, not only providing an easier means of administration, but also eliminating painful injections. The objective of this study was to compare the sedative effects of intravenous (IV) dexmedetomidine with OTM detomidine gel in dogs undergoing jugular intravenous catheter placement. We hypothesized that OTM detomidine gel would provide equivalent sedation as IV dexmedetomidine in dogs. Six healthy adult dogs were administered either 0.125 mg/m2 dexmedetomidine IV or 0.5 mg/m2 detomidine gel OTM in a randomized blinded crossover design. Level of sedation was assessed using a pre-determined scoring system by a single blinded observer during jugular vein catheterization. Jugular catheters were placed successfully in 6/6 dogs that received IV dexmedetomidine and 4/6 dogs that received OTM detomidine. Dogs that received IV dexmedetomidine had a greater global sedation score prior to catheterization (p=0.002); however there was no difference in total sedation score between the two groups (p=0.1). No adverse effects were observed in any dog at any time point. Oral- transmucosal detomidine gel at a dose of 0.5 mg/m2 provided equivalent sedation as IV dexmedetomidine in healthy dogs undergoing jugular vein catheterization and can be considered as an alternative to injectable sedatives. Further studies are necessary to characterize the bioavailability and distribution of detomidine gel in dogs when administered via the OTM route.

Funding Source: Internal funding from the Clinical Pharmacology Laboratory

41 EFFECTS OF MS 222 ON ELECTRORETINOGRAPHY (ERG) IN KOI FISH

Bailey KM, Hempstead JE, Tobias JR, Borst LB, Clode AB, Posner LP NCSU CVM

Tricaine methanesulfonate (MS-222) is an immersion anesthetic commonly used and approved for use in fish. MS-222 is reported to be retinotoxic in fish, frogs, and humans although there is little evidence to support that claim. We hypothesized that koi fish exposed to MS-222 for 20 minutes daily for 13 consecutive days would not have changes indicative of retinal damage in either the amplitude of ERG b-waves, or retinal histology. Eighteen koi fish (Cyprinus carpio) were utilized in the study. Prior to MS-222 exposure, 2 fish were euthanized and the eyes were submitted for histology to be used as controls. The remaining fish were anesthetized with MS-222 at concentrations varying from 125-200 ppm for 20 minutes each day for up to 13 consecutive days. An ERG was performed on both eyes; of each fish, on day 1, following which two fish were euthanized and eyes submitted for histology. On days 7 and 13, ERG and histology were repeated. Mean b-wave amplitudes for each eye on days 1, 7, and 13 were 17.6, 25.3, 16.1 microvolts (OD), and 15.9, 18.2, 15.3 microvolts (OS) respectively. Using a Kruskal-Wallis one way ANOVA with p value of ≤ 0.05 determining significance, consecutive b-wave amplitudes from the same eye were compared, with no significant differences among days 1, 7 and 13 (OD; p=0.40, OS; p=0.84). No histologic changes indicative of retinotoxicity were identified. This study suggests that exposure of koi fish to clinically relevant amounts of MS-222 should not adversely affect retinal structure or function.

42 ACTIVATION OF THE TGF-Β SIGNALING PATHWAY IN A DMN-INDUCED FISH MODEL OF HEPATIC FIBROSIS

A. J. Van Wettere,1, 4 S. W. Kullman,2 D. E. Hinton,3 J. M. Law 4

1 Center for Comparative Medicine and Translational Research, NCSU CVM, Raleigh, NC, USA 2 Department of Toxicology, NCSU, Raleigh, NC, USA 3 Nicholas School of the Environment, Duke University, Durham, NC, USA 4 Department of Population Health and Pathobiology, NCSU CVM, Raleigh, NC, USA

Despite a wealth of information in humans and rodents, the mechanism of hepatic fibrosis is poorly understood in fish. In mammals, hepatic stellate cell (HSC) transdifferentiation into a fibrogenic myofibroblast-like phenotype is a key event and the transforming growth factor beta (TGF-β) pathway is a critical mediator of this process. In this study, we examined the changes in the mRNA expression of tgfb1, TGF-β receptor I (tgfbr1) and II (tgfbr2), and smad3 during development of hepatic fibrosis in a Japanese medaka fish model of hepatic injury. Immunohistochemistry was used to localize the cell producing TGF-β1. We also assessed hepatic stellate cell activation using muscle specific actin immunohistochemistry and collagen content using Masson’s trichrome-stained liver sections. Three- month-old medaka were exposed to dimethylnitrosamine (DMN) in the ambient water for two weeks to induce hepatic fibrosis. Fish were euthanized at 2, 4, 6, or 10 weeks after exposure. Gene expression was determined using quantitative RT-PCR and correlated with histology. Levels of tgfb1, tgfbr2 and smad3 mRNA were significantly upregulated during development and progression of hepatic fibrosis. Expression level of tgfbr1 remained unchanged. TGF-β1 was mainly expressed in bile preductular epithelial cells. Increase in tgfb1, tgfbr2 and smad3 mRNA expression coincided with activation of HSCs and deposition of extracellular matrix. Collectively these data support a role for the TGF-β pathway in mediating the development of fibrosis during chronic liver injury in the medaka fish model.

Funding Source(s): • Van Wettere is supported by Ruth L. Kirschstein national research service award T32 RR024394 as part of NCSU’s comparative medicine and translational research training program • NCSU’s Comparative Medicine and Translational Research pilot project grants 2010

43 MAST CELL CORTICOTROPIN-RELEASING FACTOR RECEPTORS PLAY OPPOSING ROLES IN MEDIATING STRESS-INDUCED BARRIER DYSFUNCTION

Amelia Gibson, Adam Moeser NCSU CVM, Comparative Biomedical Sciences

It is well known that psychological stress can influence the development and severity of gastrointestinal diseases. Previous research has shown that stress causes breakdown of intestinal barrier function that involves release of corticotropin releasing factor (CRF) and activation of intestinal mast cells (MCs); however, the pathway by which CRF influences MC activation remains unclear. Here we investigated the role of MC CRF receptors 1 and 2 (CRF1 and CRF2) in a model of stress-induced intestinal barrier dysfunction. MC-deficient mice (W-sh/ W-sh) were repleted with bone marrow-derived mast cells (BMMCs) from wild type (WT), CRF1 KO, or CRF2 KO mice. Following the successful repletion of BMMCs, mice were subjected to 3 hours of restraint stress (RS). Colonic transepithelial electrical resistance (TER) and paracellular flux of FITC-dextran (FD4) were measured in Ussing Chambers as indices of barrier function. There was a significant increase (p=0.003) in the rate of FD flux in WT mice subjected to RS but not in W-sh/ W-sh mice. Repletion of W-sh/ W-sh mice with WT BMMCs restored stress- induced barrier dysfunction (p=0.01). Repletion of W-sh/ W-sh mice with CRF1 KO BMMCs ameliorated stress-induced increases in FD4 flux. In contrast, repletion of W-sh/ W-sh mice with CRF2 KO BMMCS exacerbated stress-induced barrier dysfunction compared with WT BMMC controls (p<0.05). These data definitively show that MC CRF1 mediates stress-induced intestinal barrier dysfunction while CRF2 may play a critical protective role. These opposing roles of CRF receptors may help in identifying novel preventative and therapeutic targets to mitigate stress- related GI disorders.

Funding source: National Institutes of Health (NIH), K08DK084313-01(AJM)

44 THE DEVELOPMENT OF A LARGE ANIMAL MODEL FOR TRANSLATIONAL STUDIES OF INTESTINAL DISEASE FOCUSING ON INTESTINAL STEM CELLS

Liara M. Gonzalez1, Scott Magness2, Jorge Piedrahita3, and Anthony T. Blikslager1 Department of Clinical Sciences1, Molecular Biomedical Sciences3, North Carolina State University, Raleigh, NC, USA and Department of Medicine2, University of North Carolina, Chapel Hill, NC, USA

A large animal model is needed to transition knowledge gained from murine studies to the treatment of patients with gastrointestinal (GI) disease. It is imperative that advances made in understanding the reparative role of intestinal stem cells (ISCs) be transferred from bench to bedside. Currently, no large animal model exists that clearly labels the distinct cells populating the GI mucosa. Without this information, the effects of injury to this barrier, integral to patient survival, cannot be well studied. Our hypothesis is that the pig is an ideal translational model for the study of the ISC niche. Intestinal tissue from healthy 8 week-old Yorkshire cross pigs was collected. Tissue for immunofluorescence was fixed in 4% paraformaldahyde, transferred to 30% sucrose solution and embedded in optimal cutting temperature media. Tissue for qualitative and quantitative analysis of gene expression utilizing RT-PCR and qRT-PCR were snap frozen in liquid nitrogen. Preliminary results demonstrated positive staining for: sex determining region Y-box 9, antigen identified by monoclonal antibody Ki67, lysozyme, Mucin 2, chromagranin A, gastrin, somatostatin, villin, carbonic anhydrase and epithelial cell adhesion molecule. Antibodies developed for murine intestine cross react with swine tissue. The suitability of the pig as a model for further GI disease research depends on available tools such as antibodies/markers. This will establish the platform for development of transgenic pigs and elucidating effects of disease on ISCs. This will provide insight into the possibility of manipulating cell function therapeutically with a goal of improving mucosal repair processes.

Funding Source: NC TraCS Pilot Grant Program

45 EARLY WEANING IMPAIRS GUT MUCOSAL DEFENSES AND EXACERBATES CLINICAL DISEASE IN F18 E. COLI INFECTION

Brittney L. McLamb, Chad Stahl, Adam J. Moeser

NCSU CVM: Department of Population Health and Pathobiology

The objective of this study was to investigate the impact of weaning age on susceptibility and severity of F18 E. coli enteric disease. Yorkshire piglets weaned at 15-16 d (Early Weaned, EW), 17-19 d (middle weaned, MW) and 20-22 d (Late weaned, LW) were individually housed, given ad libitum access to water and food, and assigned to one of six experimental treatments (n=12 piglets/treatment): 1)EW; 2)EW + E.coli; 3)MW; 4)MW + E.coli; 5)LW; 6) LW + E.coli. At 28 days of age, piglets were orally inoculated with 1x109 CFU of F18 E. coli and fecal scores (2x daily for 4 days) and intestinal barrier function (Day 4 post-challenge) was measured. Histological analyses were done on fixed intestinal tissues and neutrophil counts were performed as an index of intestinal inflammation. In response to F18 E. coli challenge, LW pigs exhibited reduced fecal scores throughout the trial (p<0.05), and less intestinal injury as indicated by lower (p<0.05) FITC dextran intestinal permeability in intestinal tissues mounted on Ussing chambers compared with EW and MW pigs. E. coli challenge caused increased neutrophil infiltration into intestinal tissues that was more pronounced in LW pigs (p<0.01). These data suggest that LW ameliorated intestinal barrier dysfunction and clinical disease severity in the presence of an E.coli challenge. Clinical and physiologic differences between weaning age groups may be due to the enhanced ability of the LW pig to mount an appropriate innate immune response, characterized by tissue neutrophil infiltration

Funding sources: National Pork Board and NIH (K08DK084313)

46 CRF-MEDIATED MAST CELL ACTIVATION AND INTESTINAL BARRIER INJURY IS MEDIATED BY THE ENTERIC NERVOUS SYSTEM

Laura L Riggs, Beth L. Overman, Adam J. Moeser North Carolina State University, College of Veterinary Medicine

Psychological stress is an important predisposing factor for many inflammatory, infectious, and functional intestinal diseases of humans and veterinary species. Previous studies show corticotropin-releasing factor (CRF) is a central mediator in stress-induced intestinal barrier dysfunction and this response is mediated by intestinal mast cell (MC) activation; however the precise mechanisms remain poorly understood. The objective of this study was to determine the role of enteric neuronal input on MC activity in CRF-induced intestinal barrier dysfunction. Porcine ileum was mounted on Ussing chambers and treated with CRF (0.5µM) in the presence of either a neuronal or MC inhibitor. Intestinal barrier function, measured as mucosal permeability to 4kDa FITC dextran (FD4), was measured over 180 minutes. Treatment with CRF significantly (p <0.001) increased FD4 flux compared with controls. Pre-treatment with the MC stabilizer drug cromolyn (104M) prevented increases in gut barrier dysfunction induced by CRF (p <0.05). Pre-treatment with tetrodotoxin (TTX, 10-7M), a neuronal sodium channel blocker, reduced baseline intestinal permeability and CRF-induced intestinal permeability (p<0.001), indicating that intact enteric neuronal signaling is required for both homeostatic and pathophysiologic intestinal barrier regulation. Histological analysis revealed that TTX prevented MC degranulation under baseline and CRF-stimulated conditions suggesting that enteric nerves modulate intestinal barrier function via control of MC activation rather than direct neuronal signaling to the epithelium, as previously hypothesized. Overall, these data provide evidence that CRF’s deleterious effects on the intestinal barrier require intact neuronal-MC communication; therefore, these findings have strong relevance to the development of therapeutic strategies for stress-related GI diseases.

Funding Source: Merial Summer Scholars Program

47 EVALUATION OF DOPPLER AS A TECHNIQUE TO DETECT CHANGES ASSOCIATED WITH ASCENDING BACTERIAL PLACENTITIS IN THE MARE

Jason M. Heitzman, C. Nicholas Buchanan, C. Scott Bailey, Cassandra Bare, Renan D. Sper, Kate Archibald, Mike Whitacre North Carolina State University, College of Veterinary Medicine

Doppler ultrasound has been shown to sensitively predict pregnancy complications in women. It represents an attractive modality for detection of equine placentitis early in the course of disease, whereas grey-scale ultrasonography depends on physical alterations of the combined thickness of the uterus and placenta (CTUP) and clinical signs are insensitive. In the current study, we hypothesized that mares with experimentally-induced placentitis would have lower Resistance and Pulsatility Indices than uninfected mares.

Twelve pregnant pony mares were randomly assigned to two groups (CONT and INOC). All mares were monitored by physical examination, greyscale ultrasonography and Doppler ultrasonography from gestational day 250 until parturition. Mares in Group INOC received 1x107 colony forming units of Streptococcus equi zooepidemicus between 280-295 days gestation. Treatment was initiated in inoculated mares when CTUP values exceeded published normal values.

Placentitis was diagnosed in 5/6 inoculated mares within 3 days based on CTUP (Mean±SD:1.83±0.7). One mare failed to develop placentitis after inoculation and one mare in Group CONT developed spontaneous placentitis. These mares were eliminated from the statistical analysis. CTUP was significantly different between groups on the day of diagnosis (1.05 vs. 0.45; P=0004). CTUP further varied by gestational age (250-270, 270-300, 300-330, and 330+ days) in both groups (P=0.0045 : INOC and P<0.00001 : CONT). In contrast, Resistance and Pulsatility Indices were not different between groups prior to or after inoculation and did not vary with gestational age.

These findings do not support the hypothesis that Doppler ultrasonography is a sensitive means of diagnosing equine placentitis in experimentally-induced pony mares.

48 ELASTOGRAPHIC EVALUATION OF THE EQUINE DISTAL FORELIMB

Meghann Lustgarten, W. Rich Redding, Rafael Labens, Gabriela S. Seiler NCSU CVM

Elastography is an ultrasound technique that evaluates mechanical properties of tissues, such as stiffness, by measuring the displacement of ultrasound echoes before and after compression. Elastography has shown promise in evaluating musculoskeletal injuries in people. Tendon and ligament injuries are common in equine athletes, but currently only morphology, not mechanical properties, of these structures can be evaluated. The purpose of this study was to determine feasibility and repeatability of elastography for imaging tendons and ligaments of the equine distal forelimb, as well as to establish the normal elastographic appearance of the digital flexor tendons and suspensory ligament branches. Forelimbs (n=20) of 18 horses without evidence of lameness, and with sonographically normal tendons and ligaments were included. Elastographic images of the digital flexor tendons, and the branches of the suspensory ligaments were obtained in longitudinal and transverse planes by two sonographers. The study was repeated with the legs in different positions, including non weight bearing. Images were evaluated qualitatively and quantitatively. Inter-operator (68%), inter-observer (79%), and intra-observer repeatability (83%) were acceptable. Tendons and ligaments were significantly softer when non weight bearing (P<0.001). There were no significant differences between different weight bearing positions. Normal tendons were mostly hard with the flexor tendons becoming progressively harder distally, and the suspensory branches softer superficially (P<0.01). Elastography is a feasible, non invasive and repeatable method for evaluation of the equine digital flexor tendons and suspensory branches. The normal elastographic appearance of these structures can be used as a baseline for investigation of injuries.

49 THE CANINE MHC CLASS I ALLELE, DLA-88*50801, PRESENTS PEPTIDES OF VARYING LENGTHS WITH FOUR KEY ANCHOR RESIDUES

Peter Ross, Jennifer Holmes, Greg Gojanovich, Keith Miller, and Paul Hess NCSU CVM and Immunology Program

Cytotoxic T lymphocytes (CTL) surveil the body’s cells through recognition of short peptide epitopes of cytosolic proteins bound within Major Histocompatibility Complex (MHC) class I molecules displayed on the cell surface and eliminate cells presenting non-self peptides. The binding of a peptide to an MHC allele depends upon a few key residues within the peptide sequence, which is present only in ~1-2% of peptides. Identification of this motif allows one to select candidate epitopes for a given antigen that may be recognized by CTL. While several studies have determined the binding specificities of numerous class I alleles in both humans and mice, no such study has been performed in the domestic dog. The principal goal of this study was to determine the peptide binding motif of a prevalent MHC class I allele, Dog Leukocyte Antigen (DLA) -88*50801. We hypothesized that a conserved binding motif could be identified through sequencing of endogenously presented peptides eluted from DLA-88*50801. To accomplish this goal, a plasmid encoding the DLA-88*50801 gene with an attached affinity tag was stably transfected into a canine cell line (DH82). A lysate of ~8 x 109 cells was affinity purified to obtain DLA-88*50801 protein. The bound peptides were eluted from the MHC binding groove utilizing low pH and heat, isolated by size exclusion and affinity purification, and subjected to tandem mass spectrometry analysis to obtain individual peptide sequences. The binding motif was determined through manual alignment of the sequences. This study provides essential information to study CTL responses in canines.

50 CLINICAL ANATOMY OF THE BEARDED DRAGON (POGONA VITTICEPS) E. Wayne, B. Waffa, M. Stoskopf College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606 US

Pet retiles are popular pets in the United States, and there is a growing demand for veterinarians capable of providing quality health care for these species. Basic anatomy data is lacking in the scientific literature even for popular companion species such as the Bearded Dragon (Pogona Vitticeps). This project obtained the data needed to create a detailed illustrated anatomical reference for P. Vitticeps, highlighting clinically relevant landmarks.

Studies were conducted using 11 frozen adult animals culled and donated by commercial breeders . Five (5) of the specimens were chosen for detailed dissection and imaging based on size, body condition, sex, and overall condition of the carcass. One thawed specimen was dissected for orientation and to inform the imaging of future specimens. After being thawed in a refrigerator for 12-16 hours the other four of these specimens were imaged using digital radiography, Computed Tomography (CT), and Magnetic Resonance Imaging (MRI) . At the completion of imaging, each specimen underwent detailed dissection. Weights, dimensions, location, and appearance of internal structures were recorded and photographed.

After the dissections were completed, illustrated anatomical plates detailing internal anatomy and landmarks for tissue sampling and venipuncture were created. Particular attention and detail was paid to reproductive structures to guide clinicians considering spaying and/or neutering procedures. These illustrated plates were combined with digital radiography, CT, and MRI images to create a more complete reference guide for clinicians to use when working up a case.

Funding Sources:George H. Hitching’s New Investigator Award in Health Research NCSU CVM Teaching Innovation Grant

51 DEVELOPMENT OF A QUESTIONNAIRE TO ASSESS CHRONIC PAIN ASSOCIATED WITH FELINE DEGENERATIVE JOINT DISEASE.

Benito J 1, DePuy V 2,1, Davidson GS 3, Zamprogno H 1, Thomson A 1, Simpson W 4, Gruen ME 5,1, Roe S 1, Hardie E 1, Hansen B 1, Lascelles BDX 1 1. Comparative Pain Research Laboratory (CPRL) & Center for Comparative Medicine and Translational Research. Dept. of Clinical Sciences. NCSU CVM. 2. Bowden Statistical Consulting, Raleigh, NC 3. Clinical Pharmacy Services, NCSU CVM. 4. Morrisville Cat Hospital, Morrisville, NC. 5. Animal Behavior Service. Dept. of Clinical Sciences. NCSU CVM.

No approved drug therapies are available for the treatment of chronic pain in the cat in the US, partly due to the fact that there are no validated assessment tools. We hypothesized that an appropriately developed subjective owner-completed instrument to assess chronic feline DJD-associated pain (Feline Musculoskeletal Pain Index [FMPI]) would prove reliable and have responsiveness and criterion validity. The constructed FMPI readability was explored with four different readability tests. 45 client- owned cats were involved in this study – 20 normal, and 25 with DJD. The FMPI sensitivity and validity was explored through a double blinded, stratified, randomized, placebo-controlled crossover clinical study over a 10 week period in 25 cats. Meloxicam was used to effect pain relief, and FMPI subjective data were compared to objective outcome measures (accelerometry). The constructed 21 question FMPI had a 6th grade readability score. Reliability (Cronbach’s alpha 0.90 and 0.93 respectively) and repeatability were good (ICC values over 0.8) for normal and DJD affected cats. The FMPI showed positive strong responses for placebo and treatment; however no treatment effects were detected under a linear mixed model. There were no treatment effects on objectively measured activity. Using a backwards-stepping regression model, percent of meloxicam target dose administered, temperament, and total FMPI score at baseline were the covariates that had most effect on FMPI. Controlling for significant covariates, most positive effects (FMPI and accelerometry) were seen for the placebo treatment. FMPI showed a good internal consistency, however neither responsiveness nor criterion validity were detected.

Funding Source(s): Morris Animal Foundation; Novartis Animal Health Global Fellowship Program.

52 CHANGES IN VON FREY QUANTITATIVE SENSORY THRESHOLDS AS A RESULT OF TOTAL HIP REPLACEMENT IN THE TREATMENT OF COXOFEMORAL JOINT OSTEOARTHRITIS

Bennett AD, Roe S, Marcellin-Little D, Lascelles BDX Comparative Pain Research Laboratory (CPRL) & Center for Comparative Medicine and Translational Research, Department of Clinical Sciences, NCSU CVM.

Peripheral input to the spinal cord from chronic pain may be associated with central nervous system sensitization (CS) resulting in altered sensory processing. Quantitative sensory testing (QST) uses reactions to applied stimuli (such as mechanical thresholds) to test for secondary hyperalgesia and CS. Total hip replacement (THR) is a surgical procedure that eliminates OA-associated hip pain. By decreasing peripheral sensory input, THR may allow CS to decrease, reflected in QST changes. The purpose this study was to evaluate the correlation between von Frey mechanical QST and hip pain, and to determine if THR resulted in altered QST over time. THR surgery was performed on 47 dogs using the BFX® THR technique. Static and dynamic ground reaction forces (limb use measures) were measured prior to THR and at 3, 6, and 12 months postoperatively. A digital von Frey filament device was used to measure mechanical QST at the same time points. Preoperatively, there was no significant correlation between degree of limb use and von Frey thresholds (correlation coefficients < 0.3; not statistically significant). All limb use parameters were significantly improved by 3 months postoperatively (p<0.001). When comparing von Frey QST pre versus post-operatively, there was no significance at 3 months (p= 0.26) or 6 months (p=0.85), but at 12 months post operatively von Frey QST were significantly increased (p= 0.03). Dogs where a chronic pain source was eliminated (by THR) were less sensitive to mechanical stimulation. This suggests that central sensitization decreased after 12 months of adaptation.

Funding Source(s): Comparative Pain Research Laboratory

53 COMPARISON OF THERMAL THRESHOLD LATENCIES BETWEEN NORMAL DOGS AND DOGS WITH HINDLIMB OSTEOARTHRITIC ASSOCIATED PAIN

Williams MD, Kirkpatrick AE, Hash J, Thomson A, Kwan T, Benito J, Lascelles BDX Comparative Pain Research Laboratory (CPRL) & Center for Comparative Medicine and Translational Research, Department of Clinical Sciences, NCSU CVM.

Chronic pain may be associated with central nervous system sensitization (CS) and result in altered sensory processing, including secondary hyperalgesia. CS may result in increased pain and may require particular analgesics to allow effective pain management. Quantitative sensory testing (QST) uses reactions to applied stimuli (such as heat) to test for secondary hyperalgesia and CS. It is unknown if CS occurs in dogs with osteoarthritis (OA). The objectives were to determine whether thermal threshold latencies (TTL) could be measured in client-owned dogs, were repeatable from week to week, and were different between normal dogs and dogs with hindlimb OA. A high-powered light source delivered a thermal stimulus to digital pad III. Time to withdrawal in seconds (TTL) was measured. Five tests were performed on each hindlimb at each time point. Clinically normal dogs (n=30) and dogs with OA-associated hindlimb pain (n=30) were tested on two occasions 2 weeks apart. Data were evaluated using paired and unpaired comparisons. Thermal thresholds were successfully measured in 34/34 client-owned dogs without prior training. Week to week TTL did not vary significantly in normal (p=0.953) or OA dogs (p=0.716). No significant difference was found between TTL of the control group (15.64s +/- 2.99s) and OA group (18.30s +/- 5.60s) (p =0.086), however TTL were seen in the OA group. TTL can be measured in client owned dogs with no training, and are repeatable from week to week. Further data is needed to know if OA causes an alteration of TTL.

Funding Source(s): Comparative Pain Research Laboratory

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