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AAVLD Strategic Plan Updated August 7, 2019

Vision The AAVLD is a world leader in advancing the discipline of veterinary diagnostic laboratory science to promote global animal health and One Health.

Mission The AAVLD promotes continuous improvement and public awareness of veterinary diagnostic laboratories by advancing the discipline of veterinary diagnostic laboratory science. The AAVLD provides avenues for education, communication, peer-reviewed publication, collaboration, outreach, and laboratory accreditation.

Motto: Advancing veterinary diagnostic laboratory science

Core values The AAVLD is committed to these core values: • Continuous improvement • Engagement of members • Effective communication • Collaboration • Support of One Health

Goals 1. Advocate for the role of veterinary diagnostic laboratories in One Health by engaging in development of animal health initiatives, policies, and dissemination of surveillance information. 2. Foster continuous improvement of diagnostic laboratories through accreditation and continuing education activities while encouraging discovery and innovation in veterinary laboratory diagnostic sciences. 3. Strengthen communication with members and promote their continued professional growth.

Our membership spans more than 32 countries worldwide. Join us today and discover for yourself the benefits and resources that AAVLD provides to its members.

https://www.aavld.org https://www.facebook.com/AAVLD American Association of Veterinary Laboratory Diagnosticians

The American Association of Veterinary Laboratory Diagnosticians (AAVLD) is a not-for-profit professional organization.

AAVLD Officers, 2020 President Deepanker Tewari, Harrisburg, PA President-elect Shuping Zhang, Columbia, MO Vice-president Jerry Saliki, Stillwater, OK Secretary-Treasurer Kristy Pabilonia, Fort Collins, CO Immediate Past-President Keith Bailey, Urbana, IL

AAVLD Executive Director David H. Zeman, Brookings, SD Executive Director Assistant Reda Ozuna, Visalia, CA

AAVLD Executive Board, 2020 President Deepanker Tewari, Harrisburg, PA President-elect Shuping Zhang, Columbia, MO Vice-president Jerry Saliki, Stillwater, OK Secretary-Treasurer Kristy Pabilonia, Fort Collins, CO Immediate Past President Keith Bailey, Urbana, IL Northeast Robert Gibson, Durham, NH North Central Eric Burrough, Ames, IA Northwest Kevin Snekvik, Pullman, WA Southeast Debbie Reed, Hopkinsville, KY South Central Amy Swinford, College Station, TX Southwest Ashley Hill, Davis, CA Canada Provincial Neil Pople, Winnipeg, MB Canada Federal, ex-Officio Maria Perrone, Ottawa, ON NVSL, ex-Officio Suelee Robbe-Austerman, Ames, IA AVMA, ex-Officio Mike Murphy, Stillwater, MN

AAVLD Business Office PO Box 6396, Visalia, CA 93290 Phone 559-781-8900 Fax 559-781-8989 Email: [email protected] Website: www.aavld.org Facebook: www.facebook.com/AAVLD/ Acknowledgments

The success of AAVLD depends on the contributions of so many individuals that it is always hard to single out any one person or group. That being said, I would like to thank all participants who make the 63rd AAVLD Annual Meeting (virtual) possible. I applaud all presenters who are willing to summarize their work and share the results with fellow diagnosticians and trainees. I must recognize the AAVLD Plenary Session and the USAHA-AAVLD Keynote speakers for their leadership and insightful presentations. Program Committee members and moderators deserve special thanks for reviewing abstracts and hosting the live Q&A sessions. The exhibitors and sponsors have once again showed their strong support for our organization.

On behalf of the entire AAVLD executive team, I want to thank Reda Ozuna for the excellent administrative support, and Kaylin Taylor for the flawless meeting coordination. Finally, a heartfelt thank you belongs to the Executive Director, Dr. Dave Zeman who has worked tirelessly to organize this virtual meeting.

The pandemic will only make us more creative, resilient and unified.

Sincerely, Shuping Zhang BVSc, MS, PhD, DACVM ****************************************************************************** Program Committee

Shuping Zhang Chair Jerry Saliki Co-chair

Jianfa Bai Chien-che Hung Keith L. Bailey Marcia Ilha Eric Burrough Christina Loiacono Cat Barr Tomy Joseph Beate Crossley Suresh Kuchipudi Laura Goodman Akhilesh Ramachandran Erin Goodrich Kevin Snekvik Tamara Gull Deepanker Tewari Ben Hause Ines Walther Ashley Hill Jianqiang Zhang Stephen Hooser Yan Zhang

Scientific Session Moderators

Jiafa Bai Tomy Joseph Keith Bailey Suresh Kuchipudi Cat Barr Mani Lejueune Beate Crossley Hemant Naikare Laura Goodman Solomon (Wole) Odemuyiwa Erin Goodrich Orhan Sahin Tamara Gull Kevin Snekvik Ashley Hill Ines Walther Stephen Hooser Jianqiang Zhang Chien-che Hung Yan Zhang Rosalie Ierardi

****************************************************************************** Please note: Abstracts published in these proceedings were peer reviewed by the members of the Program Committee for data supporting conclusions to be presented, and were edited into a consistent format. Full manuscripts were not evaluated and readers should contact the author for referral to a full presentation of data or for permission to use, copy, or distribute data contained in an abstract. 2020 Trainee Awardees

Athey, Jillian University of Illinois at Urbana-Champaign Cardia Caserta, Leonardo Cornell University Carpenter, Molly Colorado State University Cheng, Ting-Yu Iowa State University Croce, Andeliene North Carolina State University De Luca, Eliana University of Georgia Elbert, Jessica University of Georgia Gonzales Viera, Omar University of California-Davis Henao-Diaz, Alexandra Iowa State University Hengy, Miranda Ohio State University Hille, Matt University of Nebraska-Lincoln Kalantari, Allen University of Georgia-Tifton Navarro, Mauricio University of California-Davis Niedringhaus, Kevin University of California-Davis O'Toole, Alicia University of Georgia Poeta Silva, Ana Paula Iowa State University Schumacher, Loni Iowa State University Yang, Tzushan North Carolina State University Zheng, Zhiyi Iowa State University

2020 Awards Committee

Dr. Keith Bailey, Chair Dr. Steve Hooser Dr. Pat Halbur Dr. Francois Elvinger Dr. Brett Webb

The AAVLD Foundation with funds received from our members and committees have provided over $30,000 annually to support the advancement of our discipline and the development of the next gen- eration of veterinary laboratory diagnosticians. Thank you to all donors! The AAVLD Foundation Committee 2019 AAVLD Organizational Awardees

E.P. Pope Award Dr. Dave Steffen

Distinguished Service Award Dr. Francisco A. Uzal

Outstanding Performance Award for Diagnostic Services Stacy Pollock

Life Membership Award Dr. Grant Maxie Dr. Daniel P. Shaw

Best Oral Presentation Giovani Trevisan

Best Poster Presentation Grazieli Maboni

2019 AAVLD Committee Awards

BIOMIC Award for Excellence in Diagnostic Microbiology Sponsored by Giles Scientific Dr. John Dustin Loy

Richard L. Walker Best Classical Bacteriology Oral Presentation Award Sponsored by Anaerobe Systems Sreenidhi Srinivasan

J. Lindsay Oaks Best Molecular Oral or Poster Presentation Award Sponsored by BioMerieux Sai Narayanan

Brenda Love Best Classical Bacteriology Poster Presentation Award Sponsored by VMRD Molly Carpenter

Diagnostic Pathology Slide Seminar Resident/Graduate Student Award Bianca R. Pfisterer, Marta Mainenti, Abigail Finley

2019 Joint AAVLD/ACVP Pathology Awards

AAVLD/ACVP Diagnostic Pathology Resident/Graduate Student Award Awarded by AAVLD Vanessa J. Oakes

ACVP/AAVLD Diagnostic Pathology Resident/Graduate Student Award Awarded by ACVP Tzushan Yang blank page Table of Contents

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AAVLD Plenary Session Friday, October 16, 2020 Emerging Diseases with Significant Impact on Public Health and Animal Health 3

AAVLD Scientific Sessions Thursday/Friday, October 15-16, 2020 Thursday Serology/Toxicology Live Q&A Serology/Toxicology 7

Thursday Epidemiology Live Q&A Epidemiology 19

Thursday Molecular & Bioinformatics Live Q&A Molecular & Bioinformatics 25

Thursday Virology Live Q&A Virology 41

Thursday Bacteriology Live Q&A Bacteriology 51

Thursday Pathology Live Q&A Pathology 65

Friday Bacteriology Poster Live Q&A Bacteriology Poster 85

Friday Epidemiology Poster Live Q&A Epidemiology Poster 95

Friday Molecular & Bioinformatics Molecular & Bioinformatics Poster Live Q&A Poster 99

Friday Parasitology Poster Live Q&A Parasitology Poster 105

Friday Virology Poster Live Q&A Virology Poster 109

Friday Serology Poster Live Q&A Serology Poster 115

Friday Toxicology Poster Live Q&A Toxicology Poster 119

Friday Pathology Poster Live Q&A Pathology Poster 123

AAVLD Author Index 129 AAVLD Keyword Index 133

AAVLD Annual Conference Proceedings 1 AAVLD Virtual Conference blank page Emerging Diseases with Significant Impact on Public Health and Animal Health

Friday, October 16th, 2020 11:00 am – 1:00 pm

Moderators: Shuping Zhang and Jerry Saliki

U.S. SARS-CoV-2 Animal Diagnostics and Laboratory Collaborations

Mia Torchetti DVM, MS, PhD, Director Diagnostic Virology Laboratory, National Veterinary Services Laboratories Christina Loiacono DVM, PhD, Dip. ACVP, Coordinator National Animal Health Laboratory Network, National Veterinary Services Laboratories

The first detection of an animal in the U.S with COVID-19 was confirmed at the National Veterinary Services Laboratories (NVSL) on April 4th, 2020, in a clinically ill tiger at the Bronx Zoo in New York. Since then, the USDA has worked diligently to support the testing of animals in the US for SARS-CoV-2 to both learn more about the virus and provide information to help better understand the role animals play in the transmission of SARS-CoV-2. The NVSL including the National Animal Health Laboratory Network (NAHLN) and the APHIS One Health Office collaborate closely with other Federal partners to provide a One Health approach in order to better understand this emerging threat and the role animals may play in its spread. This talk will describe the diagnostic work that has been done at NVSL to confirm the presence of SARS-CoV-2 in animals, provide some characterization of the virus present in the US, and share how the NAHLN in collaboration with NVSL and other federal agencies is offering diagnostic support.

AAVLD Annual Conference Proceedings 3 AAVLD Virtual Conference

Emerging Diseases: Antibody Responses to SARS-CoV-2 James E. Crowe, Jr., M.D., Director Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN Antibodies to the SARS-CoV-2 spike protein appear to be the principal determinants of immunity to the virus. We have isolated thousands of human monoclonal antibodies from the B cells of humans who have recovered from natural infection. We will discuss detailed studies that reveal the genetic, molecular and structural basis for potent inhibition of virus replication.

AAVLD Annual Conference Proceedings 4 AAVLD Virtual Conference

Emerging Diseases: Antibody Responses to SARS-CoV-2 Influenza at the Human-Animal Interface James E. Crowe, Jr., M.D., Director Richard Webby, PhD. Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN World Health Organization Collaborating Center for Studies on the Ecology of Influenza, St Jude Children’s Research Hospital, Memphis, TN. Antibodies to the SARS-CoV-2 spike protein appear to be the principal determinants of immunity to the virus. We have isolated thousands of human monoclonal antibodies from the B cells of humans who have recovered from natural infection. We will discuss detailed studies that The past two decades has seen an increasing awareness of the importance of zoonotic influenza reveal the genetic, molecular and structural basis for potent inhibition of virus replication. and also an increasing case count. Highly pathogenic avian influenza viruses have spread to many regions of the world and swine influenza viruses have increased in diversity and spawned a pandemic. These trends have led to renewed attempts to assign risk to animal influenza viruses and to implement potential control strategies. At the global scale, collaboration between animal and human health sectors has led to development of formal risk assessment algorithms that have guided preparedness activities and identified areas where gaps exist in data. Identifying ways to fill these gaps must remain a top priority for all partners with expertise to contribute.

AAVLD Annual Conference Proceedings 5 AAVLD Virtual Conference Streptococcus zooepidemicus in Swine: Natural and Experimental Infection Rachel J. Derscheid, DVM, PhD, DACVP In September of 2019, high death loss was noted at a swine buying station in Ohio. Tissues were submitted to Iowa State Veterinary Diagnostic Laboratory (ISUVDL. This submission and a subsequent submission from this site identified Streptococcus zooepidemicus as the cause of acute mortality in swine of various ages. A subsequent case from Tennessee was submitted to the ISU VDL and also identified S. zooepidemicus as the cause of acute mortality. The isolate from this case was utilized in developing a challenge model to better understand disease dynamics. This talk will share the findings of our diagnosticians in the submitted cases as well as our research findings thus far.

AAVLD Annual Conference Proceedings 6 AAVLD Virtual Conference Serology/Toxicology Live Q&A Thursday, October 15, 2020

Moderators: Tomy Joseph and Stephen B. Hooser

1:35 PM Performance of commercial Mycoplasma hyopneumoniae serum antibody-ELISAs # * † Ana Paula Poeta Silva, Ronaldo Magtoto, Henrique Almeida, Aric McDaniel, Precy Magtoto, Rachel Derscheid, Maria Merodio, Franco Ferreyra, Igor Gatto, Dave Baum, Maria Jose Clavijo, Bailey Arruda, Jeffrey Zimmerman, Luis G. Gimenez-Lirola...... 9

1:40 PM Continual diagnostics improvement - the case of the PRRS oral fluid ELISA # * † Alexandra Henao-Diaz, Min Zhang, Luis G. Gimenez-Lirola, Phillip Gauger, Dave Baum, Maria Jose Clavijo, Marisa Rotolo, Esteban Ramirez, Rodger Main, Jeffrey Zimmerman ...... 10

1:45 PM Laboratory performance of a commercial PRRSV oral fluid ELISA # * † Alexandra Henao-Diaz, Ronaldo Magtoto, Ana Paula Poeta Silva, Luis G. Gimenez-Lirola, Dave Baum, Jeffrey Zimmerman...... 11

1:50 PM Determining the seroprevalence of Anaplasma marginale infected beef herds in Georgia ◊ Lee Jones, Hemant K. Naikare, Roy D. Berghaus, Allen Aref Kalantari...... 13

1:55 PM Development and evaluation of a Turkey coronavirus Enzyme-Linked Immunosorbent Assay (ELISA) using S1 spike protein Yuko Sato, Mohamed El-Gazzar, Ronaldo Magtoto, Luis G. Gimenez-Lirola...... 14

2:00 PM CVM Vet-LIRN feed contaminant survey 2012 to 2019 Stephen B. Hooser, Robert H. Poppenga, Renate Reimscheussel, Sarah Nemser...... 15

2:05 PM Multi-class veterinary drug screening and quantitation by high resolution mass spectrometry (HRMS) using the Orbitrap Exploris 120 mass spectrometer Dwayne Edward Schrunk, Laura Burns...... 16

2:10 PM Thevetia bicornuta toxicosis of goats Michael Filigenzi, Francisco A. Uzal, Robert H. Poppenga ...... 17

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 7 AAVLD Virtual Conference blank page Performance of commercial Mycoplasma hyopneumoniae serum antibody-ELISAs # * † Ana Paula Poeta Silva1, Ronaldo Magtoto1, Henrique Almeida2, Aric McDaniel1, Precy Magtoto3, Rachel Derscheid1, Maria Merodio1, Franco Ferreyra1, Igor Gatto2, Dave Baum1, Maria Jose Clavijo1, Bailey Arruda1, Jeffrey Zimmerman1, Luis G. Gimenez-Lirola1 1Iowa State University, Ames, IA; 22Escola de Agricultura e Ciências Veterinárias, Universidade Estadual de São Paulo, Sao Paulo, Brazil; 3Pampanga State Agricultural University, Pampanga, Philippines

Mycoplasma hyopneumoniae (MHP) ELISAs are routinely used for surveillance in swine production systems. This study evaluated six commercial MHP serum ELISAs using samples of known infection status: (1) M hyo ELISA SK 108 BioChek, (2) INgezim M. hyo COMPAC, ©Eurofins Ingenasa, (3) M. hyo Ab Test, IDEXX Laboratories Inc., (4) ID Screen® Mycoplasma hyopneumoniae Indirect, IDvet, (5) CIVTEST® SUIS MHYO, Laboratorios HIPRA, S.A., and (6) IDEIA™ Mycoplasma hyopneumoniae EIA kit, ©Oxoid Limited. Serum samples (n = 680) of known status were collected from 50 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs randomly allocated to 5 treatments: (1) negative control, (2) M. flocculare (strain 27399), (3) M. hyorhinis (strain 38983), (4) M. hyosynoviae (strain 34428), and (5) MHP (strain 232). Serum (2X per week) and oral fluid (daily) samples were collected through 56 days post-inoculation (DPI). MHP PCR testing of oral fluids and lung tissue confirmed productive infection in the MHP group and freedom from MHP infection in other groups. Analysis of ELISA performance at various cutoffs found that the manufacturers’ recommended cut-offs were generally diagnostically specific, i.e., 4 ELISAs produced no false positives on samples of known MHP-negative status. A cumulative analysis of overall misclassification error rates (false positives and false negatives) found that 4 ELISAs performed similarly, although one assay produced more false positives whereas the remaining 3 required a longer time-to-detection in MHP-inoculated animals. Although the temporal antibody response in MHP-inoculated pigs was highly variable, e.g., the first positive responses were observed on 21 DPI, monitoring MHP antibody remains a useful tool for establishing the status of commercial pig populations. However, opportunities for improving time-to-detection and overall diagnostic sensitivity should be pursued. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 9 AAVLD Virtual Conference Continual diagnostics improvement - the case of the PRRS oral fluid ELISA # * † Alexandra Henao-Diaz1, Min Zhang2, Luis G. Gimenez-Lirola1, Phillip Gauger1, Dave Baum1, Maria Jose Clavijo1,3, Marisa Rotolo3, Esteban Ramirez4, Rodger Main1, Jeffrey Zimmerman1 1Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA; 2Statistics, Iowa State University, Ames, IA; 3PIC, Nashville, TN; 4Keken, Merida, Mexico

PRRSV surveillance requires an easily collected specimen and accurate diagnostic tests. Oral fluid (OF) is the most common surveillance specimen in the US1 and both RT-PCR and OF ELISA have been adapted to the matrix. Detection of PRRSV RNA provides the advantage of early detection; PRRSV antibody provides the advantages of lower cost and longer duration of detection2. In routine surveillance, unexpected positives cause disruption in the work flow on the farm and undermine client confidence in the laboratory. Hence, diagnostic specificity (DX-SP), not diagnostic sensitivity (DX-SE), is the major consideration. Under experimental conditions, the DX-SE and DX-SP of the PRRS OF ELISA were estimated at ~100%2 using the manufacturer cut-off S/P 0.4. Herein, we evaluate the DX-SP of the PRRS OF ELISA using field samples and provide alternative cutoffs (S/P 0.6, 0.8, and 1.0) for surveillance. Two sets of OFs samples were used: Set 1: 596 OFs of known negative and positive status. Set 2: 1574 OFs from presumed PRRSV-naïve sites. Set 1 samples were generated under experimental conditions and Set 2 consisted of samples submitted for routine testing at the Iowa State University Veterinary Diagnostic Laboratory. All samples were tested on the PRRS oral fluid Ab ELISA (IDEXX Laboratories Inc.) and the data analyzed using non-parametrical statistical procedures: - DX-SE and DX-SP analyzed by receiver operating characteristic curve (ROC) analysis (Set 1); - ELISA S/P by pig age analyzed using linear regression and Tukey’s box plot (Set 2); - Effect of alternative cut-offs on positivity rates analyzed using Cochran’s Q (Sets 1, 2) Set 1: As shown in Figure 1, at a cut-off of S/P ≥ 0.4, DX-SE and DX-SP were both > 99.4%. Higher S/P cut-offs did not improve DX-SP because of the limited number of negative samples (n = 167), but likewise had minor impact on DX-SE. Set 2: Figure 2 shows the 99th percentile S/P response by pig age or category. “Extreme S/P outliers” (n = 48; Tukey’s box plot) were associated with specific age categories, i.e., 9, 24, 25 weeks of age and gestation. Table 1 provides DX-SP estimates (with and without “extreme S/P outliers”) at alternative cut-offs. Routine PRRSV surveillance demands near-perfect diagnostic specificity, even at the cost of diagnostic sensitivity. Using a cut-off of S/P ≥ 1.0 significantly reduced false positives (Table 1) and minimally affected DX-SE (Fig 1). This cut-off is recommended for the interpretation of PRRS OF ELISA results from presumed negative sites. Notably, extreme S/P responses were associated with PRRSV-naïve pigs of specific ages and/or in gestation. Work in progress is expected to identify the cause(s) of this non-specific response. References 1. Trevisan G, et al. PLoS One. 2019;14:1–16. 2. Henao-Diaz A., et al. Res. Vet. Sci. 131, 173–176 3. Henao-Diaz A, et al. Res Vet Sci. 2019;125:113–8. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 10 AAVLD Virtual Conference Laboratory performance of a commercial PRRSV oral fluid ELISA # * † Alexandra Henao-Diaz, Ronaldo Magtoto, Ana Paula Poeta Silva, Luis G. Gimenez-Lirola, Dave Baum, Jeffrey Zimmerman Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA

Introduction: “Robustness is a measure of the reproducibility of (binary) test results under the variation in conditions normally expected from laboratory to laboratory and from analyst to analyst”1. Herein, we evaluated the robustness of a commercial PRRSV oral fluid ELISA (IDEXX Laboratories, Inc.) Materials and methods: As fully described elsewhere2, individual oral fluids were collected from 12 pigs from -7 to 42 days post-vaccination and sample aliquots (2ml) were kept at -80°C. “True negatives”, defined as ≤ 7 DPV (n = 141) and “true positives” defined as ≥ 12 DPV (n = 368) were tested in 6 testing events defined by 3 variables (LAB = laboratory; TECH = technician; LOT = ELISA kit manufacturing lot). (1) LAB 1 TECH A LOT I (2) LAB 1 TECH A LOT II (3) LAB 1 TECH A LOT III (4) LAB 1 TECH B LOT IV (5) LAB 2 TECH A LOT V (6) LAB 2 TECH C LOT V For numerical results (S/P), the diagnostic performance was evaluated by ROC analyses. For binary results (positive defined as S/P ≥ 0.4), logistic mixed regression analyses were used to evaluate the effect of each variable on the results. Cochran’s Q was used in the following comparisons: - Variation - pairwise comparisons of all testing events vs true status - Intermediate-technician precision - (5) vs (6) - Intermediate-kit-lot precision - (1) vs (2) vs (3) - Repeatability - (1) vs (2) vs (3) - Reproducibility - (1, 2, 3) vs (5) Results: Testing event │ 1 │ 2 │ 3 │ 4 │ 5 │ 6│ ROC-AUC │ 1.00 │ 0.994 │ 0.996 │ 1.00 │ 0.997 │ 0.996 │ Pos (true 368) │ 368 │ 367 │ 370 │ 369 │ 367 │ 365 │ Neg (true 141) │ 141 │ 142 │ 139 │ 140 │ 142 │ 144 │ Dx-Se │ 100% │ 99.5% │ 100% │ 100% │ 99.5% │ 98.9% │ Dx-Sp │ 100% │ 99.3% │ 98.6% │ 99.3% │ 99.3% │ 99.3% │ None of the variables (LAB, TECH, or LOT) had a significant effect on binary results (logistic regression, p > 0.05); the positivity rates at all testing events level were statistically similar to true status, and the ELISA binary results were estimated as repeatable and reproducible regardless changes in the testing variables evaluated (Cochran’s Q test p > 0.05). Conclusions: An analysis of the robustness of a commercial PRRSV OF Ab ELISA involving 2 laboratories, 3 technicians, and 5 kit lots showed the kit to be highly robust.

AAVLD Annual Conference Proceedings 11 AAVLD Virtual Conference References 1. Glossary of terms for quality assurance and good laboratory practices. United Nations (2009). 2. Henao-Diaz A, et al. (2019). Res Vet Sci. 125:113–8. Authors declare no conflicts of interest (COI). JZ and DB have consulted with IDEXX Laboratories, Inc. independent of this research. Consulting has been reviewed and approved in accordance with ISU COI policies. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 12 AAVLD Virtual Conference Determining the seroprevalence of Anaplasma marginale infected beef herds in Georgia ◊ Lee Jones1, Hemant K. Naikare2, Roy D. Berghaus1, Allen Aref Kalantari2 1Population Health, UGA College of Veterinary Medicine, Tifton, GA; 2Microbiology, Tifton Veterinary Investigational Diagnostic Laboratory, Tifton, GA

The main objective of our proposed study was to determine the seroprevalence of A. marginale infected beef herds in Georgia and to identify herd risk factors associated with A. marginale positive herds. In addition, we compared the cELISA serology and Taqman qPCR for determining the prevalence of A.marginale infected herds in Georgia. Blood samples were collected from 1059 adult beef cattle (≥ 2 years) from 33 herds. The sera samples were screened using a commercial cELISA Anaplasma antibody test kit (VMRD). Eighty-six (8.1%) of 1059 cattle and 14 (42.4%) of 33 farms had positive ELISA results. EDTA whole blood samples from a subset of corresponding seropositive samples (n=73) were tested by Taqman qPCR and 77% (56/73) were PCR positive. Out of the 406 sero-negative samples, none of those animals were found to be PCR positive for A. marginale DNA. There was almost perfect agreement by Cohen kappa statistics between PCR and cELISA (k= 0.85). Management questionnaires were completed by all owners of each herd tested. Surprisingly, 27% (9/33) of owners had not heard of anaplasmosis, 18.2% (6/33) herds had been diagnosed with anaplasmosis prior to this study, 55.2% (16/29) did not disinfect instruments between calves when dehorning or castrating, and 87.8% (29/33) of the operations used the same needle to inject more than one animal. Given the presence of Anaplasma in Georgia beef cattle and findings of the survey, educational programs on effective preventive management practices to control anaplasmosis is warranted. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 13 AAVLD Virtual Conference Development and evaluation of a Turkey coronavirus Enzyme-Linked Immunosorbent Assay (ELISA) using S1 spike protein Yuko Sato, Mohamed El-Gazzar, Ronaldo Magtoto, Luis G. Gimenez-Lirola Veterinary Diagnostic & Production Animal Medicine, Iowa State University, Ames, IA

Turkey coronavirus (TCoV) is the etiologic agent of a highly contagious enteric disease of turkeys of economic significance in the United States and worldwide. There is a lack of diagnostic tests currently available for this disease in general and a lack of suitable serologic assays in particular, which may explain the lack of knowledge of the seroprevalence of TCoV in the turkey population. The immunofluorescent antibody assay (IFA) for TCoV has been used as a reference test for serologic detection of TCoV antibodies. However, cell-based immunoassays have inherent weaknesses for routine flock surveillance and monitoring, e.g., subjective, labor-intensive, and time- consuming. Enzyme-Linked Immunosorbent Assay (ELISA) is a simpler, more cost-effective testing approach for larger groups of samples collected from different flocks, as well as in outbreak situations, where large quantities of samples need to be processed in a timely, cost-effective fashion. To date, several TCoV ELISA platforms have been described, both indirect and competitive, based on different antigen targets (e.g., structural proteins, whole virus, etc). However, the only assay currently available for the U.S. commercial turkey industry is a baculovirus- expressed N-protein competitive ELISA (cELISA) from North Carolina State (JS Guy et al, Av Dis 2002). In this study, the N-terminal portion of the TCoV spike protein (S1) was selected as antigen for the development of an indirect ELISA. The coding region of the S1 protein was synthetized, cloned (pNPM5 vector) and expressed using a mammalian expression system (HEK293 cells), and purified by affinity chromatography. The diagnostic performance (sensitivity and specificity) of the TCoV S1 indirect ELISA was assessed using serum samples collected weekly from experimentally-infected control turkeys (n = 12) through 42 days post-inoculation (dpi). Preliminary results indicated that the proposed ELISA was capable of detecting anti-TCoV specific antibody after 15 days post-inoculation with rate of detection increasing throughout the end of the study (100% detection after 35 dpi). This assay can be easily expanded and allow for large scale testing. This will allow for fast accurate surveillance and better disease prevention and control strategies. This is an ongoing project and further detailed results will be discussed at the meeting.

AAVLD Annual Conference Proceedings 14 AAVLD Virtual Conference CVM Vet-LIRN feed contaminant survey 2012 to 2019 Stephen B. Hooser1, Robert H. Poppenga2, Renate Reimscheussel3,4, Sarah Nemser3,4 1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN; 2California Animal Health and Food Safety, University of California, Davis, Davis, CA; 3Food and Drug Administration, Laurel, MD; 4Center for Veterinary Medicine, Laurel, MD

In 2007 and 2012, the FDA Center for Veterinary Medicine and the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) conducted nationwide feed contaminant surveys of veterinary diagnostic laboratories as part of its mission, “To promote human and animal health by collaborating with veterinary diagnostic laboratories to provide scientific information,…and to help CVM investigate potential problems with CVM-regulated products, including animal feeds…” The objective of this survey was to ascertain chemical and microbial feed contamination events that were diagnosed in U.S. veterinary diagnostic laboratories, including Vet-LIRN laboratories, from 2012 to 2019. Veterinary diagnostic laboratories in 47 states plus Puerto Rico and five Canadian provinces, were invited to participate. This included all Vet-LIRN and AAVLD laboratories. In the summer of 2019, an electronic survey was sent to diagnostic laboratory directors. The maintenance of confidentiality for submitted laboratory information was emphasized. It was found that for many laboratories, their LIMS databases were not designed to facilitate retrieval of feed contaminant case information for retrospective evaluation. In addition, it became apparent that for some laboratories, the large number of cases during the 7 year time period precluded case-by-case entry into the electronic survey tool. Therefore, in November, 2019, a request was sent to laboratory directors to provide less detailed information regarding feed contamination via a simplified spreadsheet. Veterinary diagnostic laboratories from across the U.S. and Canada submitted information from sample submissions requesting analysis of animal feeds for chemical or microbial contamination. Results of analysis of feeds and hay for chemical contamination revealed varying numbers of cases having livestock feed or hay contaminated with naturally occurring mycotoxins (primarily aflatoxin, DON (vomitoxin), and fumonisins), ergot alkaloids, or miscellaneous chemicals such as ionophore feed additives. In livestock hay, cases with elevated concentrations of nitrate were reported as were cases of botulism in baled hay, or of cyanide in Johnson grass. Exposure of livestock to poisonous plants in hay or pasture were also reported. In pet animal feeds, incidences of food contamination with pentobarbital, vitamin D or abnormal concentrations of thyroid hormone were reported. Microbial testing by aerobic culture diagnosed small numbers of cases with bacterial contamination by Listeria, in silage for livestock, Clostridium perfringens in livestock feed and dog food, Salmonella in silage and companion animal food, and E. coli or Bacillus in companion animal foods. These survey results will help support veterinary diagnostic laboratory national capacity/capability development, and will help guide method development and validation projects. This work was supported by HHS FDA grant 1U18FD006564-01. Vet-LIRN Feed Contaminant Survey.

AAVLD Annual Conference Proceedings 15 AAVLD Virtual Conference Multi-class veterinary drug screening and quantitation by high resolution mass spectrometry (HRMS) using the Orbitrap Exploris 120 mass spectrometer Dwayne Edward Schrunk, Laura Burns VDPAM, Iowa State University, Ames, IA

Introduction: Veterinary medicines are pharmacologically active compounds used to treat and prevent animal diseases. Residues of the drugs and metabolites can remain in foods after treatment of animals. There is considerable interest in maximizing the amount of information obtained from animal product analyses for the residues of veterinary medicines. One strategy to improve efficiency is to maximize the number of analytes that may be screened using a single analytical method. A probability of detection statistical model is used to assess the performance of the method. Commodities could then be checked rapidly to ensure compliance with regulatory maximum residue limits (MRLs), and ideally, the method would be capable of simultaneously screening for other potential residues, as well as providing semi-quantitative data. Methods: A comprehensive, multi-class veterinary drug HRMS UHPLC–MS/MS method has been recently developed using an Orbitrap Exploris 120 mass spectrometer. The method was applied to bovine muscle to demonstrate reliable results for screening of multiple classes of veterinary drugs, covering benzimidazoles, macrolides, tetracyclines, avermectins, tranquilizers, ß-lactams, non-steroidal anti-inflammatories, quinolones, coccidiostats and sulfonamides. A QuEChERS sample preparation procedure with ethylenediaminetetraacetic acid and ammonium oxalate solution coupled with acetonitrile was used to extract 5g of homogenized sample. The extract was cleaned up with dispersive solid-phase extraction (dSPE). A 2 µL portion was injected into a UHPLC coupled to a Orbitrap Exploris 120 mass spectrometer using two separate data acquisition methods, data independent acquisition (DIA) and data dependent acquisition (ddMS2). Preliminary results: Chromatographic separation, mobile phase composition, and extraction recovery optimization were performed using a 109 compound multi-class standard mixture spiked into bovine muscle matrix. The veterinary drugs were screened using DIA and ddMS2 acquisition methods. Data analysis was performed with an extraction mass tolerance of 5 ppm for both precursor and product ions. At least one fragment ion was required to confirm each analyte. A highly curated veterinary drug HRMS MS/MS spectral library was used to confirm results with high confidence, with match scores in excess of 80% for most analytes. The veterinary drugs were analyzed in bovine matrix, ten replicates of negative control muscle, ½ and 1 MRL were extracted and analyzed. For nearly all of the compounds in the method a POD of 0 was obtained for the negative control samples, while a POD of > 0.8 was obtained for those fortified at ½ and 1 MRL. Most compounds were confirmed by the presence of a fragment within 5 ppm of the expected accurate mass. To demonstrate the quantitative capabilities of the method the certified reference material BOTS-1 was analyzed, results for the eight incurred residues where within the certified range for each compound.

AAVLD Annual Conference Proceedings 16 AAVLD Virtual Conference Thevetia bicornuta toxicosis of goats Michael Filigenzi, Francisco A. Uzal, Robert H. Poppenga California Animal Health and Food Safety Laboratory System, Davis, CA

Three of 4 alpine goats developed diarrhea soon after the owner placed plant clippings into their pen. The goats were being kept on a suburban property near Palm Desert, CA. A 1 year-old female goat died suddenly approximately 5 hours after eating the plant and was submitted to CAHFS San Bernardino for a postmortem examination. At presentation, the client indicated that the goats had been fed yellow oleander. Grossly, the lungs were diffusely congested and edematous. There were several subendocardial petechiae and ecchymoses over the left papillary muscle of the heart. The rumen was distended with a mixture of grain and fragments of dark green elongate leaves. Microscopically, multifocal, acute myocardial necrosis, multifocal subendocardial hemorrhages, and diffuse pulmonary edema and congestions were noted. Coccidia oocysts were noted in the feces. No bacterial pathogens were isolated from the lungs or liver. Incidentally, C. perfringens (not typed) and Paeniclostridium sordellii were isolated from the small intestine and colon. PCR for Salmonella was negative and a liver heavy metal screen was unremarkable. Rumen contents were negative for cyanide. Rumen contents were initially analyzed for oleandrin and peruvoside using LC-MS/MS. Peruvoside was detected but testing for oleandrin, the cardiac glycoside found in Nerium oleander was negative. Subsequent analysis of the rumen contents using LC-high resolution MS detected peruvoside, neriifolin, and cerberin. These three toxins were also detected in leaves found in the rumen of the dead goat. This was consistent with exposure to Thevetia peruviana, also known as Cascabela thevetia. The attending veterinarian subsequently submitted additional samples from the plant given to the goats for evaluation. Specimens were identified by the UC-Davis Herbarium as Thevetia bicornuta (synonum Cascabela bicournuta). Peruvoside, neriifolin, and cerberin were also identified in the leaves of the plant. These compounds, like oleandrin, are cardiac glycosides that reversibly inhibit the sodium-potassium adenosine triphosphatase exchanger (Na-K-ATPase) in cardiac myocytes resulting in higher-than-normal intracellular concentrations of sodium. High levels of intracellular sodium increase the resting membrane potential leading to the activation of voltage-gated calcium channels, which increase intracellular calcium concentrations resulting in cell death. In this case, they were considered to have been responsible for the myocardial necrosis and death occurring in this goat. The nomenclature of Thevetia/Cascabela is confusing. They are considered synonyms, although they have been categorized as separate genera by some. The common name of yellow oleander is used for multiple species which adds to the confusion. T. peruviana (or C. thevetia) is the most recognized toxic plant in the genera. To the authors’ knowledge this is the first documented case of T. bicornuta toxicosis in goats.

AAVLD Annual Conference Proceedings 17 AAVLD Virtual Conference blank page Epidemiology Live Q&A Thursday, October 15, 2020

Moderators: Erin L. Goodrich and Ashley E. Hill

2:15 PM Leveraging diagnostic laboratory data for research, lab management, clinical decision-making and surveillance through CoreOne analytics Brian J. McCluskey...... 21

2:20 PM Active regional surveillance for early detection of exotic/emerging pathogens of swine: a comparison of statistical approaches for selecting farms to be sampled # * † Dapeng Hu, Ting-Yu Cheng, Paul Morris, Jeffrey Zimmerman, Chong Wang ...... 22

2:25 PM Understanding and interpreting PRRSV diagnostics in the context of “disease transition stages” # * † Alexandra Henao-Diaz, Ju Ji, Luis G. Gimenez-Lirola, Dave Baum, Jeffrey Zimmerman...... 23

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 19 AAVLD Virtual Conference blank page Leveraging diagnostic laboratory data for research, lab management, clinical decision-making and surveillance through CoreOne analytics Brian J. McCluskey Trace First Inc., Fort Collins, CO

Rapid and convenient access to data housed in laboratory information systems for purposes other than provision of individual diagnostic results has been challenging for Veterinary Diagnostic Laboratories. Access to LIMS data often requires specialized skills in building queries thus limiting access to the data for laboratory staff and managers. CoreOne Analytics was developed to provide quick and easy access to LIMS data by all staff to support research, laboratory management, clinical and herd health decision-making, and surveillance. Trace First provides datasets in CoreOne analytics from which analyses and dashboards are created. CoreOne Analytics is powered by Amazon Quicksight. SPICE is QuickSight’s super-fast, parallel, in-memory calculation engine that pulls data from the CoreOne for Labs database into the datasets. SPICE is engineered to rapidly perform advanced calculations and serve data. The storage and processing capacity available in SPICE speeds the analytical queries that you run against your imported data and eliminates the load on the production database. Currently, datasets are available in CoreOne Analytics for Times (used for analyzing various workloads and testing efficiencies), Tests (used for analyzing counts of tests and accessions), and Test Results (used for investigating results for research, clinical decisions or surveillance). Each dashboard provided is built to include specific Controls, or data filters. The filtered data is displayed in an array of visualizations (charts, graphs, tables and maps). Visualizations, when selected, offer various options for the user to adjust and export the data for further analysis in other software. Veterinary Diagnostic Laboratory data used for purposes other than individual animal diagnoses has historically seen limited use because of real or perceived biases. To acquire the least biased data, researchers and others must recognize the potential presence of biases and consciously attempt to circumvent and minimize their effects. In addition, when evaluating the results of studies, researchers should be aware of and articulate the different types of biases, their mitigation and how this affects interpretation and use of data from diagnostic laboratories. The potential for Veterinary Diagnostic Laboratory data as a critical surveillance stream is significant and needs to be further leveraged.

AAVLD Annual Conference Proceedings 21 AAVLD Virtual Conference Active regional surveillance for early detection of exotic/emerging pathogens of swine: a comparison of statistical approaches for selecting farms to be sampled # * † Dapeng Hu2, Ting-Yu Cheng1, Paul Morris2, Jeffrey Zimmerman1, Chong Wang2,1 1Veterinary Diagnostic and Preventive Animal Medicine, Iowa State University, Ames, IA; 2Department of statistics, Iowa State University, Ames, IA

Introduction: Farm selection plays a crucial role in livestock disease surveillance. In the past, farm selection was based on conditional random sampling, i.e., selecting farms using predefined criteria (stratified) or according to a random starting point with fixed intervals (systematic). However, the growing recognition of spatial dependence in disease spread (autocorrelation) brings the validity of these approaches into question. In this study, five spatially- based statistical sampling methods were compared in terms of the probability of detection at a prevalence of 2.0% (farm level). Materials and Methods: For the comparison, the spatiotemporal spread of a contagious agent was modeled in a hypothetical region (195 km × 300 km in size) populated with 6,000 livestock farms. The disease spread model used in the study was derived from a model proposed by Ju et al. (2020; unpublished):

λf,t = λf-1,t + α ∑f'ϵNf exp(-df,f') pf',t-1 where pf, t denoted the disease prevalence in farm f at time t where f ϵ {1, 2, …, F} and t ϵ {1, 2, …, T}. Nf is the set of farms within a 5 km radius of farm f. λf,t and λf −1,t indicated the odds of being infected, α determined the degree of the spatial dependence from all other farms, and denoted the distance between farm f and f ′ (f ≠ f ′). Using the model to simulate disease spread in the hypothetical region, farm selection based on simple random sampling (SRS) and 5 “spatially balanced” sampling methods were compared in terms of the probability of detection (prevalence = 2.0% of farms): ● simple random sampling (SRS) ● generalized random-tessellation stratified (GRTS) ● local pivotal method (LPM) ● spatially correlated Poisson sampling (SCPS) ● local cube method (LCUBE) ● balanced acceptance sampling (BAS) Notably, this study did not assess the efficiency of on-farm sampling and assumed that disease classification of farms selected for sampling was 100% accurate when disease prevalence in farm f was ≥ 10%. Results: With a farm-level prevalence of 2.0% (120 positives among 6000 herds) and a sample size of 60, SCPS and LCUBE had the highest probability of including ≥ 1 positive farm(s) in the sample, i.e., > 80% for SCPS and LCUBE versus < 70% for BAS and SRS. Conclusions and Discussion: This study showed that spatially balanced sampling methods may perform better than SRS, i.e., are more likely to select a positive farm. These results are consistent with Tobler’s First Law of Geography: “Everything is related to everything else. But near things are more related than distant things.” Thus, neighboring farms are more likely to have similar disease status than distant ones and the probability of infection and the distance to infected farms are inversely proportional. In contrast to SRS, the spatially balanced sampling methods used various approaches to adjust for spatial dependency. For that reason, the spatial balanced sampling methods achieved higher probability of detection with fewer samples than SRS. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 22 AAVLD Virtual Conference Understanding and interpreting PRRSV diagnostics in the context of “disease transition stages” # * † Alexandra Henao-Diaz1, Ju Ji2, Luis G. Gimenez-Lirola1, Dave Baum1, Jeffrey Zimmerman1 1Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA; 2Statistics, Iowa State University, Ames, IA

Introduction: The proper use and interpretation of RT-PCR and ELISA diagnostics is fundamental for the control and elimination of infectious diseases. Given the continuous advancements in diagnostics and the capabilities of the assays, why do PRRSV control and elimination remain problematic? PRRSV causes a persistent infection divided into 4 “disease transition stages”1: viremia, immune response, virus sequestration in tissues, and eventual virus clearance2. However, the changes in diagnostic sensitivity and specificity that occur for specific combinations of specimen-tests as the animal pass through these stages have not been addressed. Herein we describe the PRRSV “diagnostic transition stages”1 and discuss their relationship with the “disease transition stages” Methods: A meta-analysis fitted with logistic regression performed on data from 19 refereed publications (1995 to 2018) reporting PRRSV data included 4307 longitudinal observations using three detection approaches: RNA in serum (RT-PCR), infectious virus in tissue (bioassay), and antibody in serum (ELISA) (n = 1973, 468, and 1866). A generalized mixed model with the logit link was fitted: test result was the binary response, DPE the explanatory variable, and publication the random effect. The intercepts and slopes were used to formulate the equations predicting the detection of PRRSV over time for each assay. Results: Prediction equations for PRRSV RT-PCR, ELISA, and bioassay testing at individual pig level: RT-PCR: exp(2.9277 - 0.0712 × DPE) / [1 + exp(2.9277 - 0.0712 × DPE)] Bioassay: exp(1.3729 - 0.0225 × DPE) / [1 + exp(1.3729 - 0.0225 × DPE)] ELISA: exp(2.4833 - 0.0019 × DPE) / [1 + exp(2.4833 - 0.0019 × DPE)] Figure 1 shows the predicted probability of PRRSV detection (%) by assay, over 36 weeks post-exposure. Conclusion and discussion: PRRSV detection has been partially described at the individual pig level in at least 19 publications. We aggregated and analyzed these data to characterize the PRRSV “diagnostic transition stages”. The result of the analysis (Figure 1) showed that the probability of PRRSV detection varied as a function of the specimen, the diagnostic assay, and the time post-exposure. This information provided a practical perspective on fundamental issues in diagnostics; notably, the relationship between “disease transition stages” and the “diagnostic transition stages”. Likewise, the analysis illustrated why PRRSV ELISA positive and RT-PCR negative results from the same animal(s) are not a diagnostic contradiction. Awareness of these relationships should be of help in test selection and interpretation: RT-PCR to detect viremic pigs and ELISA to detect pre-exposed pigs. These concepts broadly apply to other veterinary pathogens. Additional information in Henao-Diaz, et al. 2020. Res. Vet. Sci. 131, 173–176. http://doi.org/10.1016/j. rvsc.2020.04.023 References 1. Thurmond, M.C., 2003. J. Vet. Diagnostic Investig. 15, 501–514. 2. Lopez and Osorio. 2004. Vet. Immunol. Immunopathol. 102, 155-163 # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 23 AAVLD Virtual Conference blank page Molecular & Bioinformatics Live Q&A Thursday, October 15, 2020

Moderators: Laura B. Goodman and Jianfa Bai

2:30 PM First detection and characterization of bovine Kobuvirus from cattle with diarrhea in the United States Leyi Wang, Richard Fredrickson, Jonathan P. Samuelson, Shih-Hsuan Hsiao...... 27

2:35 PM Bovine Kobuvirus detected in multiple states in the United States Leyi Wang, Ailam L. Lim, Richard Fredrickson, Keith L. Bailey, Miranda Vieson, Patrick Roady, Wes Baumgartner...... 28

2:40 PM Fowl adenoviruses in California poultry: genotyping as an epidemiological and control tool for inclusion body hepatitis Beate Crossley, Daniel Rejmanek, Holly S. Seller, Asli Mete, Leslie Woods, Rodrigo Gallardo, Simone Stoute...... 29

2:45 PM Development and validation of real-time PCRs to detect SARS-CoV-2 in animal samples Sarah Gresch, Kelly Aukes, Tracy Lene Otterson, Sunil Kumar Mor, Stephanie Rossow...... 30

2:50 PM Detection of pseudorabies virus in oral fluid specimen using real-time PCR # * † Ting-Yu Cheng, Karen M. Harmon, Phillip Gauger, Chong Wang, Aruna Ambagala, Alexandra Buckley, Albert Van Geelen, Kelly Lager, Jeffrey Zimmerman, Luis G. Gimenez-Lirola...... 31

2:55 PM Development and validation of a reverse transcription real-time PCR assay to specifically detect PRRSGard®-like vaccine virus Gaurav Rawal, Fabian Chamba, Charles Francisco, Chad Smith, Jianqiang Zhang...... 32

3:00 PM Application of a multiplex Luminex bead assay for the detection of tick-borne pathogens Julia Livengood, Michael Hutchinson, Nagaraja Thirumalapura, Deepanker Tewari...... 33

3:05 PM Comparison of Mycoplasma hyopneumoniae DNA detection in oral fluid samples # * † ◊ Ana Paula Poeta Silva, Gabriel Storino, Franco Ferreyra, Jessica Miller, Karen M. Harmon, Phillip Gauger, Wendy Witbeck, Kent Doolittle, Silvia Zimmerman, Rachel Derscheid, Maria Jose Clavijo, Bailey Arruda, Jeffrey Zimmerman ...... 34

3:10 PM Application of PrimalSeq viral whole genome sequencing in veterinary diagnostics Brittany D. Cronk, Patrick K. Mitchell, Renee Anderson, Nathan D. Grubaugh, Chantal B.F. Vogels, Diego Diel, Laura B. Goodman...... 35

3:15 PM Whole genome sequencing of pathogens and bioinformatics analysis as a diagnostic tool: successes and challenges? Sunil Kumar Mor, Vikash Singh, Albert Rovira, Stephanie Rossow, Fabio Vannucci, Matthew Sturos, Connie Gebhart, Alex Primus, Robert Porter, Jerry Torrison ...... 36

3:20 PM Non-target RNA depletion strategies to improve sensitivity of next-generation sequencing (NGS) for diagnosing infectious agents in poultry David Suarez, Joshua Parris, Henry Kariithi, Dawn Williams-Coplin...... 37

3:25 PM Clamping tools for vaccine and wild-type PRRSV diagnostic testing Karen M. Harmon, Laura Bradner, Phillip Gauger...... 38

AAVLD Annual Conference Proceedings 25 AAVLD Virtual Conference 3:30 PM Profiling the wild resistome: an AMR detection strategy for wildlife species Rebecca L. Tallmadge, Kevin J. Cummings, Patrick K. Mitchell, Brittany D. Cronk, Renee Anderson, Lauren Palena, Sara E. Childs-Sanford, Noha Abou-Madi, Julie Siler, Maureen G. Gunderson, Martin Wiedmann, Laura B. Goodman...... 39

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 26 AAVLD Virtual Conference First detection and characterization of bovine Kobuvirus from cattle with diarrhea in the United States Leyi Wang, Richard Fredrickson, Jonathan P. Samuelson, Shih-Hsuan Hsiao Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL

Diarrhea is a leading cause of calf mortality in U.S. cattle herds, resulting in significant economic losses to cattle producers. Calves with diarrhea are often less than one month of age. Infectious causes of calf diarrhea include viral, bacterial and protozoal pathogens. Diagnostic tests are routinely utilized for common viruses such as bovine rotavirus (BRV), bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV). In April 2019, the University of Illinois Veterinary Diagnostic Laboratory utilized next-generation sequencing to test a fecal sample collected from a calf with diarrhea. A taxonomic analysis of raw FASTQ data using Kraken version 1 and MiniKraken database revealed the presence of over 15,000 sequence reads of bovine kobuvirus (BKV), and a complete genome of BKV was assembled. Sequence analysis showed that U.S. BKV was related to four BKV strains from Japan, UK, Egypt, and China. Further screening BKV in 9 additional intestinal samples from necropsied calves showed that 4 out of 9 samples were positive for BKV by real-time RT-PCR. These 4 BKV positive samples were negative for BRV, BCoV, and BVDV, and histopathological observation of small intestines revealed that two of these BKV positive cases with diarrhea had necrotizing enteritis with villus atrophy and fusion, suggesting a primary viral infection. This is the first time that the BKV has been detected in diarrheal calves in the U.S.

AAVLD Annual Conference Proceedings 27 AAVLD Virtual Conference Bovine Kobuvirus detected in multiple states in the United States Leyi Wang1, Ailam L. Lim2, Richard Fredrickson1, Keith L. Bailey1, Miranda Vieson1, Patrick Roady1, Wes Baumgartner1 1Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL; 2Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin-Madison, Madison, WI

Bovine kobuvirus (BKV) is a single-stranded, positive-sense RNA virus, belonging to the species Aichivirus B of the family Picornaviridae. BKV was first identified in 2003 as a laboratory cell culture contaminant in Japan. Since then, BKV has been reported in many countries including Thailand, Hungary, Netherlands, South Korea, Italy, Brazil, China, Egypt, and Turkey. In 2008, BKV was reported in one- to seven-week-old calves with diarrhea in Thailand. More recently, three additional studies reported the detection of BKV in feces from one- or two-month- old calves with diarrhea in Brazil, South Korea and Turkey. After our first detection of BKV in the U.S. in 2019, we initiated a survey for BKV in bovine fecal samples collected from different states, using a real-time RT-PCR assay. We detected BKV in bovine feces from multiple U.S. states including Arkansas, Illinois, Michigan, New Mexico, Ohio, Oklahoma, and Wisconsin. The majority of positive samples were from calves 1-2 weeks of age. Sequence analysis of BKV strains originating from different states showed they had 91-94% identities with each other at the complete genome levels. Expanded surveillance for BKV is needed to determine the true prevalence of this virus in U.S. cattle.

AAVLD Annual Conference Proceedings 28 AAVLD Virtual Conference Fowl adenoviruses in California poultry: genotyping as an epidemiological and control tool for inclusion body hepatitis Beate Crossley1, Daniel Rejmanek1, Holly S. Seller2, Asli Mete1, Leslie Woods1, Rodrigo Gallardo3, Simone Stoute1 1California Animal Health and Food Safety Laboratory, UC Davis, Davis, CA; 2Poultry Diagnostic and Research Center, University of Georgia, Athens, GA; 3School of Veterinary Medicine, UC Davis, Davis, CA

Inclusion body hepatitis (IBH) is caused by a group 1 avian adenovirus from the genus Aviadenovirus. Clinical signs are non-specific and the disease often presents with acute onset of mortality of up to 40% in affected flocks. On postmortem the livers are classically pale, swollen, friable and riddled with petechial hemorrhages. Kidneys and other organs might also be affected. Histologic lesions include acute multifocal to submassive hepatic necrosis with multifocal hepatitis and hepatocellular intranuclear inclusion bodies. Fowl Aviadenoviruses (FAdVs) affect domestic chickens, pigeons, quail and other avian species but most commonly are seen causing disease in meat type birds. Since 2011, cases in California have mostly been in commercial broilers and occasionally in layers. Over the past few years, there has been an uptake in the frequency of cases. Most recently, IBH affected multiple birds in a backyard chicken flock. Historically, serotype information has been achieved by virus isolation and partial sequencing of the Hexon gene following genotyping according to International Committee on Taxonomy of Viruses guidelines. Here we report the development of a whole genome sequencing approach utilizing a third generation sequencing technology (MinION nanopore) to gain insight into the recurrent problems seen in our commercials with possible spill-over to backyard birds. During the recent 5 years in California, we have detected a total of 199 adenoviruses by virus isolation. From those, 73 have been attributed to FAdV genotypes, C-4, D-11, and E-8 associated with IBH in commercial broiler facilities. On one premise temporarily housing egg and meat type birds FAdV C-4 isolates were detected in both poultry types. Whole genome sequencing analysis showed 100% identity between the layer and the broiler type strains in 2017. Over the subsequent 4 years, FAdV C-4 strains periodically isolated from IBH cases at the layer facility remained virtually unchanged (99.99% identity) despite the use of autogenous vaccines. This particular ranch reports continuous spikes in clinical disease. Earlier this year IBH was observed for the first time in a backyard chicken flock in northern California. Adenovirus was isolated from the liver and whole genome sequencing identified a FAdV C-4 genotype (or serotype?). The entire sequence was nearly identical (99.99%) to the strains isolated from the layer and broiler facilities. Observations from this multi-year retrospective study suggest that adenovirus-related IBH is producer and location specific. Based on the low number of evolutionary mutations found, FAdVs are shown to be very well adapted viruses.

AAVLD Annual Conference Proceedings 29 AAVLD Virtual Conference Development and validation of real-time PCRs to detect SARS-CoV-2 in animal samples Sarah Gresch, Kelly Aukes, Tracy Lene Otterson, Sunil Kumar Mor, Stephanie Rossow Veterinary Diagnostic Laboratory, University of Minnesota, St Paul, MN

Since the start of the global COVID-19 pandemic, millions of people worldwide have been infected with the causative agent, SARS-CoV-2. After confirmed positive cases were found in cats, there was an urgent need for a SARS-CoV-2 assay for animals. We developed and validated real-time PCRs that detect three portions of the SARS- CoV-2 genome; two sections of the nucleocapsid gene (N1 and N2), as well as a section of the spike gene (S). The N1 and N2 primers and probes are as published by the CDC and purchased ready-to-use from IDT. The spike gene target was created in-house since our prior work with PEDv, PDCoV, and TGEv showed greater sensitivity for detecting these targets compared to N targets. Due to the urgency of this testing, these PCRs were not multiplexed; rather three individual PCRs are run on a single plate. In order to reduce the burden on the human supply chain, we used the ThermoFisher MagMAX CORE extraction kit, and the ThermoFisher TaqMan Fast Virus 1-step PCR kit, neither of which are commonly used for human SARS-CoV-2 testing in the United States. Using serial dilutions of quantified controls for the nucleocapsid and spike genes, we determined that analytical sensitivity (ASe) is 20 viral copies/µL for N1, 2 copies/µL for N2, and 0.87 copies/µL for S. We dually spiked known concentrations of both of these controls into six different animal sample pools as well as lab-spiked and processed synthetic swabs (to mimic swabs taken from any species) to determine ASe in various sample types. Porcine fecal samples showed a 10 fold reduction in sensitivity, while porcine oral fluids showed a 100 to 1,000 fold reduction in sensitivity. The other five sample types tested (porcine oropharyngeal/tonsil swabs, lung, serum; canine serum; lab-spiked swabs) showed equal sensitivity to the quantified controls. No cross-reactivity was observed when we tested against 23 known pathogens, including porcine respiratory coronavirus, and canine coronavirus. The coefficient of variation for repeatability was <2% (n=5 each) within an assay, and was <6% between assays (n=12). The same spike gene control mentioned above was also used as an extraction control throughout this work. We obtained previously-tested RNA extracted from human swab samples from the Minnesota Department of Health and found our results matched theirs when using our assays (in 55 negative and 37 positive samples). Our SARS-CoV-2 test has demonstrated excellent analytical sensitivity, specificity, and repeatability. Spiking animal specimen pools with synthetic target, along with confirmation of human sample results, suggests diagnostic sensitivity (DSe) and diagnostic specificity (DSp) near 100%. Additional work can be done in the future to further assess DSe and DSp as clinical cases are submitted to our lab.

AAVLD Annual Conference Proceedings 30 AAVLD Virtual Conference Detection of pseudorabies virus in oral fluid specimen using real-time PCR # * † Ting-Yu Cheng1, Karen M. Harmon1, Phillip Gauger1, Chong Wang1,2, Aruna Ambagala3, Alexandra Buckley4, Albert Van Geelen4, Kelly Lager4, Jeffrey Zimmerman1, Luis G. Gimenez-Lirola1 1Veterinary Diagnostic and Preventive Animal Medicine, Iowa State University, Ames, IA; 2Department of statistics, Iowa State University, Ames, IA; 3National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Winnipeg, MB, Canada; 4Agricultural Research Service, United States Department of Agriculture, Ames, IA

Introduction: Pseudorabies virus (PRV) causes disease of the central nervous system and respiratory/reproductive signs in pigs. Many countries have eliminated PRV from domestic swine populations, but the virus remains endemic in feral swine and is occasionally introduced into commercial herds. In China, highly pathogenic PRV variants have been described, leading to concerns regarding spread to other countries. In these circumstances, detection methods for PRV surveillance, control, and elimination are still needed. The objective of this study was to evaluate the detection of PRV in swine oral fluid collected from vaccinated and/or inoculated pigs using real-time PCR assays targeting PRV gB and gE genes. Materials and methods: Samples of known PRV infection status were used to evaluate the assays and establish shedding dynamics. Nasal swabs and oral fluid samples were collected from 40 12- to 16-week-old pigs in 4 treatment groups (n = 10 per group). Group 1 - negative control (NC); Group 2 - wild-type PRV inoculation (PRV 3CR Ossabaw); Group 3 - PRV vaccination (Ingelvac® Aujeszky MLV); and Group 4 - PRV vaccination followed by PRV inoculation (PRV 3CR Ossabaw) at 21 days post vaccination (MLV-PRV). To detect the presence of PRV DNA, samples were tested using 1) gB PCR for screening PRV-positive animals and 2) gE triplex PCR for differentiation between PRV classic and PRV highly pathogenic (variant) strains. Results: The gB PCR detected PRV DNA in oral fluid samples within 3 days post inoculation. In inoculation-only pigs (Group 2), PRV gB DNA was detected in oral fluid for up to 22 days and nasal swab samples for up to 12 days. For the gE triplex PCR, inoculation-only pigs (Group 2) had fewer positives and for a shorter period of detection (10 days in oral fluid and 12 days in nasal swabs) than gB. One gB PCR false positive (n = 1) was observed in a nasal swab sample prior vaccination. No false positive results for the highly pathogenic PRV variant were observed with the triplex gE PCR in either nasal swab or oral fluid samples. Conclusions and discussion: PRV DNA was detected in oral fluid samples from PRV inoculation-only animals using real-time screening PCRs targeting gB and gE genes. The gB PCR results were consistent with a previous report on PRV detection in swine oral fluid samples from pigs inoculated with wild-type virus. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 31 AAVLD Virtual Conference Development and validation of a reverse transcription real-time PCR assay to specifically detect PRRSGard®-like vaccine virus Gaurav Rawal1, Fabian Chamba2, Charles Francisco2, Chad Smith2, Jianqiang Zhang1 1Veterinary Diagnostic and Production Animal Medicine (VDPAM), Iowa State University, Ames, IA; 2Pharmgate Animal Health, Wilmington, NC

Introduction: PRRS is an economically important disease for the swine industry. Vaccination is one prominent and efficient strategy to control PRRSV. Reverse transcription real-time PCR (RT-rtPCR) is commonly used for PRRSV RNA detection. However, with an increasing use of MLV vaccines there come challenges to interpret PCR-positive results. ORF5 sequencing or whole-genome sequencing can be conducted to distinguish vaccine-like from wild-type PRRSVs; however, sequencing is relatively expensive and time-consuming. Vaccine-specific RT-rtPCR for detection of PRRSV vaccine strains is a convenient tool to inform vaccine protocols and differentiate strains. PRRSGard® is a unique PRRSV MLV vaccine recently developed by Pharmgate whose safety and efficacy have been evaluated in experimental and field trials. Here we report development and validation of a RT-rtPCR for specific detection of PRRSGard-like vaccine virus. Materials and methods: PRRSGard® vaccine virus has a unique 23-nucleotide insertion between ORF1b and ORF2 as a genetic marker. Primers and probe spanning this region were designed. Analytical specificity, sensitivity and diagnostic performance of PRRSGard-like RT-rtPCR were evaluated and compared with a commercial PRRSV PCR (VetMAX NA&EU PRRSV PCR, Thermo Fisher Scientific). Results: PRRSGard-like and commercial PRRSV RT-rtPCR did not cross-react with any of 19 non-PRRSV swine pathogens. The Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, Prime Pac PRRS, and Prevacent PRRS vaccine viruses, as well as 33 PRRSV-2 laboratory and field isolates representing different genetic lineages, tested negative by PRRSGard-like RT-rtPCR but positive by the commercial PRRSV RT-rtPCR. The undiluted ® PRRSGard vaccine virus was positive by PRRSGard-like and commercial PRRSV RT-rtPCR with CT values of ~18 and ~17, respectively. To determine the analytical sensitivity, 10-fold serial dilutions of PRRSGard® vaccine virus (10^-1 to 10^-8, triplicate per dilution) were tested by the two RT-rtPCRs simultaneously. PRRSGard-like RT-rtPCR was able to consistently detect up to 10^-6 dilution whereas the commercial PRRSV RT-rtPCR consistently detected up to 10^-5 dilution. Diagnostic performance of PRRSGard-like RT-rtPCR was evaluated using 270 serum samples collected from an experimental PRRSGard® vaccination study. Compared to the commercial PRRSV RT-rtPCR, diagnostic sensitivity, specificity, and accuracy of the PRRSGard-like RT-rtPCR were 93.7%, 99.4%, and 97.4%, respectively. Samples with discrepant results were sequenced to confirm virus identity at ORF1b/2 and ORF5 regions. Conclusion: In contrast to a commercial PRRSV RT-rtPCR that detects all PRRSV strains, the PRRSGard-like RT-rtPCR specifically detects PRRSGard-like vaccine virus with comparable performances to the commercial PRRSV RT-rtPCR. It is a convenient tool to detect and differentiate PRRSGard-like vaccine virus from other PRRSV strains.

AAVLD Annual Conference Proceedings 32 AAVLD Virtual Conference Application of a multiplex Luminex bead assay for the detection of tick-borne pathogens Julia Livengood1, Michael Hutchinson2, Nagaraja Thirumalapura1, Deepanker Tewari1 1Pennsylvania Veterinary Laboratory, Harrisburg, PA; 2Pennsylvania Department of Agriculture, Harrisburg, PA

The reported incidence rate of tick-borne diseases has been increasing in the northeastern states of the U.S. Ticks often carry multiple organisms including one or more pathogens. Multiplex bead-based detection technology and next-generation sequencing have emerged as important tools for detecting multiple targets. We used a Luminex based xMAP® Multiflex® Mega Tick Panel for testing 297 I. scapularis ticks collected from across Pennsylvania. The panel was designed to detect 20 genetic targets covering several tick-borne bacteria and viruses. Of the 297 ticks tested, 119 ticks (40.1%) contained single microorganism and 59 ticks (19.9%) contained more than one microorganism. Overall, 59.9% of the ticks carried at least one microorganism. The microorganisms detected included Anaplasma phagocytophilum (1.7%), Babesia spp. (15.5%), Borrelia burgdorferi (45.1%), Borrelia miyamotoi (0.3%), Rickettsia spp. (21.5%) and Rickettsia parkeri (0.7%). The ticks were negative for B. afzelii, B. garinii, Ehrlichia spp., E. chaffeensis, E. canis, Powassan virus, R. rickettsii, R. japonica, R. helvetica, Ba. microti, Bartonella spp., C. burnetii, and tick-borne encephalitis virus. Our ongoing study is focused on more recent tick collections and future studies will focus on testing of animal tissues for vector-borne pathogens using the panel. The Luminex xMAP® Technology offers a novel platform that readily allows multiplexing and is ideally suited for high-throughput detection of either nucleic acid or protein targets such as pathogen genome targets or antibodies.

AAVLD Annual Conference Proceedings 33 AAVLD Virtual Conference Comparison of Mycoplasma hyopneumoniae DNA detection in oral fluid samples # * † ◊ Ana Paula Poeta Silva1, Gabriel Storino2, Franco Ferreyra1, Jessica Miller1, Karen M. Harmon1, Phillip Gauger1, Wendy Witbeck3, Kent Doolittle3, Silvia Zimmerman3, Rachel Derscheid1, Maria Jose Clavijo1, Bailey Arruda1, Jeffrey Zimmerman1 1Iowa State University, Ames, IA; 2Escola de Agricultura e Ciências Veterinárias, Universidade Estadual de São Paulo, Sao Paulo, Brazil; 3IDEXX Laboratories, Portland, ME

Control of Mycoplasma hyopneumoniae (MHP) is dependent upon detection of infected pigs using effective monitoring protocols. Oral fluids samples are routinely used in disease monitoring programs in swine production systems because it does not require animal restraint and offers an assessment of population status. However, the detection parameters of MHP DNA in oral fluids have not been clearly defined. The goal of this study was to compare 4 protocols for DNA detection in oral fluids from pigs of known MHP infection status. Oral fluid samples of known status were collected daily from 5 groups of 6-week-old pigs from day post-inoculation (DPI) -4 through DPI 59. The negative control group consisted of 3 pigs; the 4 treatment groups contained 9 pigs each. Treatment groups differed in the number of pigs inoculated with MHP (lung homogenate, 105 CFU/mL MHP 232) at time zero: 1/9, 3/9, 6/9, and 9/9 pigs. Tracheal swabs were collected twice weekly to monitor individual pig MHP infection. Two extraction and three PCR protocols were compared using oral fluids. Extraction methods included (1) MagMAXTM-96 Pathogen RNA/DNA kit, Applied BiosystemsTM, Carlsbad, CA; and (2) IDEXX RealPCR* DNA/ RNA Magnetic Bead Kit, IDEXX Laboratories Inc., Westbrook, ME. PCRs were (1a) TaqMan® Fast Virus 1-Step Master Mix (Life Technologies, Carlsbad, CA) and primer/probe described for Mhp183; (1b) TaqMan® Fast Virus 1-Step Master Mix with the addition of AmpliTaq® 360DNA Polymerase (5U/uL) (ThermoFisher Scientific), and primer/probe described for Mhp183; and (2) RealPCR* Master Mix and RealPCR* M hyo DNA Mix, IDEXX Laboratories Inc. PCRs were run to 40 cycles and performed using the Applied Biosystems® 7500 Real-Time PCR (ThermoFisher Scientific). Using Extraction 1 and PCR 1b, 18 of 20 inoculated pigs were PCR positive (tracheal swabs) for MHP DNA on DPI 3. All pigs were PCR positive for MHP DNA in tracheal swabs on or before DPI 59. For oral fluids, a comparison of positivity rates among protocols by mixed logistic regression showed significant differences among procedures, with the highest positivity rate achieved using Extraction 1 and PCR 2 (173 positives of 322 samples), followed by Extraction 1 and PCR 1b (148 of 322), Extraction 2 and PCR 2 (134 of 322), and Extraction 1 and PCR 1a (109 of 322). Significant differences among procedures in detection of MHP DNA suggest that further improvements in laboratorial methods may be possible. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant ◊ USAHA Paper

AAVLD Annual Conference Proceedings 34 AAVLD Virtual Conference Application of PrimalSeq viral whole genome sequencing in veterinary diagnostics Brittany D. Cronk2, Patrick K. Mitchell2, Renee Anderson2, Nathan D. Grubaugh1, Chantal B.F. Vogels1, Diego Diel2, Laura B. Goodman2 1Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT; 2Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY

Viral whole genome sequencing (vWGS) poses unique challenges, and a variety of methods for sample and library preparation exist. Many require isolation, which can be challenging for many viruses and can introduce additional genetic variation. Shotgun metagenomic methods that do not require viral isolation are inefficient because of the high volume of host genetic material relative to viral nucleic acids. Targeted amplicon sequencing can be done directly on sample specimens without the need for viral isolation, reducing the problems associated with other approaches. We have tested one such targeted amplicon method, PrimalSeq, which produces primers for a tiled- amplicon PCR to amplify viral genomes of interest in 400 bp fragments from cDNA. Library preparation methods followed the published protocol including cDNA synthesis, PCR generation of tiled amplicons, and a ligation based library preparation using the Kapa HyperPrep Kit. Libraries were sequenced using Illumina MiSeq 2x250bp chemistry and analyzed using bwa and iVar. After testing previously published PrimalSeq primer schemes for West Nile virus and SARS-CoV-2, we used the companion primer design tool, Primal Scheme, to generate a custom primer scheme for canine morbillivirus. Primal Scheme generated 54 primer pairs that are split into two separate primer pools to cover 15,503 bp of the 15,690 bp canine morbillivirus genome. BLAST was used to identify the most closely related canine morbillivirus genome in Genbank after mapping to the RefSeq representative genome. Mapping to this genome improved coverage to 98.7% of bases being covered by at least 1 read 94.3% by at least 10 reads, and 90.5% by at least 30 reads. Excluding the 5′ and 3′ ends outside of the primer region, these improve to 99.1%, 95.5%, and 91.6%, respectively. Areas of low and no coverage were found to primarily correspond to regions of high dissimilarity between the two reference genomes used, suggesting that genetic divergence may have inhibited primer binding and amplification in these regions. Development is ongoing to further refine the primer scheme and analysis methods to improve coverage. Veterinary and zoonotic viruses pose a serious risk to global public health. The PrimalSeq method could be a useful tool for veterinary diagnostic laboratories to aid in the characterization of virus populations circulating in animals and enhance our understanding of veterinary viral diversity. Sequencing resources for this study were provided by the Food and Drug Administration’s Veterinary Laboratory Investigation and Response Network (FDA Vet-LIRN) under grants 5U18FD006379 and 1U18FD006716.

AAVLD Annual Conference Proceedings 35 AAVLD Virtual Conference Whole genome sequencing of pathogens and bioinformatics analysis as a diagnostic tool: successes and challenges? Sunil Kumar Mor, Vikash Singh, Albert Rovira, Stephanie Rossow, Fabio Vannucci, Matthew Sturos, Connie Gebhart, Alex Primus, Robert Porter, Jerry Torrison VPM/VDL, University of Minnesota, Saint Paul, MN

Next generation sequencing (NGS) technique for obtaining whole genome sequences (WGS) of pathogens allows for an unbiased identification and characterization of variants and detection of mixed infection. The Minnesota Veterinary Diagnostic Laboratory has invested in this latest technology and is offering this test for disease diagnosis and whole genome sequencing. During 2019-20, we used NGS for WGS of bacteria and viruses from different disease conditions of poultry, pigs, cattle, fish, and wild animals. We found WGS to be helpful in characterization of novel and divergent pathogens such as a porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 3, influenza A virus, rotavirus, reovirus, and divergent picornaviruses associated with different disease conditions. Streptococcus suis and Glaesserella parasuis are the bacteria most commonly requested for WGS and characterization. The most common request was for PRRSV in pigs, mainly to differentiate vaccine-like strains from wild-type strains. In one interesting case, WGS confirmed that assembled PRRSV is a recombinant virus which has approx. 35% genome from vaccine A and 55% from vaccine B. In other PRRSV cases, deletions in ORF1a NSP2 gene were commonly detected in vaccine-like and outbreak associated strains. The whole genomes of divergent and reassortment porcine rotaviruses were assembled even if samples were positive for two or three types of rotaviruses (A, B, C). More than 100 isolates of chicken and turkey reoviruses associated with lameness and hepatitis have been sequenced. These analyses were helpful to understand differences between chicken and turkey reoviruses, source of infection in turkeys, the difference in viruses associated with arthritis and hepatitis, reassortments, virus evolution, and most importantly in the selection of vaccine candidates. A novel rotavirus causing diarrhea in calves was characterized based on WGS. The Rotavirus Classification Working Group confirmed this novel virus and assigned new genotypes as G37 and P[52]. The nearly complete genome of a fish ranavirus confirmed that virus is frog virus 3 type. NGS provides unbiased amplification of total nucleic acid within the sample using random amplification hence several challenges are associated with this method. The amount of genetic material sequenced depends on the type of virus, its titer, and overall sample quality. The WGS requested viral RNA has to compete with nucleic acid of other non-related RNA viruses, host, and bacteria. The bioinformatics analysis is critical in characterizing novel, recombination, reassortments, vaccine-like, and mixed infection cases. The advantages and challenges related to WGS will be discussed in detail.

AAVLD Annual Conference Proceedings 36 AAVLD Virtual Conference Non-target RNA depletion strategies to improve sensitivity of next-generation sequencing (NGS) for diagnosing infectious agents in poultry David Suarez, Joshua Parris, Henry Kariithi, Dawn Williams-Coplin ARS/USDA, Southeast Poultry Research Laboratory, Athens, GA

PCR and serology-based assays have long been the benchmark for diagnosing respiratory and enteric pathogens of poultry and other livestock, however, these techniques are limited in their ability to detect multiple infecting agents, must be frequently optimized to maintain sensitivity and specificity, and provide limited or no genetic information on the pathogen. In contrast, untargeted, high-throughput, next-generation sequencing can rapidly detect all infecting agents in a sample while concurrently providing genomic sequence information. Although NGS for diagnostics offers many advantages over traditional tests, one of its primary limitations is the low sensitivity to pathogens because of the abundance of host and other non-target sequence in sequencing libraries prepared from bulk RNA/DNA extracts. These non-target sequences often constitute 95-99% of final sequencing libraries. In the work presented here, we explore methods for improving sensitivity of NGS to detect respiratory and enteric pathogens in poultry from RNA extracts of field-preserved swab samples. We employed several commercial and non-commercial negative enrichment strategies to selectively deplete the most abundant rRNA reads from the host (18S, 28S, mitochondrial) and conserved portions of 16S/23S rRNA from bacteria. Standard or biotinylated DNA probes were hybridized to RNA and DNA/RNA hybrids were separated using streptavidin-coated magnetic beads or degraded with RNaseH. Depleted RNA was amplified using sequence independent, single primer amplification (SISPA), sequenced via the Illumina Miseq. Treatment reduced host signal from ~90% of total reads to ~20% using custom primers and down to ~10% using commercially available kits. This resulted in up to a 200-fold increase in viral reads in final libraries, detection of a greater number of different viral agents, and higher average genome coverage for all pathogens. Inclusion of primers to remove conserved bacterial rRNA did not impede our ability to detect potential bacterial pathogens. Both custom and commercial depletion assays added only 2 hours to NGS library preparation. Custom design offered a significant cost savings ($7-12 per sample) compared to commercially available kits ($30-50 per sample). The custom depletion strategies can be optimized for various hosts and sample types and inclusion of these enrichment steps can greatly improve sensitivity of NGS for diagnostic purposes allowing users to take full advantage of the technology.

AAVLD Annual Conference Proceedings 37 AAVLD Virtual Conference Clamping tools for vaccine and wild-type PRRSV diagnostic testing Karen M. Harmon, Laura Bradner, Phillip Gauger VDPAM, Iowa State University, Ames, IA

Porcine reproductive and respiratory syndrome (PRRS) is one of the most impactful diseases of the U.S. swine industry. Control and prevention often include herd management, biosecurity, and vaccination with a modified- live virus (MLV) PRRSV product. Swine producers frequently sequence open reading frame 5 (ORF5) to track the movement and/or introduction of new strains in their herds. However, routine Sanger sequencing is often unable to differentiate wild-type from MLV PRRSV in vaccinated herds if both are co-circulating at the time of sample collection. Advancements in Next Generation Sequencing (NGS) hold promise for such investigations but attempts to sequence a mix of two PRRSV-2 viruses in a clinical sample may be problematic unless the sequence of one PRRSV is known. Additionally, the PCR cycle threshold (Ct) value of samples often needs to be in the early-to-mid 20s in order to obtain whole genome sequences, which can be problematic for some clinical specimens. Cost and time of NGS compared to Sanger sequencing may also be a concern. Therefore, a tool which preferentially amplifies ORF5 of wild-type PRRSV in the presence of MLV vaccine virus would be highly beneficial. “PCR clamping” technology incorporates a modified oligonucleotide (“clamp”) to block PCR amplification upon binding to matching nucleic acid, increasing the likelihood of amplification of other, non-blocked sequences. The objective of this study, funded in part by the Thermo Fisher Scientific/AAVLD Innovation Grant, was to design and evaluate PCR clamping assays to suppress ORF5 amplification of PRRSV MLV vaccines and promote preferential amplification of wild-type PRRSV if also present in the sample. In vitro trials using mixtures of previously characterized PRRSV wild type and vaccine-like viruses indicated that without the clamp wild-type virus typically needed to be present at a level of at least 70% to generate wild-type sequence. When the clamp was added to the PCR reaction wild- type sequence was obtained at levels as low as 10% to 20% of the mixture. Currently two clamps are being used for inhibiting amplification of Inglevac PRRSV® MLV ORF5 sequence in the Iowa State University Veterinary Diagnostic laboratory. Clamping assays are in development for Zoetis Fostera® and Elanco Prevacent® vaccines and being used on a research basis for diagnostic testing. The clamping assays have been successful for sequencing wild-type sequences in a mix of wild-type and vaccine viruses and have also uncovered several instances of presence of dual vaccines in submitted samples. Clients also use this tool to rule out wild-type virus in vaccinated animals that test positive for PRRSV by PCR. Diagnostics which investigate vaccine and wild-type virus status can be very complicated and the clamping assays are often used in conjunction with vaccine specific PCRs as well as clinical presentation and herd health history to give a more complete scenario of sample and animal status.

AAVLD Annual Conference Proceedings 38 AAVLD Virtual Conference Profiling the wild resistome: an AMR detection strategy for wildlife species Rebecca L. Tallmadge1, Kevin J. Cummings1, Patrick K. Mitchell1, Brittany D. Cronk1, Renee Anderson1, Lauren Palena1, Sara E. Childs-Sanford2, Noha Abou-Madi2, Julie Siler1, Maureen G. Gunderson3, Martin Wiedmann3, Laura B. Goodman1 1Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY; 2Department of Clinical Sciences, Cornell University, Ithaca, NY; 3Department of Food Science, Cornell University, Ithaca, NY

The resistome is a term used to collectively refer to all antimicrobial resistance (AMR) genes. It has become clear that AMR is a One Health concern because many zoonotic bacteria harbor AMR genes. However, beyond the concern for human, domestic animal, and production animal health, it is important to note that wildlife species are also affected by bacteria that harbor AMR genes. Birds pose a particular challenge due to low fecal mass that may be recoverable. Although it is difficult to measure the impact of AMR on wildlife species, it is an imperative step in order to provide efficacious treatments and to reveal what is spread among wildlife hosts and throughout the environment. To address this knowledge gap, preliminary AMR profiling studies were performed with patient samples obtained from the Janet L. Swanson Wildlife Hospital at Cornell University. A longitudinal set of weekly samples from a Cooper’s hawk that received one month of amoxicillin/clavulanic acid treatment was investigated. In addition, surveillance for Salmonella and Listeria shedding has been conducted in order to evaluate the risk and implications of zoonotic transmission. DNA was extracted using a magnetic bead-based automated process (MagMAX CORE, Thermo Fisher Scientific). The AmpliSeq for Illumina Antimicrobial Resistance Research Panel (Illumina, Inc.) was used to profile AMR genes in these populations. This strategy generates 815 primer-subtracted amplicons to detect 478 AMR genes, which encompass 28 different antimicrobial classes. AmpliSeq amplicons from each patient sample were barcoded to facilitate multiplexing on the Illumina MiSeq platform using the MiSeq Reagent Kit v3 (600-cycle) chemistry. AMR genes were identified by mapping the reads to a reference containing all of the potential amplicons. Analysis of the longitudinal sample set revealed a shift towards beta-lactam resistance after the first week of antimicrobial treatment, followed by shifts to resistance to drug classes that had not been administered. Corresponding shifts in the bacterial microbiome composition of the samples were observed. Salmonella was cultured from 5/348 patients (1.4%; wood turtle, red fox, opossum, and two birds), and some of these isolates demonstrated phenotypic in vitro resistance to azithromycin, cefoxitin, gentamicin, and streptomycin. Listeria monocytogenes was cultured from 23/335 patients (6.9%; including birds, mammals, and reptiles). Even though this was a small data set, the results are promising and have helped to secure additional funding to continue targeted AMR profiling of wildlife species. Expansion of this dataset, including samples from patients that did not receive antimicrobial treatment, is underway. Compiled genomic and in vitro phenotypic data from the bacterial cultures will also be compared. These results will document AMR emergence among wildlife and provide insights into the scale of AMR presence in the environment.

AAVLD Annual Conference Proceedings 39 AAVLD Virtual Conference blank page Virology Live Q&A Thursday, October 15, 2020

Moderators: Jianqiang Zhang and Beate Crossley

3:35 PM Development of single well real-time RT-PCR assays to detect SARS-CoV-2 in humans and animals: a collaboration between the Iowa State University Veterinary Diagnostic Laboratory and the State Hygienic Laboratory at The University of Iowa Laura Bradner, Karen M. Harmon, Phillip Gauger, Jianqiang Zhang, Jeffrey Benfer, Wade Aldous, Michael Pentella, Rodger Main...... 43

3:40 PM Experimental infection of 5 poultry species with SARS COV-2 and MERS CoV Virus David Suarez, Erica Spackman, Darrell Kapczynski, David Swayne, Mary Pantin-Jackwood, Suzanne DeBlois, Scott Lee...... 44

3:45 PM Evaluation of the effect of various heat treatment temperatures on real-time PCR cycle threshold values and virus isolation Matthew G. Van Engen, Jianqiang Zhang, Karen M. Harmon, Phillip Gauger ...... 45

3:50 PM Evaluation of an on-site method for mass disposal of swine carcasses and virus inactivation Keith L. Bailey, Rafael Ebling, Paim Willian, Justin Turner, Jeremy Seiger, Justin Talley, Gary Flory, Caleb Whitcomb, Marta Remmenga, Matthew Vuolo, Akhilesh Ramachandran, John Gilliam, Leslie Cole, Todd Jackson, Robert Miknis, Lori Miller, Fernando Bauermann...... 46

3:55 PM Determining infectivity of pork from PEDV positive pigs of different ages # † Loni L. Schumacher, Gaurav Rawal, Ethan Aljets, Deaglan Riordan, Jinhui Zhu, Jianfeng Zhang, Wannarat Yim-im, Phillip Gauger, Drew Magstadt, Jianqiang Zhang...... 47

4:00 PM PRRSV genotyping by MinION amplicon sequencing # Leonardo Cardia Caserta, Jianqiang Zhang, Diego Diel ...... 48

4:05 PM Association of border disease virus with a high mortality outbreak amongst 3-month-old feeder lambs shipped from Colorado to New York State ◊ Elisha Frye, Gerald E. Duhamel, Erica Butler, Leonardo Cardia Caserta, Melissa Laverack, Randall Renshaw, Nancy Zylich, Mary Smith, Ed Dubovi, Diego Diel...... 49

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 41 AAVLD Virtual Conference blank page Development of single well real-time RT-PCR assays to detect SARS-CoV-2 in humans and animals: a collaboration between the Iowa State University Veterinary Diagnostic Laboratory and the State Hygienic Laboratory at The University of Iowa Laura Bradner1, Karen M. Harmon1, Phillip Gauger1, Jianqiang Zhang1, Jeffrey Benfer2, Wade Aldous2, Michael Pentella2, Rodger Main1 1Iowa State University, Ames, IA; 2State Hygienic Laboratory, University of Iowa, Coralville, IA

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China in late 2019 and rapidly spread globally causing country-wide shut downs, widespread panic, and the declaration of a worldwide pandemic. The diagnostic tool of choice to initially monitor viral spread was real-time RT-PCR. Historically, human health testing laboratories have not been called upon to perform high-throughput testing with same or next-day results. Many veterinary diagnostic labs, however, operate with these expectations on a daily basis and have expertise with large- scale testing for disease outbreaks and animal health surveillance. The Iowa State University Veterinary Diagnostic Lab (ISU VDL) partnered with the State Hygienic Laboratory (SHL) at the University of Iowa to onboard the VDL’s high-throughput testing methods at the SHL. Primer and probe sequences targeting two portions of the nucleocapsid gene (N1 and N2) of SARS-CoV-2 and the human RNase P gene (RNP) as an endogenous control were provided to the public by the Centers for Disease Control and Prevention (CDC). The CDC recommended that PCRs for each of the targets be run individually with standard cycling conditions. As testing needs rose dramatically and concern over reagent availability increased, the goal was to incorporate the three targets into a single-well triplexed assay utilizing fast thermal cycling conditions to expedite reporting of results and increase testing efficiency. In addition, a second triplex assay was validated using an exogenous internal positive control instead of the human RNP gene for potential use for non-human testing. Both assays were validated using TaqMan™ Fast Virus 1-Step Master Mix on the Applied Biosystems 7500 Fast instrument using fast cycling parameters. Quantitated viral RNA provided by Dr. Hon Ip (National Wildlife Health Center; Madison, WI) was used for initial assay development and determining limit of detection. Specificity was verified using a panel of nucleic acid extracts from 14 different animal coronaviruses and one human coronavirus provided by Dr. Diego Diel (Cornell University). The limit of detection for each of the N1 and N2 targets is less than 250 copies per milliliter for both triplexed assays. Neither assay yielded a positive result for any of the coronaviruses in the specificity panel. The single well assay was used to test a blinded panel of 93 human nasal swabs provided by SHL. Of the 93 samples, only one sample (negative upon testing and retesting at ISU VDL) yielded a result inconsistent with that provided by SHL. The source of the discrepancy is unknown but thought to be due to an error in panel preparation. The collaboration between the ISU VDL and SHL allowed for the development and implementation of a high-throughput testing process for SARS-CoV-2 in the state of Iowa, resulting in expedited and expanded testing capabilities in the public health sector as well as the veterinary community should the need arise.

AAVLD Annual Conference Proceedings 43 AAVLD Virtual Conference Experimental infection of 5 poultry species with SARS COV-2 and MERS CoV Virus David Suarez, Erica Spackman, Darrell Kapczynski, David Swayne, Mary Pantin-Jackwood, Suzanne DeBlois, Scott Lee ARS/USDA, Southeast Poultry Research Laboratory, Athens, GA

The zoonotic SARS CoV-2 virus has caused a world-wide human pandemic which has resulted in the infection of millions of people with hundreds of thousands of deaths. The Middle East Respiratory Syndrome Virus (MERS) CoV is also a zoonotic pathogen that has caused a severe respiratory disease but with relatively limited geographic spread and much fewer human cases. Both viruses are betacornaviruses and are thought to originate from a bat reservoir. Because of the novel nature of both viruses, it was important to understand what animals may pose a risk of being biological vectors of the virus, potentially amplifying the virus and increasing the spread in humans. Our laboratory inoculated chickens, turkeys, Japanese quail, Pekin ducks, and Chinese geese with both viruses at titers of 10^5 tissue culture infective dose 50 or greater and monitored the animals for 2 weeks. None of the birds showed clinical disease and viral shedding from oropharyngeal and cloacal swabs taken at days 2, 4, and 7 post-inoculation were all negative for both viruses. Serum samples have been collected and results are pending. In addition to animal challenge studies, inoculation studies of embryonated chicken eggs was performed by three routes of inoculation with two blind passages. No viable virus was detected after inoculation at any passage through 5 days of incubation. These studies support that the most common poultry species raised globally for food are not susceptible to SARS COV-2 or MERS CoV. This is the first report of experimental challenge of poultry with MERS CoV and the first report of SARS CoV-2 challenge with turkeys, quail, and geese, and supports the negative replication findings of other labs for chickens and ducks. To determine if SARS-CoV-2 host restriction is strictly due to differences in the receptor specificity, we developed avian and canine cell lines expressing the human ACE-2 gene. Preliminary results indicate that DF-1 cells, but not MDCK, expressing the hACE2 gene support replication of the SARS-CoV-2 virus. Although several laboratory animals and domestic cats have been shown to be susceptible to the virus, domestic poultry are resistant and are unlikely to contribute to the spread of the virus.

AAVLD Annual Conference Proceedings 44 AAVLD Virtual Conference Evaluation of the effect of various heat treatment temperatures on real-time PCR cycle threshold values and virus isolation Matthew G. Van Engen, Jianqiang Zhang, Karen M. Harmon, Phillip Gauger VDPAM, Iowa State University, Ames, IA

Samples submitted to diagnostic laboratories commonly undergo multiple tests that require heat treatment. In some instances, samples come from a region that may contain a foreign animal disease (FAD) agent and therefore require heat inactivation for lab submission in the United States (US). Heat treatment temperatures can vary based on assay or requirements to inactive an FAD agent and the effects of heat treatments on real time polymerase chain reaction (rt-PCR) are unknown. The objective of this study was to evaluate the impact of varying heat treatment temperatures on viral pathogens that could later undergo rt-PCR and viral viability testing. Porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), and Senecavirus A (SVA) were selected for the study. BVDV, a pestivirus, was selected as a surrogate for classical swine fever virus and SVA, a picornavirus, was a surrogate for foot-and-mouth disease virus. The PRRSV and PRV are viruses 5 of economic significance to the US swine industry. The isolates were diluted to approximately 10 TCID50/mL followed by six, 10-fold serial dilutions to represent different concentrations of virus and a range of rt-PCR cycle threshold (Ct) values. Each dilution series was created in duplicate from different stock viruses of the same strain and immediately exposed to one of five heat treatments of 72°C, 56°C, 37°C, 25°C, and 4°C for thirty minutes. After heat treatment, rt-PCR and virus isolation using appropriate cell cultures were conducted. Samples positive for virus isolation were further tested via virus titration. Regardless of virus, the rt-PCR Ct values were unaffected at 4°C, 25°C, and 37°C. Interestingly, a significant increase in rt-PCR Ct values (P<0.05) was consistently observed at high virus concentrations (low Ct values) at 72°C for PRRSV, PRV and SVA although virus was still detected at Ct values prior to the established cut-off. Heat treatment at 56°C also caused a significant increase in rt-PCR Ct values for PRV and SVA at high virus concentrations. The rt-PCR Ct values were unaffected for BVDV regardless of heat treatment. In contrast, titers determined by virus titration were significantly decreased when virus was exposed to 72°C and 56°C regardless of concentration when compared to 4°C, 25°C, and 37°C that remained unaffected. PRV was completely inactivated at 56°C and 72°C at all virus concentrations. Complete inactivation of PRRSV, BVDV, and SVA was observed only at 72°C although 56°C caused a significant decrease in virus titers compared to the lower temperatures. In conclusion, heat treatment at various temperatures and virus concentrations did not reduce detection of virus by rt-PCR at the lower temperatures evaluated, but varying effect was observed at the higher temperatures. However, viability of virus was significantly decreased when exposed to temperatures of 56°C and 72°C for 30 minutes.

AAVLD Annual Conference Proceedings 45 AAVLD Virtual Conference Evaluation of an on-site method for mass disposal of swine carcasses and virus inactivation Keith L. Bailey1, Rafael Ebling2, Paim Willian2, Justin Turner3, Jeremy Seiger4, Justin Talley3, Gary Flory5, Caleb Whitcomb6, Marta Remmenga7, Matthew Vuolo7, Akhilesh Ramachandran8, John Gilliam9, Leslie Cole10, Todd Jackson11, Robert Miknis12, Lori Miller12, Fernando Bauermann2 1Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL; 2Veterinary Pathobiology, Oklahoma State University, Stillwater, OK; 3Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK; 4Envirotech Engineering & Consulting, Inc., Enid, OK; 5Agriculture & Stormwater Program, Virginia Department of Environmental Quality, Harrisonburg, VA; 6Environmental Programs, Oklahoma Department of Agriculture, Food and Forestry, Oklahoma City, OK; 7Center for Epidemiology & Animal Health, USDA APHIS, Ft. Collins, CO; 8Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK; 9Veterinary Clinical Medicine, Oklahoma State University, Stillwater, OK; 10Emergency Coordination, USDA APHIS, Oklahoma City, OK; 11Laboratory Animal Resources, Oklahoma State University, Stillwater, OK; 12VS Strategy and Policy, USDA APHIS, Ft. Collins, CO

The introduction of African Swine Fever (ASF) into the U.S. is predicted to require the euthanasia and safe disposal of a large number of commercial pigs. The current approaches to managing swine carcasses have significant limitations. Therefore, an eco-friendly, economical, on-farm disposal method that efficiently inactivates pathogens is needed. Recently, a technique known as “Above Ground Burial” (AGB) was developed and utilizes increased microbial activity in the upper part of the soil to accelerate the decomposition process. The goals of our study were to (1) evaluate the effectiveness of AGB for the mass disposal of swine on the farm, (2) evaluate the viability of an ASF-surrogate virus (i.e. swine poxvirus) in the bone marrow over a 12-month period, (3) assess the environmental impact of the AGB method, and (4) characterize the insect activity at the burial site. For this, we euthanized 100 cull sows, divided in two groups, one week apart. The carcasses were placed in two trenches; each trench measured 6 feet wide, 22 inches deep and 115 feet long – the bottom of each trench contained 12 inches of wood shavings. Pigs were placed in the trench and the medial aspect of each femur was exposed. A portion of bone marrow was

removed to facilitate the inoculation of 10 mL of swine poxvirus with a tissue culture infectious dose (TCID50) of 106.8 per mL and the femur was sealed with a stainless-steel plug. Before the carcasses were covered with a cap of soil, the abdomens were lanced, and temperature sensors were placed at four separate locations in each trench to monitor the temperature inside the carcass cavity and at the carcass/soil interface. The carcasses were covered with soil and the site was reseeded and fenced. Game cameras with motion sensors were installed to monitor the presence of scavenger activity and document the settling of the trench and vegetative habitat. Twenty femurs (10/trench) were obtained on Days 0, 7, 14, 21, 28, 60, 90 and 180 following inoculation. The femoral plug was removed, and bone marrow contents were collected and submitted for qPCR testing and virus isolation. Soil samples were collected in 6-inch increments to a depth of 180 inches to establish background data; samples were collected in the same increments and depths at months 2, 4, 6 and 12. Sampling for arthropods utilized malaise traps (flying insects), pitfall traps (crawling insects), and direct collection (from the trenches). For femoral bone marrow samples collected to date (month 6), viable virus was only detected in samples recovered on Day 7 post-burial. All samples with viable 2 virus had a titer lower than 10 (TCID50) mL, confirming rapid inactivation of the virus in the femoral bone marrow in a 1-week period. Viral DNA was detectable by qPCR assay throughout the first 6 months of the study. To date, 6,474 arthropods have been collected and identified. These data show promising results related to virus inactivation in the AGB method.

AAVLD Annual Conference Proceedings 46 AAVLD Virtual Conference Determining infectivity of pork from PEDV positive pigs of different ages # † Loni L. Schumacher, Gaurav Rawal, Ethan Aljets, Deaglan Riordan, Jinhui Zhu, Jianfeng Zhang, Wannarat Yim-im, Phillip Gauger, Drew Magstadt, Jianqiang Zhang VDPAM, Iowa State University, Ames, IA

Since the emergence of porcine epidemic diarrhea virus (PEDV) in the U.S., some countries importing American pork voiced concerns about the presence of PEDV in meat. Previously, there was limited PEDV PCR data in muscles of neonatal pigs. We have recently evaluated the detection of PEDV in muscle of PEDV-inoculated 3-week-old (weaned) and 23-week-old (finisher) pigs as tested by a quantitative PEDV N gene-based real-time RT-PCR. Nevertheless, no data is available about infectivity of pork from PEDV-positive pigs because PCR cannot distinguish non-infectious virus from infectious virus. Since virus isolation in cell culture is not sensitive enough and previous studies have demonstrated that pig bioassay is a reliable tool to assess PEDV infectivity of product-treated feed samples or disinfectant-treated samples, we aimed to employ pig bioassay in the current study to determine if pork from PEDV infected pigs contains infectious PEDV for the first time. Ten-day-old PEDV naïve pigs (N=42) were divided into 14 groups (3 pigs/group) and orogastrically inoculated with the following inocula. Group 1: 5 virus-negative culture media; Group 2: non-S INDEL PEDV culture isolate USA/IN19338/2013 (CT=11.4, 10 TCID50/pig); Groups 3-5: three muscle homogenates (CT 26.3, 30.8 and 29.9) from three previous PEDV-inoculated weaned pigs; Groups 6-8: three muscle homogenates (CT >40 for each) from three previous PEDV-inoculated finisher pigs; Groups 9-11: three ileum homogenates (CT 22.1, 21.7 and 18.4) from the same three previously inoculated weaned pigs as described in Groups 3-5; and Groups 12-14: three ileum homogenates (CT 27.0, 24.3 and 24.2) from the same three previously inoculated finisher pigs as described in Groups 6-8. Fecal swabs were collected daily until end of study (7 dpi). All pigs in Group 1 were bioassay negative and all pigs in Group 2 were bioassay positive. All pigs inoculated with muscle homogenate either from weaned pigs (Groups 3-5) or from finisher pigs (Groups 6-8) were bioassay negative and lacked detectable PEDV in feces. All pigs inoculated with ileum homogenate from weaned pigs (Groups 9-11) were PCR positive by 2 dpi and had fecal shedding to end of study. Interestingly, among the three groups of pigs inoculated with ileum homogenates from finisher pigs (Groups 12-14), only one group (Group 13) was bioassay positive. Based on these results, pork from PEDV infected pigs does not contain infectious virus as determined by the young pig bioassay model. Information gained from this study may help address safety concerns in the market about PEDV infectivity of meat. # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 47 AAVLD Virtual Conference PRRSV genotyping by MinION amplicon sequencing # Leonardo Cardia Caserta1, Jianqiang Zhang2, Diego Diel1 1Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY; 2Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA

The global spread and constant evolution are challenges to the control of the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting the swine industry. PRRSV is divided into two species, PRRSV Type 1 (more prevalent in Europe) and Type 2 (more prevalent in North America). Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. A sensitive and specific protocol was developed with the aim to sequence a ~1600 bp region of the genome covering the complete ORF5 (envelope protein) and ORF6 (membrane protein) using MinION amplicon sequencing. Six 10-fold sequential dilutions of four different PRRSV isolates including type I and II were sequenced using the PCR barcoding amplicons protocol (Oxford nanopore Technologies, ONT) in two independent MinION runs. Aliquots of the same RNA used for the sequencing experiments were tested by RT qPCR. After 20 minutes of sequencing it was possible to detect PRRSV reads in all dilutions, showing the sensitivity of the protocol. Viral loads resulting in qPCR Cts as high as 35 and 37 for type I and II respectively, were successfully sequenced and retrieved 25 and 18 reads respectively, corresponding to the full-length amplicons. From RNA extraction to start the sequencing run, the average time to process 24 samples was approximately 17 hours. Clinical swine serum samples with Cts ranging from 15 to 35 were also tested. The sequencing resulted in reads for all the samples, enabling the classification into type I or II after phylogenetic analysis. Besides amplicon sequencing, the whole genome of seven different PRRSV isolates was obtained by random sequencing using an adaptation of the PCR Barcoding Genomic DNA protocol (ONT) and were compared to the results of a sequencing using the Miseq platform (Illumina). The average genome coverage obtained from MinION random sequencing was 99.88% and the identity between platforms was 99.61% on average. A fast turnover time, portability, repeatability and an accuracy compatible with genotyping suggests that this is a useful platform with the potential to improve the understanding of PRRSV evolution and provide important information for the control of the virus. Further optimization of protocols together with the use of faster bioinformatic pipelines will potentially decrease the turnaround time, improving an already cost-effective platform for PRRSV genotyping. # AAVLD Trainee Awardee

AAVLD Annual Conference Proceedings 48 AAVLD Virtual Conference Association of border disease virus with a high mortality outbreak amongst 3-month-old feeder lambs shipped from Colorado to New York State ◊ Elisha Frye, Gerald E. Duhamel, Erica Butler, Leonardo Cardia Caserta, Melissa Laverack, Randall Renshaw, Nancy Zylich, Mary Smith, Ed Dubovi, Diego Diel Population Medicine, Cornell University, Ithaca, NY

Border disease virus (BDV), a Pestivirus antigenically related to Bovine Viral Diarrhea (BVD) virus, causes a variety of reproductive disorders in sheep, including failure to conceive, abortion and the birth of persistently infected (PI) lambs, so called “hairy shakers” because of congenital infection leading to postnatal tremors and abnormal hair-like fleece. With the exception of a single outbreak with 50 percent mortality in intensively reared 3-5-month-old lambs in France in 1983, acute outbreaks of BDV leading to high mortality have not been reported previously. Here we describe an association between the presence of BDV and a high mortality outbreak in a group of 1,708, mixed-breed 3-month-old feeder lambs that started 6-weeks after transport from an open range pasture in Colorado to an enclosed barn in New York. At the beginning of the outbreak, lamb mortality ranged between 20 to 30 head per day, without demonstrating appreciable clinical signs. Over a 3-week period, more than 300 lambs died, resulting in an overall mortality rate of approximately 18 percent. Autopsies of 20 lambs revealed emaciation, pneumonia, and ruminal acidosis. Histologic examination of 3 lambs showed evidence of bronchopneumonia, polyserositis and sepsis. Whole genome sequencing of a cultured virus isolate from the outbreak revealed the presence of BDV. Three weeks after the initial outbreak, 12 of 30 apparently healthy lambs had serum neutralizing antibody (NA) titers (range 8-512) to the outbreak BDV isolate. By week 7 of the outbreak, 48 of 100 (48%) apparently healthy lambs showed significant BDV NA titers (range 8-3,072). This group included 29 from the initial sampling, of which 9 more had seroconverted. Based on our data, the outbreak was attributed to acute infection of susceptible lambs with BDV; however, BDV persistently infected (PI) lambs, which was not investigated, might have been the source of the initial outbreak. Transport over a long distance together with a change in diet and management, from range grazing to intensively housed, also may have contributed to rapid BDV transmission and high mortality rate over a short period. Sheep producers and small ruminant veterinarians should be aware of this BDV presentation in a large flock of lambs. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 49 AAVLD Virtual Conference blank page Bacteriology Live Q&A Thursday, October 15, 2020

Moderators: Chien-Che Hung and Tamara Gull

4:10 PM New species of Moraxella associated with clinically healthy eyes and cases of pinkeye in cattle Rebecca P. Wilkes, Jobin Kattoor, Eman Anis...... 53

4:15 PM Rapid differentiation of Moraxella bovoculi genotypes 1 and 2 using MALDI-TOF mass spectrometry profiles # † Matt Hille, Aaron Dickey, Kara Robbins, Michael Clawson, John Dustin Loy...... 54

4:20 PM Validation and use of a high-throughput, real-time Mycoplasma bovoculi PCR in infectious bovine keratoconjunctivitis samples Kris A. Clothier, Gabriele Ute Maier, Steve Reinl, John Angelos...... 55

4:25 PM MALDI SepsityperTM technology provides a more rapid but less sensitive method for diagnosis of bacteremia in veterinary patients # Miranda Hengy, Jessica Garcia, Jessica Pempek, C. Austin Hinds, Faye Hartmann, Rebecca J. Franklin-Guild, Josh Daniels, Gregory Habing, Dubraska Diaz-Campos ...... 56

4:30 PM Clostridium botulinum type A detected by MALDI-TOF mass spectrometry in bovine rumen contents after ingestion of feed contaminated by a raccoon carcass ◊ Elisha Frye, Christina Egan, Micheal Perry, Esther Crouch, Kyle Burbank, Kathleen Kelly. . . . .57

4:35 PM Diagnostic performance of a commercial cartridge-based PCR assay vs. a selective chromogenic agar to detect blaNDM-5 E. coli from animal rectal swabs Stephen Cole, Shelley C. Rankin...... 58

4:40 PM Isolation of a newly emerging atypical Brucella sp. from White’s tree frog (Litoria caerulea) Chien-Che Hung, Leyi Wang, Jennifer Langan, Martha Delaney, Caitlin Burrell, Carol Wolfgang Maddox...... 59

4:45 PM Bacteriological analysis of houseflies (Musca domestica L.) captured on a New York State dairy farm Gloria Gioia, Jamie Catherine Freeman, Carlos Santisteban, Matthias Josef Wieland, Anja Sipka, Valeria Maria Alanis Gallardo, Valentina Monistero, Jeffrey Scott, Paolo Moroni...... 60

4:50 PM Prevalence and antibiograms of Salmonella serovars isolated from diagnostic specimens of dogs and cats submitted between year 2014 to 2019 Owais Ahmed Khan, Jessie D. Monday, Russell Raleigh, Sonia W. Lingsweiler, Narayan C. Paul, Amy Swinford...... 61

4:55 PM Development of an experimental challenge model for Streptococcus equi subsp. zooepidemicus in pigs ◊ Nubia Macedo, Panchan Sitthicharoenchai, Eric Burrough, Orhan Sahin, Karen M. Harmon, Ganwu Li, Maria Jose Clavijo, Susan Brockmeier, Suelee Robbe-Austerman, Samantha Hau, Kristina Lantz, Jessica Goncalves dos Santos, Ana Paula Poeta Silva, Chelsea Ruston, Rodger Main, Rachel Derscheid...... 62

AAVLD Annual Conference Proceedings 51 AAVLD Virtual Conference 5:00 PM Bisgaard Taxon 40 associated with high mortality in seabirds: characterization and comparative analyses of virulence factors and antimicrobial resistance. # Eliana De Luca, Grazieli Maboni, Rodrigo Baptista, Kevin Niedringhaus, Nicole Nemeth, Susan Sanchez...... 63

5:05 PM Genomic islands analysis in Klebsiella pneumoniae associated with bovine mastitis # * † Zhiyi Zheng, Patrick J. Gorden, Ganwu Li...... 64

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 52 AAVLD Virtual Conference New species of Moraxella associated with clinically healthy eyes and cases of pinkeye in cattle Rebecca P. Wilkes1,2, Jobin Kattoor1, Eman Anis3 1Comparative Pathobiology, Purdue University, Lafayette, IN; 2Infectious Diseases, University of Georgia, Athens, GA; 3Pathobiology, University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked adverse impact on the animal welfare and economy of the cattle industry. Moraxella species isolated from cases of pinkeye include M. bovis and M. bovoculi. We recently showed that a significant number of Moraxella species reads could be detected from samples from clinically healthy eyes by 16S rRNA metagenomics, despite an inability to culture the bacteria from the sample. We also identified a new species of Moraxella (suggested name M. oculobovii) from the eyes of cattle with and without pinkeye through isolation and whole-genome sequencing. This strain is non- hemolytic on blood agar, is missing the RTX operon, and is expected to be a non-pathogenic strain in clinically healthy eyes. It is 100% identical to M. bovis or M. bovoculi in variable regions of the 16S gene, suggesting this new species could potentially be misidentified. Therefore, we developed a targeted NGS method using primers for Moraxella bovis, M. bovoculi, and M. oculobovii gene targets 16S-23S ribosomal RNA intergenic spacer region (ITS), ATP synthase F1, RNA polymerase subunit B, histidine kinase, phospho-N-acetylmuramoylpentapeptide transferase, and ferredoxin to evaluate swabs collected from two farms from cattle with clinically healthy eyes and from cattle with pinkeye. These farms were previously shown to have the new species of Moraxella based on culture and ITS sequencing. A preliminary study with 13 cattle (7 with pinkeye and 6 with clinically healthy eyes) was performed, and the targeted NGS results (defined as the total percentage of reads of each of the three species) from the two groups were compared by Mann-Whitney U tests. The targeted NGS assay was able to differentiate the three different species directly from DNA extracted from the swab samples, without the need for culture to separate multiple Moraxella species that may be present in the sample. All the samples, except one (a sample from a healthy cow), contained all three species. There was a statistical difference (p ≤ 0.05) with the number of reads of M. bovis detected from cattle with pinkeye versus healthy-eyed cattle. There was no statistical difference between the two groups with respect to M. bovoculi or the newly identified species, and these results were unexpected. However, considering the small sample size, additional testing is warranted.

AAVLD Annual Conference Proceedings 53 AAVLD Virtual Conference Rapid differentiation of Moraxella bovoculi genotypes 1 and 2 using MALDI-TOF mass spectrometry profiles # † Matt Hille1, Aaron Dickey2, Kara Robbins1, Michael Clawson2, John Dustin Loy1 1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE; 2U.S. Meat Animal Research Center, United States Department of Agriculture, Clay Center, NE

Moraxella bovoculi is the most frequently isolated bacteria from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK), also known as bovine pinkeye. Two distinct genotypes of M. bovoculi, genotype 1 and genotype 2, were characterized after whole genome sequencing showed a large degree of single nucleotide polymorphism (SNP) diversity within the species. To date, both genotypes have been isolated from the eyes of cattle without clinical signs of IBK while only genotype 1 strains have been isolated from the eyes of cattle with clinical signs of IBK. We used 38 known genotype 1 strains and 26 known genotype 2 strains to assess the ability of matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to accurately genotype M. bovoculi strains using mass spectrum biomarkers. Mass spectrum data was analyzed with ClinProTools 3.0 software and six models were developed that classify strain genotypes with accuracies ranging from 90.6% - 100%. Next, the spectra was manually inspected to determine highly discriminatory peaks. Using four of the most genotype-specific peaks that also exhibited high peak intensities from the six automated models, we developed a customized model (UNL assisted model) that had recognition capability, validation, and classification accuracies of 100% for genotype classification. Samples were prepared using a combination of ethanol treatment followed by extraction using formic acid and acetonitrile. When the more rudimentary, direct transfer or “smear” method was used for sample preparation, the accuracy of the UNL assisted model genotype classification remained 100%. Our results indicate that MALDI-TOF MS biomarkers can be used to accurately discriminate genotypes of M. bovoculi without the need for additional methods. # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 54 AAVLD Virtual Conference Validation and use of a high-throughput, real-time Mycoplasma bovoculi PCR in infectious bovine keratoconjunctivitis samples Kris A. Clothier1, Gabriele Ute Maier2, Steve Reinl1, John Angelos3 1California Animal Health & Food Safety Lab, U. C. Davis, Fairfield, CA; 2Department of Population Health and Reproduction, University of California, Davis, Davis, CA; 3Department of Medicine and Epidemiology, University of California, Davis, Davis, CA

Infectious bovine keratoconjunctivitis (IBK) or “pinkeye” is the most commonly reported bovine ocular disease and is an important health concern to cattle industries worldwide. While most animals recover, IBK is both an animal welfare and performance concern due to ocular pain and damage, decreased feed intake, lighter weaning weights, and permanent corneal scarring. Severe cases can lead to eye rupture and blindness. The causative agent is Moraxella bovis (M. bovis), although other agents including infectious bovine rhinotracheitis (IBR, bovine herpesvirus) and Moraxella bovoculi (M. bovoculi) have been implicated as contributing factors in IBK outbreaks. Recently, an increase in frequency and severity of IBK cases had been reported to our laboratory, particularly in herds with robust pinkeye prevention programs in which no primary pathogens could be identified. Mycoplasma bovoculi (Myco. bovoculi) has been shown to cause clinical conjunctivitis and may contribute to IBK; however, due to its fastidious growth requirements and the difficulty with species confirmation, the role it plays in IBK is not clear. The purpose of this study was to validate a high-throughput, real-time PCR for Myco. bovoculi for use in herds with IBK and to examine any association with known IBK pathogens. A previously published assay was optimized for use with Amies charcoal swabs submitted for Moraxella sp. culture. A limit of detection of 10 cfu/ml was established utilizing Mycoplasma bovoculi ATCC 29104 and two clinical Myco. bovoculi isolates. Forty-eight ocular swabs were evaluated with this PCR assay; 41 swabs came from eyes with clinical IBK. Thirty-four IBK samples (82.9%) were positive for Myco. bovoculi, while none of the swabs from eyes without lesions were PCR-positive. Of the Myco. bovoculi positive samples, 12 were positive for M. bovoculi, one for M. bovis, one for both M. bovoculi and M. bovis, and two for Mycoplasma bovis. Eight samples had no primary pathogens detected by culture, and 10 had culture plates that were overgrown with contaminants (bacterial or fungal.) Of the seven Myco. bovoculi PCR negative samples, five were positive for M. bovoculi, one for M. bovis, and one had no aerobic growth. The present study reveals that Myco. bovoculi was frequently identified in samples from clinical IBK cases and may be contributing to outbreaks in herds with previously controlled IBK.

AAVLD Annual Conference Proceedings 55 AAVLD Virtual Conference MALDI SepsityperTM technology provides a more rapid but less sensitive method for diagnosis of bacteremia in veterinary patients # Miranda Hengy1, Jessica Garcia2, Jessica Pempek2, C. Austin Hinds3, Faye Hartmann4, Rebecca J. Franklin-Guild5, Josh Daniels6, Gregory Habing2, Dubraska Diaz-Campos1 1Department of Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, Columbus, OH; 2Department of Veterinary Preventive Medicine, The Ohio State University College of Veterinary Medicine, Columbus, OH; 3Department of Food Animal Medicine and Surgery, University of Missouri Veterinary Health Center, Columbia, MO; 4Department of Microbiology and Clinical Pathology, University of Wisconsin-Madison Veterinary Care, Madison, WI; 5Animal Health Diagnostic Center, Cornell University College of Veterinary Medicine, Ithaca, NY; 6Department of Bacteriology, Colorado State University Veterinary Diagnostic Laboratory, Fort Collins, CO

Culture-based diagnosis of bacteremia in veterinary patients requires a minimum of three days, but can extend up to a week or more. Therefore, culture-based diagnoses are impractical when rapid treatment decisions are needed. In most cases, antibiotics are given empirically without confirmation of sepsis or identification of the causative agent, raising concerns over unnecessary or suboptimal antibiotic administration. A recent alternative method for diagnosing bacteremia involves utilization of a SepsityperTM kit in conjunction with matrix-assisted laser desorption/ ionization time of flight (MALDI-TOF) mass spectrometry. This method is capable of detecting and speciating bacteremia-causing organisms within 30 min of a blood culture bottle (BCB) demonstrating bacterial growth. The objective of this study was to investigate the use of Sepsityper with MALDI-TOF to detect bacteremia in a variety of veterinary patients with suspected septicemia. We hypothesized that Sepsityper would provide a more rapid diagnosis than traditional culture to media-based techniques, but that the culture-based techniques would identify bacteria in a larger proportion of positive BCBs. Aseptic blood samples were collected from veterinary patients with clinical concern for sepsis, and were used to inoculate a BCB. Blood culture bottles were monitored at least daily for bacterial growth according to standard operating procedures of the four participating diagnostic labs. Blood culture fluid from positive BCBs was analyzed by Sepsityper with MALDI-TOF and traditional culture to media- based isolation and identification methods. Chocolate agar was utilized in all participating laboratories as a standard non-selective culture media. The Sepsityper method identified bacteria in 67.1% (n=94/140) of cases with apparent bacterial growth in the BCB, while the culture-based method was significantly more successful in identifying bacteremia-causing agents at 94.3% (n=133/141). The proportion of positive blood cultures successfully diagnosed by Sepsityper did not differ significantly for the three major animal species included in the study (canine, bovine, and equine). Despite a lower sensitivity, results from the Sepsityper method were available at least 24 h sooner than culture-based methods. Because the Sepsityper method allows for a reduced time to pathogen identification, we recommend utilizing this method in conjunction with regular culture-based methods that will still be necessary for antimicrobial susceptibility testing. Utilizing Sepsityper in conjunction with culture will maximize diagnostic potential, allowing for a higher level of care to critically ill patients and a reduced potential for the development of antimicrobial resistance. # AAVLD Trainee Awardee

AAVLD Annual Conference Proceedings 56 AAVLD Virtual Conference Clostridium botulinum type A detected by MALDI-TOF mass spectrometry in bovine rumen contents after ingestion of feed contaminated by a raccoon carcass ◊ Elisha Frye1, Christina Egan2, Micheal Perry2, Esther Crouch1, Kyle Burbank1, Kathleen Kelly1 1Population Medicine, Cornell University, Ithaca, NY; 2Division of Infectious Diseases, Wadsworth Center Biodefense Laboratory, Albany, NY

Twenty-eight lactating dairy cattle in New York State showed clinical signs of anorexia, decreased milk production, decreased tongue tone, profound weakness, and recumbency over a 5-day period. Twelve of the affected cattle died and 16 recovered, but never returned to full productivity. The day after the outbreak began, a partial raccoon carcass was found in the total mixed ration (TMR). An initial differential diagnosis included botulism, ionophore toxicity, mycotoxin toxicity, nitrate poisoning, organophosphate poisoning, cyanide poisoning, vitamin E/selenium deficiency, toxic plant ingestion and metabolic disorders. Monensin and mycotoxin testing were within acceptable limits. Autopsies and ancillary testing were performed on 5 cattle. Aqueous humor nitrate concentrations, serum cholinesterase activity and liver selenium concentrations were all within normal limits. A chemistry panel on one cow showed elevated electrolytes, liver enzymes, and creatine kinase. Anaerobic culture, real-time PCR for Clostridium botulinum toxin genes, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for C. botulinum neurotoxin detection was performed on rumen contents and liver samples. Results confirmed the presence of C. botulinum type A in the rumen contents of two cattle, and C. botulinum type C in the liver of one cow. An unusual postmortem lesion suggesting a toxic myopathy was identified in 4 of the carcasses, the significance of which is unknown. This presentation will focus on the diagnostic challenges of suspect botulism cases in bovines, the significance of C. botulinum Type A in the northeastern United States, and the ability to detect preformed neurotoxin using MALDI-TOF MS. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 57 AAVLD Virtual Conference Diagnostic performance of a commercial cartridge-based PCR assay vs. a selective chromogenic agar to detect blaNDM-5 E. coli from animal rectal swabs Stephen Cole, Shelley C. Rankin UPenn, Philadelphia, PA

Introduction: There have been few reports of carbapenemase producing Enterobacteriaceae (CPE) isolated from companion animals. Therefore, little is understood about fecal colonization in dogs and cats. During an outbreak of blaNDM-5 positive Escherichia coli at the veterinary teaching hospital (VTH) of the University of Pennsylvania, School of Veterinary Medicine, we determined that there are no validated methods to screen companion animals for fecal colonization. The aim of this study was to compare the diagnostic performance of a multiplex PCR assay (Xpert Carba-R, Cepheid) with selective chromogenic agar culture (CHROMID Carba, Biomerieux) to detect carbapenem resistant organisms (CROs) in rectal swabs from dogs and cats. Methods: A 0.5 McFarland saline suspension of blaNDM-5 E. coli, reference isolate 24213-18, was serially diluted and used to spike assay swabs (Cepheid Collection Device, Copan) that were pre-inoculated with CPE-negative canine feces. The PCR assay was run in duplicate to determine the limit of detection (LoD). To evaluate the PCR for fecal screening of animals, double-headed rectal swabs were collected from animals at the time of hospital admission and again upon discharge. One swab was processed on the Cepheid GeneXpert. The other swab was streaked to a CHROMID Carba plate, incubated at 37C and examined at 24 and 48 hours. Presumptive CR-E. coli colonies were tested for carbapenemase production by modified carbapenem inhibition method (mCIM). Results: The LoD determined for blaNDM-5 E. coli was 50 CFU/swab. A total of 970 (519 admission and 451 discharge) rectal swabs from 552 animals (134 cats and 418 dogs) were screened. Eleven (1.1%) specimens from 6 dogs were positive for the blaNDM gene. Nine corresponding cultures were positive for E. coli which were mCIM positive. Two additional cultures, from the same dog, were positive for Enterobacter cloacae which was mCIM positive. Conclusions: The LoD of blaNDM with the Xpert Carba-R assay was not inferior to the manufacturer-claimed LoD for NDM (74 CFU/swab). When the Xpert Carba-R PCR assay was compared to culture on a CHROMID Carba plate, 100 % concordance was observed. Both methods detected CROs and could be used to screen animals for fecal colonization.

AAVLD Annual Conference Proceedings 58 AAVLD Virtual Conference Isolation of a newly emerging atypical Brucella sp. from White’s tree frog (Litoria caerulea) Chien-Che Hung1, Leyi Wang1, Jennifer Langan2, Martha Delaney1, Caitlin Burrell2, Carol Wolfgang Maddox1 1Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL; 2University of Illinois at Urbana-Champaign, Urbana, IL

Brucellosis is an important zoonotic disease with significant public health threat. Recently, an atypical Brucella species has been associated with infections in frogs and toads, highlighting its significance as a newly emerging bacterial pathogen in this amphibian. Here, we report a case of Brucella infection in a White’s tree frog (Litoria caerulea) from a zoological collection. The adult female frog presented due to weight gain and bloating with acute coelomic distention. Ultrasound identified a large quantity of coelomic fluid containing marked flocculent material. A sample of the fluid was aspirated under ultrasound guidance and submitted to the Veterinary Diagnostic Laboratory of University of Illinois for aerobic and anaerobic culture. A non-hemolytic Gram-negative bacterium with short rod morphology was isolated. Bacterial identification by MALDI-ToF suggested Brucella melitensis with score of 2.32. However, multiplex PCR results showed that the PCR amplification pattern of the isolate did not match to other typical Brucella sp. Sequence analysis of 16S rRNA indicated that the strain was 100% identical to the atypical Brucella sp. isolated from several species of frogs. Furthermore, whole genome SNP-based phylogenetic tree clustered the isolate with other atypical Brucella sp. strains isolated from frogs and humans. Following identification of a Brucella sp. from the coelomic fluid, the animal was euthanized due to zoonotic disease concerns. Gross and microscopic evaluation identified disseminated granulomatous inflammation involving many organ systems consistent with an infectious process resembling other reported cases of atypical brucellosis. This atypical Brucella sp. also causes infections in humans, bringing attention to zoonotic potential and public health concern regarding this newly emerging bacterial pathogen.

AAVLD Annual Conference Proceedings 59 AAVLD Virtual Conference Bacteriological analysis of houseflies (Musca domestica L.) captured on a New York State dairy farm Gloria Gioia1, Jamie Catherine Freeman2, Carlos Santisteban1, Matthias Josef Wieland1, Anja Sipka1, Valeria Maria Alanis Gallardo1, Valentina Monistero3, Jeffrey Scott2, Paolo Moroni1,3 1Department of Population Medicine and Diagnostic Sciences - Animal Health Diagnostic Center -QMPS, Cornell University, Ithaca, NY; 2Department of Entomology, Cornell University, College of Agriculture and Life Sciences, Ithaca, NY; 3Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Lodi, Italy

Houseflies are one of the most common non-biting muscoid insects identified as hosts for a large number of bacteria. Bacteria can be carried on the surface of the fly’s exoskeleton and in their alimentary canal from which the bacteria are disseminated through defecation or regurgitation. Severe housefly infestations on dairy farms may result in economic losses attributable to stress for animals, decreased milk production, increased bacterial counts in milk and potential occurrence of transmitted diseases. The goal of this study was to evaluate the bacterial community associated with houseflies captured on a dairy farm in NY State. A total of 101 flies were collected at 3 farm sites: 27 flies from the hospital pen, 42 from calf hutches and 32 from the milking parlor. The species and gender of each fly were determined through external morphology observation under a stereomicroscope resulting in 53 females and 48 males. Bacteriological analyses were performed through aerobic and Mycoplasma cultures from both the external surfaces and internal parts of each fly. For each case, samples were streaked for isolation after a pre-enrichment step in Todd Hewitt broth for 6 hours at 37°C and in Mycoplasma Hardy broth for 72 hours at 37°C with 5% CO2. For the external surface, each fly was washed by immersion in 1X PBS, the washing suspensions were inoculated into the media reported above. For the testing of internal parts, each fly was homogenized using 5 sterile zirconium beads with a diameter of 1.7 mm and a bead-beating step of 2 minutes at 7,000 rpm. After the enrichment steps, each sample was streaked on different culture media. Agar medium used for the isolation of aerobic organisms included: TSA with 5% sheep blood and 0.1 % esculin, MacConkey, Edwards with 5% sheep blood, Vogel Johnson and Prototheca Isolation Media. All these media were incubated for 48 hours at 37°C. For Mycoplasma isolation, we used Modified Hayflick agar incubated 7 days at 37C with 5% CO2. Isolated bacteria were identified by MALDI- TOF or Sanger sequencing. A total of 394 bacteria were isolated and identified, 47% from females, 53% from males. The majority of bacteria were found in the internal parts of the flies, 74% versus 26% isolated from the external surfaces. The distribution of isolates from the 3 farm sites was: 42% hospital pen, 33% calf area and 25% from the milking parlor. We isolated 26 different organisms identified at the species level that can be sources of mastitis, some of which are also considered contagious. An example of this category was 1 Mycoplasma arginini isolated from the internal part of one fly captured from the hospital pen. In the category of pathogens with impact on human health, we identified 5 organisms that are considered foodborne pathogens. The results of this study confirmed that houseflies carry a high bacterial diversity which includes either organisms that can be associated with animal infections or that can be a concern for public health.

AAVLD Annual Conference Proceedings 60 AAVLD Virtual Conference Prevalence and antibiograms of Salmonella serovars isolated from diagnostic specimens of dogs and cats submitted between year 2014 to 2019 Owais Ahmed Khan1, Jessie D. Monday2, Russell Raleigh1, Sonia W. Lingsweiler3, Narayan C. Paul3, Amy Swinford4 1Bacteriology and Serology, Texas A&M Veterinary diagnostic Laboratory, Amarillo, TX; 2VETERINARY DIAGNOSTICIAN, Texas A&M Veterinary Diagnostic Laboratory, AMARILLO, TX; 3Bacteriology, Texas A&M Veterinary Diagnostic Laboratory, College Station, TX; 4Associate Director, Texas A&M Veterinary Diagnostic Laboratory, College Station, TX

Salmonellosis is an important disease of animals causing morbidity and mortality in various age groups. Commonly reported veterinary clinical manifestations include diarrhea, dehydration, pneumonia, septicemia, abortion and death. Foodborne salmonellosis is an important zoonotic disease attributed to consumption of contaminated meat, eggs and dairy products. Shedding of Salmonella by asymptomatic dogs and cats and its close contacts with household members poses a public health risk. This retrospective study conducted at Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) aimed to determine the prevalence and antibiotic resistance patterns of Salmonella serovars among clinical cases of dogs and cats across Texas and neighboring states during 2014-2019. A total of 95 (95/1099, 8.64%) and 19 (19/320, 8.43%) Salmonella isolates were isolated from 1,099 and 320 clinical samples of dogs and cats, respectively, using standard Salmonella isolation method. These samples were comprised of fecal, enteric and non-enteric tissue samples including liver, lungs, gall bladder and organs swabs. Among the submitted samples, 1,310 were from Texas, and 109 from other states. Salmonella serotyping results were obtained from NVSL and antibiotic susceptibility tests were conducted using commercial MIC plates and Kirby-Bauer disk diffusion methods. Interpretations of antibiotic susceptibility test results were performed according to CLSI guidelines. A total of 37 different serovars were isolated from dogs, and the most prevalent serovars were S. Newport, 19 (21.34%); S. Anatum, S. Rubislaw, S. Infantis each 6 (6.75 %); S. Heidelberg and S. Montevideo each 5 (5.61%). In cats 16 different serovars were isolated and prevalent serovars were S. Typhimurium, 4 (15.38%); S. Newport, 3 (11.54%) and S. Enteritidis, S. Infantis and S. Javiana, 2 (7.69%). All tested isolates of Salmonella in dogs (64 out of 95) and cats (13 out of 19) were sensitive to fluoroquinolone and imipenem. Dog isolates were highly resistant to rifampin (100%) clindamycin (93.8%), erythromycin (93.8%) and penicillin (82%). Similarly, all cat isolates were resistant to rifampin, clindamycin, erythromycin, and penicillin. Salmonella from dogs were also resistant to amikacin (11.3%), amoxy-clav (6.3%), ampicillin (11.3%), cephalothin (10%), ceftiofur (6.2%), chloramphenicol (7.9%), gentamicin (12.7%), tetracycline (21.6%) and trimethoprim- sulphamethoxazole (4.8%). In cats, amikacin (38.5%), ampicillin (7.7%), cefazolin (38.5%), cefalexin (55%) and gentamicin (38.5%) resistant were also observed. The present study demonstrated the distribution of Salmonella serovars across different regions of Texas and antibiotic susceptibility of these serovars and possible risk of human infections in close contact individuals to drug resistant pathogens.

AAVLD Annual Conference Proceedings 61 AAVLD Virtual Conference Development of an experimental challenge model for Streptococcus equi subsp. zooepidemicus in pigs ◊ Nubia Macedo1, Panchan Sitthicharoenchai1, Eric Burrough1, Orhan Sahin1, Karen M. Harmon1, Ganwu Li1, Maria Jose Clavijo1, Susan Brockmeier2, Suelee Robbe-Austerman3, Samantha Hau4, Kristina Lantz5, Jessica Goncalves dos Santos1, Ana Paula Poeta Silva1, Chelsea Ruston1, Rodger Main1, Rachel Derscheid1 1VDPAM, Iowa State University, Ames, IA; 2USDA, Ames, IA; 3Diagnostic Bacteriology and Pathology Laboratory, NVSL, Ames, IA; 4ARS, USDA, Ames, IA; 5APHIS, USDA, Ames, IA

Introduction: In swine, Streptococcus equi subsp. zooepidemicus (SEZ) is not commonly recognized as a significant pathogen in the US although it occasionally causes cervicitis, metritis, mastitis, arthritis, and septicemia. However, sporadic outbreaks of SEZ in sows and feeder pigs with high mortality due to septicemia were reported in Canada in the spring and summer of 2019 and in the USA later that fall. The clinical signs included lethargy, weakness, high fever and rapid mortality affecting 30-50% among exposed populations. SEZ was isolated from internal organs of affected pigs and other potential causes were ruled out as part of the extensive diagnostic evaluation and testing on the cases submitted. Little is known on the pathogenicity of SEZ in vivo and most studies were based on in vitro models. Therefore, the objective of this study was to establish an animal experimental model to advance our knowledge about the pathogenicity and diagnosis of SEZ in pigs. Study design: Ten 6-week-old conventional crossbred pigs were individually identified and assigned to three groups. All pigs tested negative for PRRSv, Mycoplasma hyopneumoniae and influenza. Streptococcus suis was isolated from the upper respiratory tract (URT) of all pigs. SEZ was not isolated before inoculation. Two pigs were randomly assigned to the control group (G1) and four pigs were assigned to each challenge groups. At day 0, challenged pigs received either 3 mL of 7 x 108 CFU/mL (high dose – G3) or 3 mL of 7 x 105 CFU/mL (low dose – G2) of the 2019 SEZ US outbreak strain, intranasally. The control pigs received 3 mL of saline. Results: After inoculation, SEZ was isolated from URT of 3 pigs from G3 and from one pig from G2. Control pigs tested negative throughout the study. All 4 pigs from G3, and 2 pigs (50%) from G2 were euthanized due to severe clinical signs at 1 and/or 2 DPI. The remaining two challenged pigs from G2 also showed clinical signs at 8 DPI and were euthanized. Overall, clinical signs included high fever (>105°F), coughing, nasal secretion, dyspnea and vomiting. Even though the disease manifested more acutely in pigs from G3, all pigs had lesions, including marked cranioventral lung consolidation, mild splenomegaly, mild hepatic congestion, and enlarged tracheobronchial lymph nodes. SEZ was isolated from 7 out of 8 inoculated pigs from blood samples and tissues, such as lung, heart, liver, spleen, and submandibular lymph. Besides SEZ, lungs samples were positive for S. suis (4 pigs) and Glaesserella parasuis (2 pigs). Summary: This new challenge model using the highly virulent SEZ swine strain to infect conventional 6-week-old pigs mimicked the rapid onset of clinical signs and high mortality of sows and feeder pigs observed during the fall of 2019. Therefore, this model will be extremely useful for future studies of the pathogenicity of SEZ in swine, as well as a model for testing control measures and diagnostic tools. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 62 AAVLD Virtual Conference Bisgaard Taxon 40 associated with high mortality in seabirds: characterization and comparative analyses of virulence factors and antimicrobial resistance. # Eliana De Luca1, Grazieli Maboni1, Rodrigo Baptista2, Kevin Niedringhaus3,4, Nicole Nemeth3, Susan Sanchez1 1Athens Veterinary DIagnostic Laboratory, University of Georgia, Athens, GA; 2Institute of Bioinformatics, University of Georgia, Athens, GA; 3Southeastern Cooperative Wildlife Disease Study, Depts. of Pathology and Population Health, University of Georgia, Athens, GA; 4Department of Pathology, Microbiology and Immunology, University of California-Davis, Davis, CA

The Pasteurellaceae family is often associated with fatal infectious diseases in birds. [WU1] Several new species and taxa within this family have been reported in avian hosts, however their epidemiology and the pathogenic role are not completely understood. Here, we describe the phenotypic and genomic characterization of Bisgaard taxon 40 (Pasteurellaceae), which was implicated in a mass mortality event affecting terns (Thalasseus sandvicensis and Sterna hirundo) in Florida, USA. The isolates were biochemically characterized, and 16S rRNA gene sequence comparison with representative Pasteurellaceae species was performed. Furthermore, whole genome sequencing was performed by MiniOn Nanopore and Illumina sequencing platforms. To achieve the whole genome of this isolate, sequence reads obtained by MinION were coupled with those originating from the Illumina platform employing a hybrid assembly approach. VFDB database was used to identify the virulence factors. Antimicrobial susceptibility test was performed by broth microdilution with Trek Sensititre. Since there are no guidelines to define MICs for Bisgaard taxon 40, interpretative criteria derived from Pasteurella multocida were employed. Growth on blood agar showed β-hemolytic, shiny, circular, whitish-cream colonies, with catalase and oxidase positive reactions. Urease and indole tests were negative. Acid was formed from D-glucose, D-mannose, D-maltose, D-fructose, D-galactose, D-cellobiose. A phylogenetic analysis of the complete 16S rRNA gene sequence placed the organism on a distinct branch within the Pasteurellaceae family, alongside Pasteurella testudinis, and Bisgaard taxon 14 and 32, which were previously associated with disease in birds. The nucleotide identity of our strain and above Pasteurellaceae members ranged between 90.8% and 92.5%. Antimicrobial resistance was detected for amoxicillin-clavulanate, chloramphenicol and tetracycline. Major bacterial virulence genes were identified in this strain, including adhesins, capsule, hemolysin β, heat shock protein, immunoglobin A1 protease, and type IV pilus protein. This is the first whole genome sequence reported for this microorganism. The role of this bacterium in the event mortality as a primary or opportunistic pathogen remains unclear. Comparative genomic analyses with other pathogens belonging to the Pasteurellaceae family are the object of current investigations. [WU1]That is are already well known, so we can save some words # AAVLD Trainee Awardee

AAVLD Annual Conference Proceedings 63 AAVLD Virtual Conference Genomic islands analysis in Klebsiella pneumoniae associated with bovine mastitis # * † Zhiyi Zheng, Patrick J. Gorden, Ganwu Li VDL (Veterinary Diagnostic Lab), Iowa State University, Ames, IA

Genomic islands (GIs) are gene clusters in bacterial genome that appear to be obtained by horizontal gene transfer, granting bacteria extra phenotype features to either better occupy a new niche or to be a pathogen. GIs or specifically pathogenicity islands (PAIs) are widely present in the genomes of many bacterial pathogens including mastitis E. coli. However, research on GI has rarely been done in Klebsiella pneumoniae (Kp), one of the most common bacterial pathogens causing the costly disease bovine mastitis in dairy industries worldwide. In the previous study, we performed a bacterial genomic epidemiological study with 81 strains of mastitis Kp. To better explore the distribution and function of GIs in mastitis Kp strains, 8 bovine sourced Kp strain (7 strains from bovine mastitis milk and 1 from retail meat) genomes were assembled into complete circular chromosomes and putative GIs were identified by using a combination of several methods. As our result presents, each mastitis strain contained 25-27 GI-like regions (mean = 26, SD = 0.81), while the meat strain contained up to 35. A GI-like region pool was constructed based on seven mastitis strain chromosomes, 63 GI-like regions were predicted as putative GIs, in which 46 were finally defined as GIs following a length cutoff at 8Kbp or at least 8 genes. These 46 GIs were then aligned with 81 previous bovine mastitis strains, 30 publicly available human pathogenic Kp strains, and 31 dairy farms environmental Kp strains. Comparison outputs revealed 6 GIs with relatively higher prevalence in mastitis strains, with either metabolic, fitness, symbiosis or putative virulence gene clusters harbored. Notably, One GI was found with putative pyocin coding genes, which might be one of the virulence factors in assisting Kp strains to compete with other bacterial pathogens. Several other GIs were further identified with complete gene clusters encoding carbohydrate-specific enzyme II (EIIs), which are essential components of bacterial phosphotransferase system functioning in obtaining carbohydrates to adapt different environment. Besides, genes associated with type VI secretion system as well as phages were also identified within some GIs. Moreover, some other GIs were found either prevalent in human/environmental strains or in all three sourced Kp groups rather than only prevalent in mastitis strains. The phenotypic and functional assays of selected GIs are under conducting in our lab currently. As a widely existing environmental bacteria, Kp is active in communication at genetic level within and beyond species. GIs as parts of mobile acquired genes possibly play a crucial role in forging Kp into a bovine mastitis pathogen. We hope our current and future studies will lead to a better understanding of epidemiology and pathogenicity of virulent Kp strains causing bovine mastitis. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 64 AAVLD Virtual Conference Pathology Live Q&A Thursday, October 15, 2020 Pahtology Live Q&A

Moderators: Kevin R. Snekvik and Keith L. Bailey

5:10 PM Veterinary diagnostic laboratory design: challenges and solutions Alexander Penn Clinton ...... 67

5:15 PM Insights into pathogenesis and treatment for SARS-CoV-2 infection in small animal models at Colorado State University ◊ Tawfik A. Aboellail, Anna Fagre, Savannah Rocha, Ron Tjalkens, Tony Schountz...... 68

5:20 PM The benefits, considerations, and outcome of transitioning from traditional light microscopy to digital pathology in a diagnostic laboratory Maritza Anguiano, Alexis Templeton, Erin Edwards, Gabriel Gomez, Jay Hoffman...... 69

5:25 PM Novel approach of total allowable error determination for quantitative laboratory test validation and performance: example of an immunoassay (Immulite 2000 Xpi) for serum and urine cortisol in dogs Jeremie KORCHIA, Kathleen Freeman ...... 70

5:30 PM Diagnosis of rabbit hemorrhagic disease virus 2-related disease in southern California, May 2020 Javier Asin, Akinyi C. Nyaoke, Janet D. Moore, Viviana Gonzalez-Astudillo, Deana Clifford, Beate Crossley, Francisco A. Uzal...... 71

5:35 PM Tissue distribution and pathology of Rabbit Hemorrhagic Disease Virus-2 with in-situ hybridization via RNAScope # † Alicia Dawn O’Toole, Jian Zhang, Laura Artz Williams, Fawzi Mohamed, Corrie Brown...... 72

5:40 PM Encephalitis associated with avian reovirus in commercial broilers in Mississippi # Tzushan Sharon Yang, Brittany Baughman, Natalie Armour, Martha Pulido-Landinez, Heidi Huffman Rose, Alejandro Banda...... 73

5:45 PM Infectious bronchitis virus prevalence, characterization and strain identification in California backyard chickens # + * † Omar Antonio Gonzales Viera, Emily R. Blair, Rodrigo Gallardo, Ozge Erdogan-Bamac, Alejandra Figueroa, Daniel Rejmanek, Beate Crossley, Asli Mete ...... 74

5:50 PM Bovine mammary gland involution measured by histology and morphometry following intramammary administration of casein hydrolysate together or compared with other treatments at dry-off David J. Wilson, Justine E. Britten, Chad Clancy, Kerry A. Rood...... 75

5:55 PM The comparative pathology of enterocolitis in horses Francisco A. Uzal, Fabio Mendonca, Mauricio Navarro...... 76

6:00 PM Tyzzer’s disease in horses: a retrospective analysis # + * † Mauricio Navarro, Juan Garcia, Karina Fresneda, Francisco Uzal...... 77

6:05 PM A suspected case of copper toxicosis in a warmblood foal Scott D. Fitzgerald, Leah Stein, John Philip Buchweitz...... 78

AAVLD Annual Conference Proceedings 65 AAVLD Virtual Conference 6:10 PM Pyogranulomatous dermatitis in a dog caused by Paralagenidium karlingii: a case report # + * † Jessica Elbert, Dayle McClintock, Frane Banovic, Michaela G. Austel, Kaori Sakamoto ...... 79

6:15 PM Fatal fly bait toxicity in a young dog + Mayra Frances Tsoi, John Philip Buchweitz, Dalen W. Agnew...... 80

6:20 PM Intravascular large B-cell lymphoma in a cat + * † Andeliene Vienna Croce, Luke Borst, Crystal Lindaberry, Keith Linder...... 81

6:25 PM Diagnostic trends of five swine endemic bacterial pathogens using data from the Iowa State University Veterinary Diagnostic Laboratory (2010-2019) # * † ◊ Ana Paula Poeta Silva, Kent Schwartz, Bailey Arruda, Jessica Goncalves dos Santos, Nubia Macedo, Orhan Sahin, Eric Burrough, Karen M. Harmon, Christopher Siepker, Phillip Gauger, Panchan Sitthicharoenchai, Michael Rahe, Drew Magstadt, Alyona Michael, Pablo Pineyro, Rachel Derscheid, Rodger Main, Eduardo Fano, Maria Jose Clavijo...... 82

6:30 PM Streptococcus gallolyticus detected in cases of swine infectious endocarditis from 2016-2020 Panchan Sitthicharoenchai, Michael Rahe, Eric Burrough...... 83

6:35 PM Yersiniosis in a blackbuck antelope doe: case report and disease review # + * † Jillian Marie Athey, Keith L. Bailey, Kristina Ellis, Chien-Che Hung, Amy K. Stevenson, Paul Marx...... 84

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 66 AAVLD Virtual Conference Veterinary diagnostic laboratory design: challenges and solutions Alexander Penn Clinton Perkins&Will, Houston, TX

There is a generation of numerous aging veterinary diagnostic laboratories across the country that have been ear- marked for renovation/replacement or will require it in the near future. Proper planning/design of these facilites for managing biosecurity /biorisk /biosafety and increased workflow efficiency will ensure the future safety of livestock populations and the humans that work/interact with them. With proper careful consideration in the planning of a new facility, modern Veterinary Diagnostic Laboratories should be able to address and resolve the biorisk/biosecurity/ and biosafety challenges of past generation buildings while also providing a more efficient workflow for quicker turnaround of results to their clients and quicker identification of potential disease risks to the livestock population. This presentation will focus on real-world examples of the integration of design and planning best-practices into modern Veterinary Diagnostic Laboratories (VDL’s) to address the shortcomings of previous generation laboratories and enhance the design of future facilities for best possible efficiency, security, and safety. Case studies will be used to discuss the common design pitfalls of existing/aged VDL’s and the design strategies of 3-4 recently-designed/ built VDL’s to overcome them, including: - The Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL), College Station, Texas - The Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL), Canyon, Texas - Washington Animal Disease Diagnostic Laboratory (WADDL), Pullman, Washington - Iowa State University Veterinary Diagnostic Laboratory (ISU VDL), Ames, Iowa Pros and cons of different design strategies will be discussed, both at a holistic building level, and more detailed operational level. Issues to be discussed may include: - Managing Biosecurity/Biosafety through building circulation strategies. - Increased Workflow Efficiency through efficient planning strategies. - Improved client experience and interface with the VDL. - Increased worker comfort throughout the building, including the labs. - Fostering increased staff interaction and collaboration. - Workplace strategies for officing of staff. - Providing facility adaptability for the future. - Consumable and sample storage approaches. - Inclusion of BSL-3 space, key factors to be considered, and facility integration. - Necropsy design and animal/carcass handling/storage. - Carcass disposal methodologies and results of different approaches. - Design and operational lessons learned from both existing and newer facilities. At the end of this presentation the attendees will have an understanding of different design approaches for new facilities that can resolve the operational challenges faced daily in older facilities, and how best to evaluate their use based on the specific needs of their institution.

AAVLD Annual Conference Proceedings 67 AAVLD Virtual Conference Insights into pathogenesis and treatment for SARS-CoV-2 infection in small animal models at Colorado State University ◊ Tawfik A. Aboellail1, Anna Fagre1, Savannah Rocha2, Ron Tjalkens2, Tony Schountz1 1Microbiology, Immuology and Pathology, Colorado State University, Fort Collins, CO; 2Cell and Molecular Biology, Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

With severe consequences to human health and search for a suitable animal model, researchers at Colorado State University developed several small animal models for the novel severe acute respiratory syndrome coronavirus 2, SARS-CoV-2 infection (Covid-19). Jamaican fruit bats (15 animals), deer mice (15) and Syrian hamsters (20)

were inoculated with 2x104 TCID50 of SARS-CoV-2 (Washington State isolate 2019-nCoV/USA-WA1-F6/2020). Animals were humanley euthaniized at 3-, 6-, and 14 days post infection (dpi) for bats and deer mice and at 6 dpi for the hamsters. In the deer mice animal model, histological examination of hemiskulls revealed that in early stage of the disease at 3 dpi, the virus propagated to the brain in a retrograde axonal transmission along olfactory and trigeminal pathways. Disruption to the blood brain barrier (BBB) followed at 6 dpi resulting in mild suppurative inflammation that mainly manifested in the cortex of the frontal lobe with status spongiosus at the area of lateral sulcus nucleus in the brain stem and other nuclei at the thalamus and hypothalamus. Researchers and clinicians should be aware of a wide spectrum of neuropathologies that could precipitate clinical manifestations of parasympathetic and sympathetic roots such as epiphora, trigeminal neuralgia, confusion, and more consistently hyposmia/anosmia and hypogeusia/ageusia. In Syrian hamsters, the virus replicated efficiently in the lungs precipitating severe histioneutrophilic bronchointerstitial pneumonia mimicking human disease. Highly potent fully human neutralizing antibody clone (AvGn-B), constructed from yeast display libraries against SARS-CoV-2, significantly reduced viral loads in infected lungs when treated at 1- and 3- dpi with a low dose of 1mg/hamster and a high dose of 2.5mg/hamster. At the high dose, the antibody conferred more protection diminishing macrophage infiltration into lungs of infected hamsters. Deer mice and Syrian hamsters emerged as useful models to understand the complex relationship between neuropathology and immune system of Covid-19 patients and for evaluation of vaccines, immunotherapies and antiviral compounds. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 68 AAVLD Virtual Conference The benefits, considerations, and outcome of transitioning from traditional light microscopy to digital pathology in a diagnostic laboratory Maritza Anguiano, Alexis Templeton, Erin Edwards, Gabriel Gomez, Jay Hoffman Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX

Digital pathology is transforming and revolutionizing the field of modern pathology. In a diagnostic setting, digital slide scanners have the potential to enhance laboratory workflow processes, save costs and efforts through consolidation and centralization of histology laboratories, and improve output and distribution of slides to pathologists with ultimate benefit to the client. Successful integration of a digital slide scanner into a diagnostic laboratory requires careful planning, preparations, and adaptations. After many considerations, the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) recently purchased and successfully integrated a digital slide scanner into our routine histopathology workflow. The TVMDL system consists of four laboratory sites that are geographically separate and employs 12 anatomic pathologists. With the acquisition of a digital scanner, we were able to consolidate tissue processing/histology to the main laboratory site (College Station, TX), where we currently process approximately 320 histology slides per day, including routine H&E and special stains. Our samples in histopathology vary significantly in size and type of tissue. Based on our initial research, there were several aspects of digital pathology and scanning systems that we needed to consider. The primary concerns for the digital scanner included slide capacity, scan time, magnification, and image quality. Additionally, there were obstacles within the laboratory that needed to be addressed, such as equipment and hardware upgrades for slide labeling, coverslipping, computer workstations, and archival of digital files. Finally, we had to consider the training and management of laboratory personnel to accommodate the additional steps of scanning and slide delivery to the pathologists. The complete integration of digital pathology at TVMDL took about six months. Following the purchase of a digital slide scanner, we made several upgrades to other lab equipment, and we modified processing schedules. We coordinated with the IT departments within our agency and university system to solve online storage and distribution issues. All lab personnel received training in scanning slides, and all pathologists verified and trained in the imaging software. Today, we have revised our workflow to ensure that we are still meeting the same turnaround time as with traditional light microscopy. In this presentation, we will discuss the steps and considerations to be taken when purchasing a scanner and the impacts digital pathology had on our entire processing workflow to provide more information to other laboratories considering digital pathology.

AAVLD Annual Conference Proceedings 69 AAVLD Virtual Conference Novel approach of total allowable error determination for quantitative laboratory test validation and performance: example of an immunoassay (Immulite 2000 Xpi) for serum and urine cortisol in dogs Jeremie KORCHIA1, Kathleen Freeman2 1Clinical Pathology, TVMDL, Bryan, TX; 2Clinical Pathology, SYNLAB-VPG, Exeter, United Kingdom

Total error goals can be used as a tool in the process of assay validation. The TEa goal represents the amount of allowable error to assure sufficient quality for the intended use of the results by setting a limit for the combined imprecision and bias, that is tolerable in a single measurement to ensure clinical usefulness. When no guidelines for TEa exist for a given measurand, the observed total error (TEo), calculated from the imprecision and bias observed in validation studies, can be used as starting point to develop guidelines for TEa since TEo provides an estimate of the technically achievable performance. Veterinary endocrinology expert guidelines for TEa are currently lacking. Our objective was to use the information obtained from the validation of cortisol in canine serum and urine to establish recommendations for TEa for serum and urine cortisol. In addition to using the traditional approach of determining the sigma metric and QC rules from a predetermined TEa, we reversed the approach and determined the TEa from the QC validation with conditions of high error detection and low false rejection. In spiked serum, imprecision increased exponentially, from ≈ 2% to 20%, with the decreasing cortisol concentration, whereas the spiking-recovery bias was minimal across the reportable range (< 7%). The resulting TEo increased, from ≈ 10% to 43%, with decreasing cortisol concentration. In contrast, the control materials (QCMs) had TEo of 8.5% and 14.5%. In spiked urine, imprecision increased exponentially at the low end of the decreasing cortisol concentration, remaining < 3% above 2.5 μg/dL, to ≈ 7% to 30% at lower concentrations. The spiking-recovery bias increased exponentially, from ≈ 3% to 70% with the decreasing cortisol concentration. The resulting TEo varied from ≈ 7% to 100%, with decreasing cortisol concentration. The QCMs TEo was 20% (both levels). In both serum and urine, TEo at 1.4 μg/dL was ≈30% (0.98 – 1.82 μg/dL) and TEo at 20 μg/dL was ≈20% (16 – 24 μg/dL); serum cortisol interpretation is improved by the consideration TEo at the corresponding thresholds (1.4 and 20 μg/dL). The reverse-approach for determination of TEa showed that the observed performance is controllable with a simple

rule (1.2.5S) and 2 levels of control material at TEa of 20-35% for serum and 24-35% for urine. Use of a multirule (13S/22S/R4S or 13S/22S/R4S/41s and larger numbers of control materials (n = 2-6) allow statistical QC to be applied at a lower TEa with a high Ped and low Pfr, but was not considered practically implementable in a veterinary commercial laboratory. Sigma metrics for serum and urine cortisol at the TEa goals determined using the reverse approach ranged from 4.8 to 5.09.

AAVLD Annual Conference Proceedings 70 AAVLD Virtual Conference Diagnosis of rabbit hemorrhagic disease virus 2-related disease in southern California, May 2020 Javier Asin1, Akinyi C. Nyaoke1, Janet D. Moore1, Viviana Gonzalez-Astudillo1, Deana Clifford2, Beate Crossley3, Francisco A. Uzal1 1California Animal Health and Food Safety Laboratory, San Bernardino Branch, University of California, Davis, San Bernardino, CA; 2Wildlife Investigations Lab, California Department of Fish and Wildlife, Rancho Cordoba, CA; 3California Animal Health and Food Safety Laboratory, Davis Branch, University of California, Davis, Davis, CA

Rabbit hemorrhagic disease (RHD) is a highly contagious, frequently fatal disease of rabbits caused by a single- stranded RNA virus of the genus Lagovirus, family Caliciviridae. This disease was classically associated with rabbit hemorrhagic disease virus (RHDV), which affects mostly wild and domestic European rabbits (Oryctolagus cuniculus), causing a peracute, fulminant, necrotizing hepatitis with secondary disseminated intravascular coagulation and multisystemic hemorrhages. In 2010, a new variant of this virus, called rabbit hemorrhagic disease virus 2 (RHDV2; also known as RHDVb or RHDV2/b), appeared in France and rapidly spread to other countries, frequently replacing the classic RHDV variants that were circulating in those locations. RHDV2-related disease has a similar clinic-pathologic presentation to classic RHD, except that it has a slightly longer incubation period and clinical course, and a much broader host range, which includes, in addition to European rabbits, several hare species (Lepus spp.), cottontail rabbits (Sylvilagus spp.), and possibly other lagomorphs. An outbreak of RHDV2- related disease is currently occurring in southwest United States since March 2020, with cases confirmed in New Mexico, Arizona, Texas, Colorado, Nevada and, most recently, California. On May 7, 2020 a pregnant, female black-tailed jackrabbit (Lepus californicus) was submitted by the California Department of Fish and Wildlife to the San Bernardino lab of the California Animal Health and Food Safety Laboratory System. The animal was found dead in an area where 10-20 wild rabbit carcasses had appeared in the previous days. Necropsy revealed congested and hemorrhagic nasal turbinates and lungs, mild reticular pattern in the liver, and hemorrhages in the uterine serosa. Histologically, there was periportal to massive hepatic necrosis, perifollicular splenic necrosis, and microthrombosis of pulmonary alveolar and renal glomerular capillaries. A presumptive diagnosis of RHD was made based on gross and microscopic lesions. The Foreign Animal Disease Diagnostic Laboratory at Plum Island confirmed the presence of RHDV2 by RT-PCR in liver of this animal. Since then, and as of June 14, 2020 (current date), additional submissions of wild and domestic lagomorphs have been processed, and RHDV2 has been detected in two additional black-tailed jackrabbits and one desert cottontail (Sylvilagus audubonii). The gross lesions in the liver may be very subtle and not indicative of the profound hepatic necrosis observed histologically. This is the first diagnosis of RHDV2-related disease in California and it is an evolving situation, thus more information about the disease, affected species, and epidemiology will be available for the AAVLD meeting next fall.

AAVLD Annual Conference Proceedings 71 AAVLD Virtual Conference Tissue distribution and pathology of Rabbit Hemorrhagic Disease Virus-2 with in-situ hybridization via RNAScope # † Alicia Dawn O’Toole1, Jian Zhang3, Laura Artz Williams4, Fawzi Mohamed2, Corrie Brown3 1Comparative Biomedical Sciences, University of Georgia-Athens, Statham, GA; 2Foreign Animal Disease Diagnostic Lab, USDA-APHIS, Plum Island, NY; 3College of Veterinary Medicine, Department of Pathology, University of Georgia-Athens, Athens, GA; 4Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA

Rabbit Hemorrhagic Disease Virus-2 (RHDV-2) is an emerging pathogen of both domestic and wild rabbits in the United States. Isolated reports in domestic rabbits began in September 2018, but it was not until March 2020 that natural infections were reported in both black-tailed jackrabbits (Lepus californicus) and desert cottontail rabbits (Sylvilagus audobonii) in New Mexico. Since then, positive cases have been detected in Arizona, California, Colorado, Nevada, and Texas. The tissue distribution and pathology of this virus have been described utilizing conventional hematoxylin and eosin staining as well as immunohistochemistry. As with Rabbit Hemorrhagic Disease Virus-1 (RHDV-1), RHDV-2 is hepatotropic, causing massive hepatic necrosis, loss of clotting factors, hemorrhage, and disseminated intravascular coagulation. Lymphoid depletion of the spleen, Peyer’s patches, sacculus rotundus, ceca, mesenteric lymph nodes, and thymus as well as splenic necrosis within red pulp have also been described. Immunohistochemical staining has identified viral antigen within hepatocytes as well as macrophages of the liver, spleen, alveoli, kidneys, and small intestine, however there are many limitations with this method, including high background staining and poor sensitivity, which may not accurately represent viral activity. New in-situ hybridization techniques for RNA viruses are proving effective tools not only for locating virus, but for quantification of viral load within various tissues. RNAScope is a method of in-situ hybridization that utilizes a “double-z” probe with extremely high specificity that allows for considerable amplification of detected RNA. Formalin-fixed, paraffin-embedded tissues from multiple outbreaks in domestic rabbits in Washington State, as well as experimental infections of Eastern Cottontail rabbits from the Foreign Animal Disease Diagnostic Laboratory (Plum Island, NY) are currently being processed an analyzed via RNAScope in situ hybridization, and have yielded highly specific staining with little to no background. This technique may better characterize viral pathogenesis while comparing subtle differences across species without the need for special preservation techniques, and with the added benefit of combination-techniques (dual-channel staining, combination with immunohistochemistry) to elucidate the immune response and fine-tune vaccine strategies. # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 72 AAVLD Virtual Conference Encephalitis associated with avian reovirus in commercial broilers in Mississippi # Tzushan Sharon Yang1, Brittany Baughman3, Natalie Armour2, Martha Pulido-Landinez2, Heidi Huffman Rose4, Alejandro Banda2 1North Carolina State University, Raleigh, NC; 2Poultry Research and Diagnostic Laboratory, Mississippi State University, Pearl, MS; 3College of Veterinary Medicine, Mississippi State University, Starkville, MS; 4Mississippi Veterinary Research and Diagnostic Laboratory, Mississippi State University, Pearl, MS

Avian reovirus is an important pathogen within the commercial poultry industry, and is often associated with significant economic impact, especially in broilers. Typical lesions caused by the virus include tenosynovitis/ arthritis, myocarditis, multiorgan lymphoid depletion, and enteritis. Neurologic disease linked to reovirus is rarely reported with natural infection in chickens. This report describes histologic brain lesions associated with reovirus infection in commercial broiler chickens in Mississippi. Affected birds ranged from 27-40 days old, and exhibited severe neurologic signs including torticollis and head tremors. Necropsy confirmed severe inflammation in the brain in 26 birds submitted from affected houses. The histologic lesions were most severe in the brainstem and cerebellar white matter, characterized by robust mononuclear perivascular cuffs, neuronal degeneration and necrosis, sometimes accompanied by microglial nodules, and neuropil vacuolation with axonal degeneration. Similar lesions were found occasionally in the cerebral cortex and spinal cord. Affected birds often had concurrent lesions characteristic for avian reovirus, such as lymphonodular tenosynovitis, pericarditis or myocarditis, and widespread lymphoid aggregates in multiple organs. Fresh brain tissues were PCR positive for avian reovirus and reovirus was isolated from pooled brain samples. Other major causes for neurologic disease in chickens were ruled out based on further diagnostic testing. While the histologic findings from these cases can mimic other common viral encephalitides in chickens, the lesion distribution and inflammatory pattern appeared to be consistent within our cases. These findings suggest that avian reovirus should be considered as a possible cause for chickens with similar neurologic presentations and histologic features. # AAVLD Trainee Awardee

AAVLD Annual Conference Proceedings 73 AAVLD Virtual Conference Infectious bronchitis virus prevalence, characterization and strain identification in California backyard chickens # + * † Omar Antonio Gonzales Viera1, Emily R. Blair2, Rodrigo Gallardo3, Ozge Erdogan-Bamac4, Alejandra Figueroa3, Daniel Rejmanek5, Beate Crossley5, Asli Mete1 1Anatomic Pathology, California Animal Health adn Food Safety, Davis, CA; 2College of Agriculture and Environmental Sciences, University of California, Davis, Davis, CA; 3Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, Davis, CA; 4DIstanbul University- Cerrahpasa Faculty of Veterinary Medicine, Department of Pathology, Istanbul, Turkey, Davis, CA; 5California Animal Health and Food Safety Laboratory System, Davis, Davis, CA

Infectious bronchitis virus (IBV) is a highly contagious gamma coronavirus that has a detrimental impact on the poultry industry around the world. Industrial systems use vaccination strategies to mitigate the problems, but with variable success. In California, IBV studies have focused on commercial broilers and layers, neglecting the increased detection of IBV in backyard flocks (BYF). Herein, we investigated the prevalence and characterization of IBV in every backyard chicken submitted for necropsy to the Northern (Davis) and Southern (San Bernardino) branches of the California Animal Health and Food Safety Laboratory System from January through March of 2019. All case reports were analyzed for a) demographic and geographic data and cause(s) of mortality, b) presence of IBV in the trachea, kidney and cecal tonsils by qRT-PCR and immunohistochemistry (IHC), c) histologic lesions in the trachea and kidneys, and d) sequence data and strain identification of positive cases. A chicken was considered positive when at least one of the tissues was positive by qRT-PCR (Ct<35). A total of 192 chickens were submitted to both laboratories and the birds came from all over the state. There was no age or breed prevalence, and the vast majority of the submissions are females. Samples for qRT-PCR were available from 171 chickens and 30.4% (n=52) were positive for IBV. In the Davis and San Bernardino laboratories, the percentages were 16.9% (21/124) and 65.9% (31/47), respectively. Cecal tonsil tissue was the tissue with the highest viral load and percentage of IBV detection (n=47) in comparison with the kidney (n=19) and trachea (n=8). This denotes the chronicity of infection in BYF. The positive PCR cases correlated well with the IHC results and histopathology revealed that IBV was clinically significant in at least 10 birds with respiratory and/or renal lesions. From the sequence analysis of 22 IBV isolates, 14 cases were most similar to CA1737 (91% - 96% identity), one bird harbored an IBV strain with 99.9% similarity to Conn 46, two were most similar to Cal99 and ArkDPI with 89% identity, respectively. Since vaccination is not a common practice in backyard chickens, the above identified strains might represent either exposure to a circulating vaccine virus or a field challenge. Five birds had strains that did not have substantial matches to any of the reference strains. Those strains showed the closest relationship to Cal557 (81% - 84% identity), which suggests that IBV can evolve independently in BYF. Finally, the backyard chickens might collaterally be infected from the live attenuated vaccines administered in commercial poultry. Concurrently, they might represent a source of virulent IBV strains to naïve commercial poultry farms in California. # AAVLD Trainee Awardee + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 74 AAVLD Virtual Conference Bovine mammary gland involution measured by histology and morphometry following intramammary administration of casein hydrolysate together or compared with other treatments at dry-off David J. Wilson1, Justine E. Britten2, Chad Clancy3, Kerry A. Rood1 1Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT; 2Udder Health Systems, Layton, UT; 3National Institutes of Health Rocky Mountain Laboratories, Hamilton, MT

The dry (non-lactating) period between the end of one lactation and the next calving, and administration of dry cow treatment (DCT) at dry-off are established management practices in the dairy industry. Because of potential drug residues in milk and possible antimicrobial resistance in mastitis pathogens there is current interest in alternatives to antibiotic DCT. The study objective was measurement and comparison of quantitative histological changes during bovine mammary involution between intramammary infusion treatments administered at dry-off. Casein hydrolysate (CH) alone or with standard DCT and/or with an internal teat sealant (TS) were evaluated. A split udder design was used; one udder half (front and rear quarters) of each cow received one treatment and the contralateral udder half was administered DCT + TS as a control. Treatment groups were: CH, CH + DCT, CH + TS and CH+DCT+TS. Cows (n = 16) were non-pregnant, ≥ 300 days in milk with 4 normally lactating quarters, were randomized to be euthanized at 2 days dry (d 2) or 7 days dry (d 7), and then randomized to treatments (n = 4 per treatment group, n = 16 controls) within assigned day of euthanasia. Humane euthanasia in compliance with an IACUC protocol was performed at the Utah Veterinary Diagnostic Laboratory. Tissue samples were collected from 3 anatomic regions of each quarter: near the ventral body wall (dorsal [D]), immediately above the teat canal (ventral [V]), and vertically halfway between V and D (mid [M]). Ten photographs per histological section were made with a microscope lens camera (200X) and interalveolar stroma thickness, epithelial cell height and alveolar lumen diameter were measured using cellSens® software. A generalized linear mixed model tested for significant differences between treatment groups including from control udder halves within each cow, and within time and anatomic region, in the above 3 indicators of mammary involution; comparisons among individual treatment means used Tukey’s test. Alveolar lumen diameters within region V were smaller at d 2 and d 7 in udder halves within all 4 CH treatment groups compared to Control udder halves (P < 0.001). At d 2, thickness of interstitial stroma in all anatomic regions was greater in udder halves within all 4 CH treatment groups compared to controls (P < 0.01). Intramammary administration of CH, or any treatment that included CH, at dry-off was associated with earlier histological indications of mammary involution than control treatment. These results suggest that intramammary casein hydrolysate alone or in combination with antibiotic dry treatment or internal teat sealant may result in faster mammary involution early in the dry period.

AAVLD Annual Conference Proceedings 75 AAVLD Virtual Conference The comparative pathology of enterocolitis in horses Francisco A. Uzal1, Fabio Mendonca2, Mauricio Navarro1 1UCDavis, San Bernadino, CA; 2Federal Rural University of Pernambuco, Recife, Brazil

The etiologic diagnosis of equine enterocolitis may be challenging and relies on gross and microscopic lesions coupled with several ancillary tests. Gross and microscopic lesions of the alimentary tract of horses with enterocolitis may be very similar to each other, regardless of the etiology involved. This study was performed to determine if there are significant differences in the gross and microscopic pathology of enterocolitis produced by five of the most prevalent causes of this condition in horses, in the hope that a detailed gross and microscopic examination of tissues might help in establishing the etiology. Ninety cases of equine enterocolitis submitted to the San Bernardino laboratory of the California Animal health and Food safety laboratory were studied and their microscopic lesions compared. These included cases due to Clostridium perfringens type C (CP; n=20), Clostridioides difficile (CD; n=20), Paeniclostridium sordellii (PS; n=15), Salmonella enterica subspecies enterica serovar Typhimurium (ST; n=20) and NSAIDs intoxication (NS: n=15). Grossly, necrotizing hemorrhagic typhlocolitis was most frequently seen in cases of ST, CD and NS disease. Cases of CPC and PS presented with enteritis or colitis in similar percentages. Congestion, hemorrhage and pleocellular inflammatory infiltrate (88, 98%) followed by mucosal and submucosal necrosis (77, 85.5%) were the main microscopic lesions found in horses with enteritis or colitis produced by any of the etiological agents investigated. The necrosis was statistically more severe in cases of CPC than in cases associated with any of the other four etiologies. Ulceration and intestinal pseudomembranes were observed with similar prevalence in the small intestine and colon affected by all agents studied. Thrombosis of the lamina propria and/or submucosa was observed in approximately half of the cases of enteritis and colitis by all etiologies. Other microscopic findings that were observed in variable prevalence but with no differences between etiology, included mucosal and submucosal edema, presence of gram positive rods, crypt necrosis, dilated lymphatics, lymphoid tissue necrosis and serosal hyperemia and hemorrhage. The results of this study suggest that gross and microscopic lesions are not specific enough to establish a diagnosis of enterocolitis by any of the etiological agents included in this study.

AAVLD Annual Conference Proceedings 76 AAVLD Virtual Conference Tyzzer’s disease in horses: a retrospective analysis # + * † Mauricio Navarro1, Juan Garcia2, Karina Fresneda1, Francisco Uzal1 1University of California, Davis, San Bernardino, CA; 2INTA Balcarce, Balcarce, Argentina

Tyzzer’s disease (TD) is caused by Clostridium piliforme, a Gram-negative and obligate intracellular bacterium. The disease occurs in multiple species, but it is more frequent in laboratory animals, lagomorphs and foals. It is believed that the mode of transmission is fecal-oral, with ingestion of spores from contaminated environments. C. piliforme proliferates in the intestinal mucosa, and then disseminates to the liver and other organs. Virulence factors for this microorganism are unknown. It is commonly acknowledged that TD develops a triad of lesions, i.e. colitis, hepatitis and myocarditis, in laboratory animals species. However, this seems to be inconsistent in equine cases. Information about the distribution of lesions in horses with TD is scant and to the best of our knowledge, there are no detailed descriptions of the distribution of C. piliforme in different tissues of horses with this disease. For the present study, 26 equine cases with a diagnosis of TD were retrieved from the database of the California Animal Health and Food Safety Laboratory System. This retrospective analysis included information regarding clinical history, macroscopic and microscopic changes. In addition, for this study PCR was performed on formalin-fixed, paraffin-embedded (FFPE) tissues, targeting the 16S rRNA gene was performed. Twenty five out of 26 (96%) of the cases occurred in foals less than 45-days old, with 14/26 (54%) being foals between 15 and 30 days of age. Only 1 foal was older than 45 days old (90 days). There were equal numbers of females and males. The breeds involved were Quarter (57%), Thoroughbred (26%), Arabian (2%), Paint (1%) and Hanover (1%). Most of the cases occurred during spring (77%). The foals were found dead, with no clinical signs being observed in 11/26 cases (42%). The remaining 15/26 animals (58%) presented fever, weakness, lack of nursing, depression and/or diarrhea. Gross findings included icterus, ascites, hepatomegaly with acinar pattern, serosal hemorrhages and congestion, pulmonary edema, splenomegaly and/or fluid small and large intestinal content. Microscopically, all foals had severe, multifocal, necrotizing hepatitis with intra-hepatocytic, Gram-negative and Steiner-positive filamentous bacilli. Multifocal necrotizing myocarditis was found in 7/26 (27%) foals, whereas necrotizing colitis was observed in 9/26 (35%). These bacteria were also observed in the cytoplasm of enterocytes and myocardiocytes in 55 and 9% of the cases, respectively. PCR on FFPE tissues detected C. piliforme DNA in the liver (100%), colon (64%) and heart (18%). In conclusion, this retrospective analysis demonstrated that the so called triad of lesions is not a characteristic of TD in horses. # AAVLD Trainee Awardee + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 77 AAVLD Virtual Conference A suspected case of copper toxicosis in a warmblood foal Scott D. Fitzgerald, Leah Stein, John Philip Buchweitz Veterinary Diagnostic Laboratory, Michigan State University, Lansing, MI

A 4-month-old thoroughbred-cross filly foal was euthanized following a 3-week history of respiratory signs, lethargy, and a 104.6°F fever that worsened despite intensive antibacterial therapy. On postmortem examination the filly was in good body condition. The lungs were diffusely non-collapsed and rubbery, and there was subpleural emphysema within the left caudal lung lobe. The non-glandular stomach contained multiple yellow hyperkeratotic plaques, and the glandular stomach mucosa had disseminated 2-5 mm diameter red erosions. Histologic lesions included: widespread acute proximal renal tubular epithelial necrosis; diffuse hepatic congestion with prominent Kupffer cells, mild intrahepatocellular cholestasis, moderate numbers of intrahepatocellular rhodamine positive granules, and multifocal hepatocellular necrosis; widespread alveolar edema and mild histiocytosis, expansion of interlobular septa by edema and low numbers of perivascular eosinophils, mild BALT hyperplasia and low numbers of eosinophils within bronchi. General aerobic and Mycoplasma cultures of the lung had no bacterial growth. The liver cultures grew only rare E. coli, and PCR for Leptospira was negative. PCR for EHV-1, EHV-4, and EIV were all negative. Fecal examination revealed many Parascaris equorum eggs, few strongyle type eggs, and an adult Anoplocephala tapeworm. Mineral analysis by ICP-MS revealed a critically elevated liver copper concentration of 519.25 ug/g, and a mildly elevated renal copper concentration of 25.9 ug/g. The exact cause of the gross and histologic pulmonary changes remains uncertain. The heavy antibacterial therapy could have masked a bacterial infection. The heavy intestinal parasite infestation and presence of eosinophils within the lungs raises concern for parasite larval migration. More importantly, the remarkably high liver copper, mildly elevated renal copper, multifocal hepatocellular necrosis, and extensive acute proximal renal tubular necrosis are strongly suggestive of acute copper toxicosis. Horses are considered resistant to copper toxicity, with only a handful of cases reported in the literature. In this case, no pre-existing liver damage, or evidence of pyrrolizidine alkaloid hepatopathy was found, and the owner and referring veterinary knew of no copper supplementation or exposure. The gastric lesions may be suggestive of oral ingestion of caustic copper sulphate, which has shown to cause both natural and experimentally induced copper toxicosis in horses from Yugoslavia.

AAVLD Annual Conference Proceedings 78 AAVLD Virtual Conference Pyogranulomatous dermatitis in a dog caused by Paralagenidium karlingii: a case report # + * † Jessica Elbert1, Dayle McClintock2, Frane Banovic2, Michaela G. Austel2, Kaori Sakamoto1 1Department of Pathology, University of Georgia, Athens, GA; 2Department of Small Animal Medicine and Surgery, University of Georgia, Athens, GA

An approximately 8-year-old, neutered male, mixed breed dog presented to the University of Georgia Veterinary Teaching Hospital’s Dermatology service in October 2019 for evaluation of multifocal to coalescing, erythematous plaques and nodules with mild crusting over the right thorax of four months duration. The dog suffered from chronic atopic dermatitis for which it was given cyclosporine, ketoconazole, and topical steroids for six months. Fine needle aspirate of the masses upon presentation revealed pyogranulomatous inflammation, with no microbial organisms. Four, 8-mm punch biopsies of the masses were taken and submitted to the University of Georgia Department of Pathology’s Biopsy Service for histological evaluation. Affecting approximately 90% of examined sections, the dermis and panniculus were multifocally infiltrated and expanded by high numbers of degenerate and viable neutrophils, epithelioid macrophages, and reactive fibroblasts, as well as scattered lymphocytes, plasma cells, eosinophils, and multinucleated giant cells (pyogranulomatous dermatitis), frequently admixed with a finely granular, lightly basophilic, myxomatous matrix. Multifocally throughout the tissue and centrally located within dense inflammatory aggregates were numerous, transverse and cross-sections of 8-10 micron in diameter, myceloid, septate, presumptive hyphae, with nonparallel walls and nondichotomous acute and right angle branching, which were positive using both a standard GMS stain and a modified GMS stain optimized to detect oomycetes. The overlying epidermis was mildly acanthotic with minimal orthokeratotic hyperkeratosis and a locally extensive aggregate of eosinophilic, amorphous, homogenous material admixed with pyknotic and karyorrhectic cellular debris, inflammatory cells, hemorrhage, keratin, and low numbers of coccobacilli (serocellular crust). Fungal culture of the affected tissue was negative. Conventional pan-oomycete PCR and Lagenidium PCR was performed on formalin-fixed, paraffin-embedded tissue targeting the PCR amplicon of the internal transcribed spacer (ITS) region. The pan-oomycete and Lagenidium PCR yielded a positive and negative result, respectively. The product from the pan-oomycete PCR was purified using the QIAquick PCR purification kit and sequenced by Sanger method at the AVDL. This yielded a sequence 100% homologous to Paralagenidium karlingii available in the BLAST database. Paralagenidium karlingii is an exceedingly rare oomycete pathogen, previously recognized as a cause of cutaneous lesions in four dogs from the southeastern USA as well as from a single human patient from the midwestern United States. While opportunistic fungal organisms and Pythium spp. are more frequently recognized and diagnosed in dogs, paralagenidiosis due to Paralagenidium karlingii is an important differential diagnosis for canine mycotic/ oomycotic dermatitis and cellulitis. # AAVLD Trainee Awardee + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 79 AAVLD Virtual Conference Fatal fly bait toxicity in a young dog + Mayra Frances Tsoi, John Philip Buchweitz, Dalen W. Agnew Pathobiology and Diagnostic Investigation, Michigan State University, Lansing, MI

An 18-month-old female intact Great Pyrenees dog was found seizing and had blue liquid oozing from her rectum. This dog was euthanized and submitted for necropsy to the Veterinary Diagnostic Laboratory at Michigan State University. Necropsy revealed that the esophagus, stomach, and entire intestinal tract was full of bright blue, viscous liquid mixed with brown, spongy ingesta. The fur around the perineum was stained light blue. The blue coloration was subtly dissimilar to the blue-green coloration of common rodenticides. There were no significant histopathologic findings. A general organic compound screen by gas chromatography-mass spectrometry (GCMS) identified a mixture of methomyl and Z-9-tricosene, consistent with the active and coactive ingredients, respectively, in fly bait. Methomyl is a carbamate insecticide that acts by inhibiting cholinesterase enzymes, resulting in overstimulation of the nervous system causing muscle tremors, respiratory muscle paralysis, and death. It is highly toxic to humans and mammals when ingested, has an oral lethal dose (LD50) of 25mg/kg in rats, and has an extremely rapid onset of clinical signs (reported to occur within 15 minutes). It is rapidly metabolized, excreted, and does not accumulate in tissues, which can make identification difficult in chronic cases of intoxication. Methomyl is used as both an agricultural and residential insecticide but has been misused to poison wildlife and other perceived pests, often in combination with sweeteners such as colas. In one report, fly bait dissolved in a caffeinated drink was identified via GCMS in the stomach contents of a deceased feral cat, which was interpreted as intentional misuse of fly bait. In our case, no additional compounds were identified to suggest “baiting” and no source for the fly bait was found, further suggesting complete consumption by the dog. As such, it remains unclear whether the poisoining was accidental or intentional. Given the gross similarity between this poison and certain rodenticides, these compounds should be considered in the differential diagnosis for acute neurologic death associated with blue colored ingesta. + AAVLD/ACVP Pathology Award Applicant

AAVLD Annual Conference Proceedings 80 AAVLD Virtual Conference Intravascular large B-cell lymphoma in a cat + * † Andeliene Vienna Croce, Luke Borst, Crystal Lindaberry, Keith Linder Population Health and Pathobiology, North Carolina State University College of Veterinary Medicine, Raleigh, NC

A 13-year-old spayed female domestic shorthaired cat was presented with a history of progressive vomiting, hyporexia, right hind limb lameness, hypertension, and intermittent neurologic signs including anisocoria and a left head tilt. Abdominal ultrasound revealed bilateral renal cortical infarctions, a splenorenal shunt, and diffuse thickening of the small intestinal muscularis. Endoscopic biopsies of the intestinal mucosa captured mild chronic lymphoplasmacytic enterocolitis. The cat failed to respond to medical management and euthanasia was elected following a grand-mal seizure with development of acute cortical blindness. Histologically, numerous vessels within the central nervous system and multiple other tissues were occluded by a pleomorphic population of large neoplastic round cells, occasionally accompanied by thrombosis and fibrinoid vascular necrosis. Neoplastic cells in the cerebral vasculature and bone marrow displayed strong membranous and weak to moderate cytoplasmic immunoreactivity for CD20 with no immunolabeling for CD3. The absence of a primary extravascular mass, lack of neoplastic cells in circulation on a blood smear, tropism for the central nervous system, and immunohistochemical results supported a diagnosis of intravascular lymphoma. Intravascular large B-cell lymphoma is a rare subtype of lymphoma, which to our knowledge, has not been previously reported in a cat. + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 81 AAVLD Virtual Conference Diagnostic trends of five swine endemic bacterial pathogens using data from the Iowa State University Veterinary Diagnostic Laboratory (2010-2019) # * † ◊ Ana Paula Poeta Silva1, Kent Schwartz1, Bailey Arruda1, Jessica Goncalves dos Santos1, Nubia Macedo1, Orhan Sahin1, Eric Burrough1, Karen M. Harmon1, Christopher Siepker1, Phillip Gauger1, Panchan Sitthicharoenchai1, Michael Rahe1, Drew Magstadt1, Alyona Michael1, Pablo Pineyro1, Rachel Derscheid1, Rodger Main1, Eduardo Fano2, Maria Jose Clavijo1 1Iowa State University, Ames, IA; 2Boehringer Ingelheim, Atlanta, GA

Streptococcus suis (SS), Glaesserella parasuis (GPS), Mycoplasma hyorhinis (MHR), Actinobacillus suis (AS), and Mycoplasma hyosynoviae (MHS) are considered the top 10 bacteria that affect pig production. Disease diagnosis of these agents is challenging due to their commensal ecology, lack of virulence-specific, and polymicrobial interactions. The goal of this study was to describe the frequency of disease diagnosis using data from the ISU-VDL. Disease diagnoses were based on pathologists’ assessment by evaluating clinical history and testing results. Field cases (2010-2019) associated with disease diagnosis for the body systems (cardiovascular, musculoskeletal, nervous, respiratory, or systemic) were included. Preferred specimens for etiological diagnosis included: central nervous system tissues, fibrin, heart, joint, kidney, liver, lung, pleura, and spleen. Over a 10-year period, 42,884 cases were associated with one of the interested body systems. From those, 8,744 cases received a final disease diagnosis as at least one of these five bacteria. Either SS or GPS diagnoses were associated with 16% of all cardiovascular cases; 16% of all musculoskeletal cases were given either a SS or MHS diagnosis. For nervous and respiratory cases, 30% and 6% were given SS diagnosis. In systemic cases, 11%, 10%, and 4% were diagnosed with SS, GPS, and MHR. Disease diagnosis of these agents increased cumulatively 20% per year. Lung and CNS samples were frequently used to diagnose SS, while lung and fibrin were used to diagnose GPS, MHR, and AS. MHS was diagnosed using joints. The lesions associated with SS disease were serositis, bronchopneumonia, and meningitis. Lesions associated with GPS were serositis and bronchopneumonia. Lesions associated with MHR diagnosis were serositis, arthritis, and sepsis. For AS diagnosis, lesions included bronchopneumonia and sepsis. MHS diagnosis was only associated with arthritis. Results demonstrate an increase in the annual diagnoses for all agents, except for MHS. Factors as improved diagnostic protocols and tests, increased awareness and number of submissions, or changes in antimicrobial use might contribute to the increase. Lungs were frequently submitted for SS, GPS, MHR diagnosis; however, their use for systemic disease diagnosis requires caution due to their commensal nature in the respiratory system, compared to systemic sites typically targeted by pathologists (joint, brain, spleen, etc). The anatomic location for sampling, coupled with proper animal selection, offers a more comprehensive assessment for the contribution of these bacteria to systemic disease. It is critical to evaluate their impact on pig production and to close significant gaps to develop improved control and prevention strategies. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant ◊ USAHA Paper

AAVLD Annual Conference Proceedings 82 AAVLD Virtual Conference Streptococcus gallolyticus detected in cases of swine infectious endocarditis from 2016-2020 Panchan Sitthicharoenchai, Michael Rahe, Eric Burrough Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA

Vegetative valvular endocarditis (VVE) in swine is a manifestation of systemic bacterial infection. Common bacterial causes of this lesion vary between animal species with Erysipelothrix rhusiopathiae, Streptococcus suis, Streptococcus spp. and Trueperella pyogenes classically reported in pigs. A retrospective analysis of diagnostic case results from submissions to the Iowa State University Veterinary Diagnostic Laboratory from 2016-2020 was conducted to evaluate the frequency of isolation of different bacteria from the heart valves of swine with VVE. A total of 286 cases with VVE were identified and 218 of these had a potential pathogen recovered by routine bacterial culture of affected heart valve. Bacterial pathogens isolated include Streptococcus suis (55.08%), Streptococcus equisimilis (10.17%), Streptococcus gallolyticus (5.56%), Erysipelothrix rhusiopathiae (2.54%), Actinobacillus spp. (2.54%), other Streptococcus spp. (1.98%), Trueperella pyogenes (0.85%), Enterococcus faecalis (0.56%), Vagococcus fluvialis (0.28%) and Staphylococcus aureus (0.28%). The substantial number of S. gallolyticus isolated from VVE cases was surprising as there are no prior reports in the scientific literature of S. gallolyticus (formerly Streptococcus bovis) associated with swine VVE. This pathogen has long been associated with human infective endocarditis as well as a potential role in the development of human colonic cancer. S. gallolyticus is part of Lancefield group D streptococci and has been described as a commensal flora in gastrointestinal tract of mammalian and avian species. In poultry, pigeon and turkey poults are most susceptible to disease resulting in septicemia with fewer reports in chickens, geese and ducks. In dairy cattle, it has been reported sporadically as the cause of mastitis. Reports of S. gallolyticus isolation in swine are limited with previous isolations from tonsils of normal piglets and a fecal sample. This is the first report of isolation from the heart valve and demonstrates a frequent association with swine VVE. From our data set, S. gallolyticus was detected in the VVE lesion of pigs 6-13 weeks of age whereas E. rhusiopathiae was isolated in an older age group of 12-20 weeks. Future studies on factors predisposing to the development of systemic infection with S. gallolyticus in swine, lesion manifestation in other organs, and reproduction of the disease in an experimental setting are needed to better understand the ecology and impact of this bacterial infection in growing pigs.

AAVLD Annual Conference Proceedings 83 AAVLD Virtual Conference Yersiniosis in a blackbuck antelope doe: case report and disease review # + * † Jillian Marie Athey1, Keith L. Bailey1, Kristina Ellis1, Chien-Che Hung1, Amy K. Stevenson1, Paul Marx2 1Veterinary Diagnostic Laboratory, University of Illinois Urbana-Champaign, Urbana, IL; 2Marx Veterinary Services, Arthur, IL

A three-year-old, blackbuck doe (Antilope cervicapra) that died unexpectedly was submitted to the Veterinary Diagnostic Laboratory at University of Illinois Urbana-Champaign for postmortem examination. The animal was maintained on a wildlife preserve. At necropsy, an approximately 35 cm segment of distal small intestine was red to purple and firm with gradual demarcation from the adjacent, normal intestine. On cut surface, the intestinal wall was transmurally dark red to purple; the intestinal wall layers were not grossly evident. The lumen of the affected section contained a friable clump of sloughed mucosa admixed with bloody fluid. Delicate strands of fibrin were identified on the omentum, ventral body wall, and serosal surface of the affected small intestine. On histologic examination, the affected segment had transmural necrosis, hemorrhage and multiple, discrete bacterial colonies within the mucosa, submucosa, and serosa. The bacteria were occasionally intracellular and morphologically were short, slightly plump rods. Bacterial colonies with morphologically similar bacteria were also found in a lymph node associated with the spiral colon, liver, adrenal cortex, and within pulmonary and renal vasculature. Aerobic culture of the liver yielded heavy growth of Yersinia enterocolitica. Anaerobic culture of the affected intestine recovered moderate growth of Clostridium sp., which was considered normal intestinal flora. The death of this doe was attributed to acute yersiniosis. Yersinia enterocolitica is a globally distributed, zoonotic pathogen that causes disease in sheep, cattle, goats, pigs, and ruminants and has been recently associated with an outbreak of sudden deaths in alpaca. Yersiniosis should be considered as a cause of acute mortality in captive cervids. Early identification of this disease is important to prevent widespread outbreaks within affected herds. # AAVLD Trainee Awardee + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant

AAVLD Annual Conference Proceedings 84 AAVLD Virtual Conference Bacteriology Poster Live Q&A Friday, October 16, 2020

Moderators: Chien-Che Hung and Orhan Sahin

1:30 PM A single site equivalency study for non-fastidious Gram negative veterinary organisms: comparing the newly developed ARIS HiQ system to the existing Legacy ARIS 2x Dylan Staats, Chris Lewis, Nicole Holliday, Cindy Knapp, Scott Killian, Cory Pike, Brooke Olson, Thomas Fritsche...... 87

1:35 PM Novel bovine Actinobacillus suis-like strain identified by whole genome sequencing ◊ Rebecca J. Franklin-Guild, Patrick K. Mitchell, Craig Altier, Renee Anderson, Laura B. Goodman, Anil J. Thachil ...... 88

1:40 PM A retrospective analysis (2015-2019) of microbial etiologies associated with bovine respiratory disease (BRD) suspect cases from diagnostic laboratory submissions in Georgia, USA. # * Allen Aref Kalantari, Yung-Yi Mosley, Hemant K. Naikare...... 89

1:45 PM Antimicrobial susceptibilities of bacterial bovine respiratory disease (BRD) pathogens isolated from veterinary diagnostic laboratory samples: 2015-2019 # * Allen Aref Kalantari, Cynthia Watson, Yung-Yi Mosley, Hemant K. Naikare...... 90

1:50 PM Species distribution, antimicrobial susceptibility, and genomics of Campylobacter from seven commercial dog breeders in the midwestern United States Orhan Sahin, Swanand Sathe, Yue Yin, Nada Pavlovic, Zuowei Wu, Megin Nichols, Dawn Sievert, Paul Plummer, Qijing Zhang...... 91

1:55 PM Septic listeriosis in a cotton-top tamarin (Saguinus oedipus) Chien-Che Hung, Shih-Hsuan Hsiao, Leyi Wang, Carol Wolfgang Maddox...... 92

2:00 PM Retrospective survey of respiratory and gastrointestinal bacterial disease of captive white-tailed deer submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) Gabriel Gomez, Erin Edwards, Narayan C. Paul, Kellie Richardson, Terry S. Hensley, Josue Diaz-Delgado ...... 93

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 85 AAVLD Virtual Conference blank page A single site equivalency study for non-fastidious Gram negative veterinary organisms: comparing the newly developed ARIS HiQ system to the existing Legacy ARIS 2x Dylan Staats1, Chris Lewis1, Nicole Holliday1, Cindy Knapp1, Scott Killian1, Cory Pike2, Brooke Olson2, Thomas Fritsche2 1R&D, Thermofisher Scientific, Oakwood Village, OH; 2Marshfield Clinic Research Institute, Marshfield, WI

Introduction: The Thermo Scientific™ Sensititre™ ARIS HiQ™ System is a benchtop automated read and incubation system (ARIS) for antimicrobial susceptibility testing developed to replace the legacy ARIS 2X instrument. Upgraded to include a new, user friendly touchscreen LCD interface, increased plate capacity and an improved heating system, the ARIS HiQ has recently been both CE marked and FDA cleared for the testing of non-fastious Gram positive, Gram negative and fastidious specimens. Specimens acquired from veterinary labs were tested utilizing standard veterinary plate designs on both the ARIS 2X and the ARIS HiQ. Results were then evaluated for essential and categorical agreement based on the CLSI VET 08 document. Materials and methods: 100 stored clinical Gram negative organisms were tested on the automated Sensititre™ Companion Animal Gram Negative COMPGN1F Vet AST Plate. These specimens included 20 Escherichia coli, 7 Enterobacter cloacae, 23 Klebsiella species, 5 Moraxella species, 10 Pasteurella species, 10 Proteus mirabilis, 15 Pseudomonas aeruginosa and 10 other non-enteric specimens. Evaluated plates were inoculated in duplicate according to the manufacturer’s instructions. Reproducibility was conducted using 10 Gram negative isolates incubated and read in the ARIS HiQ instrument over three days in triplicate. QC organisms were determined by the plate being used at the time of testing and were run a minimum of 20 times. Results: Comparisons of the essential and categorical agreements of the results obtained were as follows. Gram negative organisms isolated from canine patients had an average total essential agreement (EAT) of 99.8% (1005/1007 results) an average evaluable agreement (EAE) of 99.3% (289/291) and an average categorical agreement (CAT) of 98.8% (322/326). Isolates from equine patients had an EAT of 99.6% (246/247 results) an EAE of 98.6% (72/73) and a CAT of 97.9% (47/48). Isolates from feline patients had an EAT of 99.3% (283/285 results) an EAE of 97.3% (71/73) and a CAT of 100.0% (50/50). Gram negative isolates from other species had an EAT of 100.0% (361/361 results) and an EAE of 100.0% (81/81). Overall for this study Gram negative isolates showed an EAT of 99.7% (1895/1900), an EAE of 99.2% (514/518) and a CAT of 99.3% (419/422) Reproducibility results showed agreement rates greater than 95% for all organisms tested. Conclusions: Based on the results obtained, the ARIS HiQ was determined to be equivalent in performance to its predecessor the ARIS 2X for the purposes of testing Gram negative veterinary isolates but with a significantly improved touchscreen LCD interface, increased (100) plate capacity and updated functionality and handling improvements.

AAVLD Annual Conference Proceedings 87 AAVLD Virtual Conference Novel bovine Actinobacillus suis-like strain identified by whole genome sequencing ◊ Rebecca J. Franklin-Guild1, Patrick K. Mitchell1, Craig Altier1, Renee Anderson1, Laura B. Goodman1, Anil J. Thachil2 1Population Medicine and Diagnostic Sciences, AHDC Cornell University, Ithaca, NY; 2Rollins Animal Disease Diagnostic Laboratory, NC State College of Veterinary Medicine, Raleigh, NC

Bovine disease caused by pathogenic strains of Actinobacillus species are typically associated with Actinobacillus lignieresii. Over a period of eighteen months, eight bovine cases from seven unique locations were submitted to the New York State Veterinary Diagnostic Laboratory for testing that resulted in the isolation of a similar Actinobacillus organism from sites including lung (4), kidney (2), joint fluid (1) and lymph node (1). All the isolates showed strong similarities to both Actinobacillus equuli and Actinobacillus suis by Bruker Daltonics MALDI-TOF MS Biotyper and the use of conventional morphological and biochemical methods based on standard references. Neither of these organisms has been previously described in literature as being pathogenic in bovine cases. Additional testing was performed, including Sensititre TM automated identification panel, 16s rDNA sequencing, infB sequencing and whole genome sequencing (WGS). All methods other than WGS were unable to distinguish between Actinobacillus equuli subspecies haemolyticus and Actinobacillus suis. WGS showed that these cases were more closely related to Actinobacillus suis than Actinobacillus equuli, however they cluster together into their own clade when compared to porcine Actinobacillus suis samples. The isolates also have an average nucleotide identity (ANI) of ~0.99 compared to one another as opposed to ~0.97 compared to the other Actinobacillus suis genomes and ~0.94 compared to Actinobacillus equuli. These data suggest that this organism may be a novel subspecies of Actinobacillus suis. This study was supported by the Food and Drug Administration’s Veterinary Laboratory Investigation and Response Network (FDA Vet-LIRN) under grants 5U18FD006379 and 1U18FD006716. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 88 AAVLD Virtual Conference A retrospective analysis (2015-2019) of microbial etiologies associated with bovine respiratory disease (BRD) suspect cases from diagnostic laboratory submissions in Georgia, USA. # * Allen Aref Kalantari, Yung-Yi Mosley, Hemant K. Naikare University of Georgia, Tifton, GA

Bovine respiratory disease (BRD) is a global health concern and the number one disease of stocker, backgrounder, and feedlot cattle in North America. It is multifactorial in origin and causes pneumonia. The objective of this study was to investigate and categorize the common etiologic agents associated with BRD from necropsy respiratory submissions (fresh/fixed lung tissues or whole carcasses) presented to the University of Georgia, Tifton Veterinary Diagnostic and Investigational Laboratory (TVDIL) between 2015 and 2019. In all, 375 cases of respiratory illnesses in cattle were included in this study to determine the type of bacterial infections (diagnosed by isolation and identification, PCR or sequencing methods) and viral infections (diagnosed by virus isolation, PCR, sequencing, antigen-capture ELISA and fluorescent antibody test methods). Each individual case was reviewed to capture details such as age, sex, breed, farm origin/location, specimen types, and the most common infectious etiologies (six bacterial species and 5 respiratory viruses) that were diagnosed from the laboratory investigations. Over 68% of 375 cases were from female cattle, while only 25% were from male cattle and 7% cases were unknown. Bacterial agents were detected from 80% of the cases (n=299) and viral agents were detected from 26% cases (n= 99). A single pathogen was detected from 31% cases (n=118), while 2 or more concomitant associations of infectious agents were identified from 36% cases (n=133). From the remaining 33% cases, other opportunistic and potential pathogenic bacteria and viruses (different than the 11 common BRD etiologies) and environmental contaminants were identified. The type and percentages of bacterial agents detected were: Mannheimia haemolytica (21%, n=79), Mycoplasma spp/M. bovis (24%, n=91), Pasteurella multocida (23%, n=88), Histophilus somni (12%, n=44), Bibersteinia trehalosi (2%, n=7) and Trueperella pyogenes (15%, n=56). The viral agents identified were Bovine Viral Diarrhea Virus (BVDV) (13%, n=50), Bovine Respiratory Syncytial Virus (BRSV) (4.5%, n=17), Parainfluenza type 3 Virus (PI3) (1%, n=5), Coronavirus (4%, n=16) and Infectious Bovine Rhinotracheitis Virus (IBR) (2.5%, n=9). For bacterial agents, the percentage of total positive cases detected from 2015 to 2019 were: M. haemolytica (15 - 29%); Mycoplasma spp/ M. bovis (15 – 33%); P. multocida (17 – 29%); H. somni (5 – 16%); T. pyogenes (11 – 19%) and B. trehalosi (0 – 4%). For viral agents, the percentage of total positive cases were in the range of 6 – 17% for BVD, 1 – 8% for BRSV, 2 – 9% for Coronavirus, 0 – 6% for IBR and 0 – 4% for PI3 respectively. Interestingly, P. multocida, M. haemolytica and Mycoplasma spp/M. bovis were the most commonly diagnosed bacterial BRD agents, whereas BVDV, BRSV, and Bovine coronavirus were the three most commonly diagnosed viral pathogens from BRD cases in Georgia. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 89 AAVLD Virtual Conference Antimicrobial susceptibilities of bacterial bovine respiratory disease (BRD) pathogens isolated from veterinary diagnostic laboratory samples: 2015-2019 # * Allen Aref Kalantari, Cynthia Watson, Yung-Yi Mosley, Hemant K. Naikare University of Georgia, Tifton, GA

The antimicrobial susceptibility patterns of bovine respiratory disease (BRD) pathogens vary regionally; also within the same region they alter over time. So, constantly updating our knowledge on their antimicrobial susceptibility plays an important role in determining therapeutic outcome. The primary objective of this study was to analyze the antimicrobial susceptibility patterns of three frequently associated BRD bacteria (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) cultured from bovine respiratory cases submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory (TVDIL), University of Georgia from 2015 to 2019. A total of 186 isolates which consisted of 67 Mannheimia haemolytica (MH), 76 Pasteurella multocida (PM) and 43 Histophilus somni (HS) were tested for in vitro susceptibility using Kirby-Bauer disk diffusion method. MH was least resistant to Trimethoprim/Sulfa (13.4%, 9/67) and Ceftiofur (15.2%, 10/66) and was highly resistant to Tulathromycin (90%, 54/60) and Tetracycline (70.7%, 29/41). PM was least resistant to Ceftiofur (2.8%, 2/71) and Enrofloxacin (3.9%, 3/76) and was highly resistant to Neomycin (47.4%, 36/76) and Spectinomycin (41.2%, 21/51). HS showed the lowest resistance to Ceftiofur (2.4%, 1/41), Florfenicol (5%, 2/40) along with highest resistance for Neomycin (58.1%, 25/43), Tilmicosin (52.9%, 9/17) and Spectinomycin (52.4%, 11/21). The overall resistance pattern of antibiotics to these pathogens was the highest for Neomycin (53.1%) followed by Tulathromycin (49.4%) and Tetracycline (48.8%). Notably, the highest susceptibility was seen with Ceftiofur (82.8%) and Florfenicol (80.1%). Over the 5-year period, there were 28.9%, 20.9% and 7.5% of PM, HS and MH isolates that were not resistant to any of the tested antimicrobials. HS which had 20.9% resistant isolates against 3 tested antimicrobials, showed the highest percentage of resistant isolates to multiple tested antimicrobial drugs. This was followed by 16.4% MH resistant isolates for 3 tested antibiotics. Also, no bacterial isolates revealed any resistance to all combination of tested antimicrobials. Interestingly, 64.2%, 22.4% and 34.9% of MH, PM and HS isolates were multidrug resistant (≥ 3 antimicrobial class), respectively. This retrospective study demonstrated the antimicrobial resistance patterns of the common BRD bacterial pathogens isolated from veterinary diagnostic laboratory samples in Georgia. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 90 AAVLD Virtual Conference Species distribution, antimicrobial susceptibility, and genomics of Campylobacter from seven commercial dog breeders in the midwestern United States Orhan Sahin1, Swanand Sathe1, Yue Yin2, Nada Pavlovic2, Zuowei Wu2, Megin Nichols3, Dawn Sievert3, Paul Plummer1,2, Qijing Zhang2 1Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA; 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA; 3Centers for Disease Control and Prevention, Atlanta, GA

Multidrug-resistant Campylobacter from infected puppies has been implicated in human disease outbreaks in the United States. Despite the emergent role of puppies and dogs in transmitting zoonotic Campylobacter, little is known about the overall burden of Campylobacter in dogs in commercial breeding facilities. To help close this knowledge gap, we conducted a prospective study on Campylobacter occurrence in US commercial dog breeding facilities. Fecal samples were collected from seven breeders in Iowa, Ohio and Indiana, which covered 13 dams and 106 puppies. Repeated sampling at intervals of 10-14 days was performed for 12 of the 13 litters. Among the 430 fecal samples cultured for Campylobacter, 16 (3.7%) were positive for Campylobacter jejuni and 42 (9.8%) were positive for Campylobacter upsaliensis, yielding a total of 32 C. jejuni and 58 C. upsaliensis isolates for analysis. All C. jejuni positive samples were collected from a single breeder in Iowa, while the C. upsaliensis isolates were from four different breeders in Iowa and Ohio. Antimicrobial susceptibility testing indicated that all C. jejuni isolates were susceptible to the nine antibiotics tested, including azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin. Of the 58 C. upsaliensis isolates, 53 were susceptible to all the antibiotics tested while resistance to nalidixic acid (n= 5), ciprofloxacin (n= 2), and gentamicin (n= 2) was detected at low levels. Two isolates displayed co-resistance to ciprofloxacin, gentamicin, and nalidixic acid. Genetic analysis of C. jejuni isolates by pulsed-field gel electrophoresis (n= 32) and whole-genome sequencing (WGS) (n= 11) revealed that all the C. jejuni isolates belonged to a single genotype, while the C. upsaliensis isolates (n= 6) were genetically diverse as determined WGS. Additionally, WGS revealed that the two C. upsaliensis isolates with an elevated ciprofloxacin MIC harbored the C257T mutation in the gyrA gene, confirming the result of the MIC test. These findings provide the most recent estimates of Campylobacter in dogs in commercial breeding facilities in the midwestern U.S. Information generated from this study may inform future studies to curb the development and transmission of antibiotic-resistant Campylobacter.

AAVLD Annual Conference Proceedings 91 AAVLD Virtual Conference Septic listeriosis in a cotton-top tamarin (Saguinus oedipus) Chien-Che Hung, Shih-Hsuan Hsiao, Leyi Wang, Carol Wolfgang Maddox Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL

Listeria monocytogenes can cause diseases ranging from self-limited colitis to life-threating sepsis, meningitis, or encephalitis in humans. Non-human primates are also known to be susceptible to this bacterial pathogen. Here, we report the first case of listeriosis discovered in a cotton-top tamarin (Saguinus oedipus). A 14-year-old, male, cotton-top tamarin was submitted to Veterinary Diagnostic Laboratory of University of Illinois for necropsy with recent spike in temperature and death in conspecific. On gross examination, liver was yellow and polycystic with hints of nodular hyperplasia/regeneration. Other viscera were grossly unremarkable. Histopathological examination showed military hepatitis and splenitis with multifocal necrosis and infiltration of neutrophils, suggestive of septic infection. Bacterial culture results showed that a heavy growth of L. monocytogenes was isolated from the liver. An immunohistochemical stain using an antibody against Listeria was also conducted on the liver tissue and revealed countless intralesional, listerial coccobacilli, validating the septic infection of Listeria.

AAVLD Annual Conference Proceedings 92 AAVLD Virtual Conference Retrospective survey of respiratory and gastrointestinal bacterial disease of captive white-tailed deer submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) Gabriel Gomez1, Erin Edwards1, Narayan C. Paul2, Kellie Richardson1, Terry S. Hensley3, Josue Diaz-Delgado1 1Anatomic Pathology, Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX; 2Bacteriology, Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX; 3Diagnostic Services, Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX

Infectious diseases of the respiratory and gastrointestinal tract are the most common causes of morbidity and mortality in captive white-tailed deer (WTD; Odocoileus virginianus). However, the main etiologies, their interplay, and the associations with coinfecting agents remain poorly characterized. In order to partially fill this gap of knowledge, we reviewed the records from WTD tissue and body submissions to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) and determined the prevalence of pathogens associated with respiratory and alimentary tract disease. From March 2016 through February 2019, there were a total of 589 WTD tissue or body submissions. A total of 239 cases (40.6%) had gross and/or microscopic lesions of the respiratory system and bacterial analyses results. The total number of bacterial isolates from respiratory disease cases was 447, of which the most common included Escherichia coli (23.5%), Trueperella pyogenes (19.0%), Proteus sp. (6.3%), Bibersteinia trehalosi (5.8%), Pasteurella multocida (5.8%), and Pseudomonas aeruginosa (5.8%). Concurrent viral infection was detected via polymerase chain reaction (PCR) analysis in 40/239 (16.7%) cases (epizootic hemorrhagic disease virus [EHDv], 29; bluetongue virus [BTv], 10; and Ovine herpesvirus [OHV]-2, 1). Fungi and/or yeast were cultured in 3 cases and Mycoplasma sp. was identified in 8 cases via PCR. Respiratory bacterial disease was often accompanied with disease in other organ systems including: alimentary tract (20.1%), hepatobiliary (11.7%), urogenital (7.9%), cardiovascular (11.7%) and nervous (2.1%). Concerning alimentary tract disease, 116 cases (19.7%) had bacterial culture results that were accompanied with gross and/or microscopic evidence of infection. The total number of bacterial isolates from the alimentary tract was 327 and the most common were Escherichia coli (34.9%), enterics (33.9%), Clostridium perfringens (19.3%), and Salmonella sp. (4.9%). PCR analyses identified 15 cases with concurrent viral infection (EHDv, 9; BTv, 5; OHV-2, 1). Fungi and/or yeast were cultured from 6 cases and Mycoplasma sp. was detected in 2 cases via PCR. Bacterial infection of the alimentary tract was accompanied with disease in the following organ systems: respiratory (50.0%), hepatobiliary (17.2%), urogenital (14.7%), cardiovascular (6.9%), and nervous (5.2%). In conclusion, bacterial infections of the respiratory and gastrointestinal tract of captive WTD are common and concomitant disease in other organ systems is frequent. These data delineate baseline etiologic knowledge of and enable further characterization studies on a proposed “WTD respiratory disease complex”. Moreover, these data may aid veterinary practitioners to formulate better preventive practices and serve as diagnostic orientations for WTD.

AAVLD Annual Conference Proceedings 93 AAVLD Virtual Conference blank page Epidemiology Poster Live Q&A Friday, October 16, 2020

Moderators: Ashley E. Hill and Erin L. Goodrich

2:05 PM Concurrent infection of Streptococcus equi subspecies zooepidemicas and canine parainfluenza virus in a group of shelter dogs. Narayan C. Paul, Pamela J. Ferro, Andres Delaconcha...... 97

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 95 AAVLD Virtual Conference blank page Concurrent infection of Streptococcus equi subspecies zooepidemicas and canine parainfluenza virus in a group of shelter dogs. Narayan C. Paul1, Pamela J. Ferro3, Andres Delaconcha2 1Bacteriology and Mycology, Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX; 2Veterinary Pathology, Texas A&M Veterinary Medical Diagnostic Lab, College Station, TX; 3Virology and Molecular Microbiology, Texas A&M Veterinary Medical Diagnostic Lab, College Station, TX

Pneumonia in dogs is part of the canine infectious respiratory disease complex (CIRDC) and is a multifactorial disease typically involving predisposing factors such as stress, comingling, poor nutrition, as well as other factors. Viral infections further compromise the pulmonary defense mechanisms and facilitate colonization of deeper pulmonary tissues by bacteria. Two males and one female dogs aged between 1.5 to 3 years were submitted for necropsy at TVMDL. In all dogs, between 35% and 85% of the lungs were dark red and severely consolidated. On cut section, variable amounts of foam and fluid were found in the lumen of the trachea and airways. A gross diagnosis of acute, severe pneumonia was established. Lung samples were submitted to the bacteriology and molecular sections for culture and identification, and for the canine respiratory disease PCR panel, respectively. Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Escherichia coli were isolated from the lung of the three dogs. S. zooepidemicus is a zoonotic and commensal organism found on the tonsils, upper respiratory tract, skin and urogenital tract of horses and has been associated with respiratory tract infections in foals and young horses, but is not considered part of the normal tonsillar flora in either dogs or man. In recent years, S. zooepidemicus has caused several outbreaks of hemorrhagic pneumonia in dogs. Infection in dogs with S. zooepidemicus is highly contagious and characterized by sudden onset of fever, dyspnea and hemorrhagic nasal discharge. In addition, canine parainfluenza virus type-2 RNA was detected by rtPCR in the lung of the three dogs. Canine distemper virus, influenza A virus, canine adenovirus type 2, canine coronavirus, canine herpesvirus type 1 and Bordetella bronchiseptica nucleic acid were not detected by PCR. Canine parainfluenza virus (CPIV) is most frequently recognized as an upper respiratory pathogen. CPVI is readily transmitted within a closely confined group of dogs. Clinically, the respiratory form is characterized by fever accompanied by nasal discharge and cough. Generally, CPIV infection of the respiratory tract of dogs has a mild to moderate clinical course, but concurrent infections with other viral and bacterial pathogens including canine distemper virus, canine herpesvirus, canine adenovirus-2, Bordetella bronchiseptica and as in this case with S. zooepidemicus can lead to severe disease and death.

AAVLD Annual Conference Proceedings 97 AAVLD Virtual Conference blank page Molecular & Bioinformatics Poster Live Q&A Friday, October 16, 2020

Moderators: Yan Zhang and Tomy Joseph

2:10 PM Development of two multiplex real-time PCR assays (mRT-PCR) for detecting main pathogens associated with neonatal bovine diarrhea Christian Savard, Andre Broes...... 101

2:15 PM A panel of realtime PCR assays for the detection of Bourbon virus, Heartland virus, Trypanosoma cruzi and West Nile virus in major disease transmitting vectors Anushri Warang, Michael Zhang, Shuping Zhang, Zhenyu Shen...... 102

2:20 PM Validation of IDEXX’s RealPCR DNA master mix and Mycoplasma hyopneumoniae DNA mix for the detection of Mycoplasma hyopneumoniae in porcine samples Tracy Lene Otterson, Samantha Radermacher, Phillip Blomberg, Kelly Aukes, Michele Leiferman, Stephanie Rossow...... 103

2:25 PM Mitigation of PCR inhibition in molecular assays for detecting toxic cyanobacteria in freshwater Jian Yuan, Kyoung-Jin Yoon...... 104

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 99 AAVLD Virtual Conference blank page Development of two multiplex real-time PCR assays (mRT-PCR) for detecting main pathogens associated with neonatal bovine diarrhea Christian Savard, Andre Broes Molecular Diagnostic, Biovet, Saint-Hyacinthe, QC, Canada

Neonatal diarrhea is a significant disease in cattle due to a high rate of morbidity and mortality. Disease can be caused by several pathogens, acting either alone or in combination. Common agents include bacteria such as Escherichia coli (K99) and Salmonella spp, parasites such as Cryptosporidium spp and Giardia duodenalis, and viruses such as group A Rotavirus (RVA), Bovine Coronavirus (BCoV), Bovine Torovirus (BoTV), and Bovine Viral Diarrhea Virus (BVDV). Dealing with so many different potential etiological agents is a challenge for accurate and rapid differential diagnosis. Conventional methods such as virus isolation, bacterial culture or fecal flotation are laborious, relatively insensitive, time consuming, and costly. By contrast, real-time PCR assays are faster and potentially more sensitive and specific. Moreover, their multiplexing capacity allows further reduction of their cost. We have developed two multiplex real-time PCR (mRT-PCR) assays for detecting either main viruses (BCoV, BoTV, BVDV and RVA) or main bacteria and parasites (E.coli (K99), Salmonella spp, Cryptosporidium spp, and G. duodenalis) associated with bovine neonatal diarrhea and compared them with corresponding singleplex RT-PCR. For evaluating the viral multiplex assay, we tested 47 fecal samples with 4 singleplex PCR assays and the 4-plex RT-PCR for BCoV, BoTV, BVDV and RVA. The multiplex assay detected respectively 94.1% (16/17), 75 % (6/9), 100% (10/10) and 89.5% (17/19) of the samples which tested positive in the singleplex assays for BCoV, BoTV, BVDV, and RVA. On the other hand, 96.7 % (29/30), 100% (35/35), 100% (37/37) and 100% (28/28) of the samples which tested negative in the singleplex assays were also negative in the multiplex assay. For evaluating the multiplex assay for the bacteria and parasites, we tested 70 fecal samples with 4 singleplex PCR assays and the 4-plex RT-PCR for E. coli K99, Salmonella spp, Cryptosporidium spp, and G. duodenalis. The multiplex assay detected respectively 93.8% (15/16), 100% (15/15), 96,4% (27/28) and 90.9% (10/11) of the samples which tested positive for E.coli K99, Salmonella spp, Cryptosporidium spp, and G. duodenalis in the singleplex assays. On the other hand, 98.2% (53/54), 100% (55/55), 83.3% (35/42) and 98.3% (58/59) of the samples which tested negative in the singleplex assays were also negative in the multiplex assay. Our mRT-PCR assays demonstrated diagnostic performances quite similar to those of the commercial singleplex assays while significantly reducing the cost and the time of testing. The few discordant results between the singleplex and multiplex assays concerned samples with high Ct values, except for Cryptosporidium spp. For several of these samples, we confirmed by Sanger DNA sequencing that the multiplex positive results were real true positives. Our two mRT-PCR thus appear to be sensitive, specific and cost effective tools for detecting the main pathogen agents associated with bovine neonatal diarrhea

AAVLD Annual Conference Proceedings 101 AAVLD Virtual Conference A panel of realtime PCR assays for the detection of Bourbon virus, Heartland virus, Trypanosoma cruzi and West Nile virus in major disease transmitting vectors Anushri Warang1,3, Michael Zhang1,3, Shuping Zhang1,2, Zhenyu Shen1,2 1Veterinary Medical Diagnostic Laboratory, College Of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO; 2Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO; 3Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO

Vector borne pathogens, such as Bourbon virus, Heartland virus, Trypanosoma cruzi, and West Nile virus are a great threat to public health and/or animal health. Bourbon virus (BRBV) in Thogotovirus genus and Heartland virus (HRTV) in Phlebovirus genus are transmitted by the lone star tick (Amblyomma americanum) to humans. Trypanosoma cruzi (TCZ), the causative agent of Chagas disease, is a parasitic organism transmitted by the blood- sucking triatomine bugs to humans and dogs. West Nile Virus, a flavivirus, is mainly transmitted by Culex species of mosquitoes to humans and animals. These pathogens have been detected in Missouri and nearby states. The goal of this study was to develop a panel of Taqman real-time PCR assays for the detection of BRBV, HRTV, TCZ, and WNV in major arthropod vectors. PCR targets were selected based on nucleic acid sequences deposited in Genbank. Primers and probes were either de novo designed or selected from publications. The coverage of primers was examined in silico with BLAST. Synthetic DNA/RNA fragments (gBlocks) were used as PCR templates in initial assay development and positive controls in subsequent assay validation. For operational efficiency, the same thermocylcing profile was used for BRBV, HRTV, and WNV and a similar profile without the initial reverse transcription step was used for TCZ. PCR assays were optimized by titrating primer/probe concentrations. Analytical sensitivity of each assay was determined using serially diluted gBlock and confirmed using pathogen genomic DNA/RNA. Analytical specificity was determined by testing against pathogens from related taxa and organisms that have the same vector host. PCR analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Ct=36.01 ± 0.70), HRTV (Ct=36.57 ± 0.93), TCZ (Ct=38.83 ± 0.28), and WNV (Ct=35.5 ± 0.35), respectively. PCR sensitivities using vector genomic DNA/RNA spiked with gBlock were 100, 100, 10, and 10 copies/reaction for BRBV (Ct=33.84 ± 0.69), HRTV (Ct=36.49 ± 0.83), TCZ (Ct=38.10 ± 0.84), and WNV (Ct=34.08 ± 0.72), respectively. PCR sensitivities using vector genomic DNA/RNA spiked with pathogen genomic DNA/RNA were 100, 1000, 100, and 100 copies/reaction for BRBV (Ct=33.83 ± 0.76), HRTV (Ct=34.66 ± 0.65), TCZ (Ct=35.14 ± 0.23), and WNV (Ct=32.19 ± 0.62), respectively. No cross reactivity was observed, except that TCZ PCR showed weak cross- reactivity with high concentrations of Leishmania major which, however, is transmitted by sandfly vectors. In conclusion, the BRBV, HRTV, TCZ, and WNV PCR assays are sensitive and specific; and can be used in epidemiological study and disease prevention and control.

AAVLD Annual Conference Proceedings 102 AAVLD Virtual Conference Validation of IDEXX’s RealPCR DNA master mix and Mycoplasma hyopneumoniae DNA mix for the detection of Mycoplasma hyopneumoniae in porcine samples Tracy Lene Otterson, Samantha Radermacher, Phillip Blomberg, Kelly Aukes, Michele Leiferman, Stephanie Rossow Veterinary Diagnostic Laboratory, University of MInnesota, St. Paul, MN

Mycoplasma hyopneumoniae (M. hyo) is one of the smallest bacteria known, contains minimal genetic material, lacks a cell wall, and is ubiquitous within swine herds worldwide. M. hyopneumoniae causes bronchopneumonia in pigs and also contributes to the porcine respiratory disease complex. Outbreaks of M. hyo pneumonia can kill pigs, add treatment costs, impair growth rates and decrease feed conversion, resulting in significant economic losses for swine producers. Specific, rapid and accurate detection of M. hyo is important for identifying and controlling these outbreaks. There is always a desire to improve the sensitivity and speed of PCR tests and an opportunity arose to reevaluate the M. hyopneumoniae real time PCR test we were using. The MVDL performed a methods comparison validation of their existing real time PCR to the newly released RealPCR DNA Master Mix and M. hyo DNA Primer and Probe Mix developed by IDEXX. Using the same nucleic acid purification method, the ThermoFisher MagMAX CORE, the RealPCR M. hyo assay was assessed for analytical sensitivity (ASe), analytical specificity (ASp), repeatability, diagnostic sensitivity (DSe), and diagnostic specificity (DSp). ASe was evaluated using serially diluted, quantified M. hyo positive DNA tested over three separate days in triplicate. The ASe was found to be one fold more sensitive than the existing assay with a limit of detection of 1 copy/ml. ASp was determined by testing sixty-four bacterial and viral pathogens that may be present in swine samples. The only pathogen to amplify was the M. hyopneumoniae isolate in the panel, proving the real time PCR to be highly specific. To show repeatability, one dilution of DNA at 10 copies/ml was run on three separate testing days in triplicate. The standard deviation of the Ct values between real time PCR runs was shown to be 0.325. When one dilution was tested ten times on the same PCR plate, the standard deviation of the Ct value detected was shown to be 0.192, demonstrating a strong repeatability. DSe and DSp were determined by testing 108 total samples of various types. 52 of the samples were positive and 56 of the samples were negative on the existing assay. The IDEXX assay detected 57 positive samples and 51 negative samples compared to the in house assay. This demonstrates a 100% DSe for the IDEXX assay compared to the in house assay and a 91.8% DSp of the IDEXX assay compared to the in house assay. Repeated testing of discrepant samples showed consistent detection in samples close to the limit of detection using the IDEXX assay, and since the ASe showed an increase in sensitivity compared to the in house assay, this increase in detection was acceptable. The results of this methods comparison show that the RealPCR DNA Master Mix and the M. hyo DNA Mix from IDEXX is equivalent or better than the existing in house assay and acceptable to use for the detection of M. hyopneumoniae from swine.

AAVLD Annual Conference Proceedings 103 AAVLD Virtual Conference Mitigation of PCR inhibition in molecular assays for detecting toxic cyanobacteria in freshwater Jian Yuan, Kyoung-Jin Yoon VDPAM, Iowa State University, Ames, IA

Freshwater cyanobacteria can produce toxins that pose a lethal threat to animals when drinking contaminated water. Toxic cyanobacteria and their toxins in a pond can also reach livestock in confinement via a water supply line. Therefore, it is imperative to monitor the presence of toxic cyanobacteria in freshwater. For that purpose, PCR assays for toxigenic cyanobacteria have demonstrated the utility. However, some humic substances in freshwater can interfere with PCR performance. We explored two strategies based on previous microalgal works to mitigate PCR inhibition from waters. First, Microcystis aeruginosa cultures with and without the addition of garden soil were prepared. The concentration and purity of extracted DNA from these samples was assessed using spectrophotometry after the extraction was done by a chloroform-based extraction coupled with a genomic DNA cleanup kit (elution method), chloroform- based extraction followed by ethanol precipitation (precipitation method), or a solid phase extraction kit. The presence and absence of inhibition were then determined by performing a conventional PCR on serially diluted extracts. Second, six PCR enhancers (BSA, betaine, DMSO, glycerol, Tween 20, formamide) were compared for their ability to reduce inhibition during PCR amplification. A set of DNA solutions with known copy numbers for M. aeruginosa was prepared with and without the addition of water mixed with garden soil. The samples were tested by a qPCR specific for M. aeruginosa to assess the degree of inhibition by substances of soil origin. Third, the mitigation ability of a selected extraction method and PCR enhancer was evaluated on pond waters spiked with various cyanobacteria (Microcystis, Anabaena, Cylindrospermopsis, and Nodularia) of known concentrations using qPCRs specific for individual cyanobacteria. The elution method yielded extracts with the least impurities from all of the soil-added cyanobacterial cultures. A260/A280 and A260/A230 ratios ranged from 1.8-1.9 and 2.0-2.2, respectively, whereas those ratios of the extracts made by the commercial kit and precipitation method were 1.6-1.7 and 0.5-0.8, and 1.6-1.7 and 1.2-1.3, respectively. Nonetheless, PCR amplification of the target from the undiluted extracts could not be made, while the target was amplified from the extracts when diluted 1:10 or more. Among the six PCR enhancers, only BSA could mitigate PCR inhibitiors in the undiluted extracts to a degree, restoring approximately 10% of the expected template quantity. On the spiked pond waters, the combination of the elution method and BSA addition almost eliminated inhibition by substances of water origin; otherwise, PCR inhibition rates by the spiked waters ranged from 34% to 63%. Our study demonstrated that field surface water samples contain natural PCR inhibitors which could be eliminated by an elution method and by introducing a PCR enhancer, leading to accurate quantification of toxigenic cyanobacterial abundances.

AAVLD Annual Conference Proceedings 104 AAVLD Virtual Conference Parasitology Poster Live Q&A Friday, October 16, 2020

Moderators: Mani Lejeune and Hemant K. Naikare

2:30 PM The diversity of mites associated with mange in American black bears (Ursus americanus) in the United States # + * † ◊ Kevin Niedringhaus, Justin Brown, Mark Ternent, Michael Yabsley...... 107

2:35 PM Comparison of microscopy and multiplex real-time PCR (mRT-PCR) for the detection of intestinal protozoan parasites in cats and dogs Christian Savard, Andre Broes, Sandra Beaudoin...... 108

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 105 AAVLD Virtual Conference blank page The diversity of mites associated with mange in American black bears (Ursus americanus) in the United States # + * † ◊ Kevin Niedringhaus1,4, Justin Brown3, Mark Ternent2, Michael Yabsley4 1School of Veterinary Medicine, University of California, Davis, Davis, CA; 2Pennsylvania Game Commission, Harrisburg, PA; 3Department of Veterinary and Biomedical Sciences, Pennsylvania State University, State College, PA; 4SCWDS; College of Veterinary Medicine, University of Georgia, Athens, GA

American black bears (Ursus americanus) are iconic North American mammal that are expanding their range across many parts of North America. This range expansion provides additional opportunities for intraspecific pathogen transmission as well as transmission to humans and domestic animals. While mange was previously considered rare in black bears throughout the country, there have been increased reports of severe and fatal sarcoptic mange (caused by Sarcoptes scabiei) in bears in multiple regions, notably the mid-Atlantic and northeastern populations. However, sporadic cases of mange in bears have been reported to be caused by mites other than S. scabiei including Demodex ursi, Ursicoptes americanus, and a newly-reported Chorioptes species. This report summarizes the basic natural history that is currently known for these pathogens, specifically related to bears, including geographical location, summary of lesions and severity of disease, and the importance of these pathogens at the population level through a brief review of the current literature. Finally, this study discusses diagnostic assays and key identification features for these pathogens and emphasizes the role of skin scrapes, which are currently the best method to detect and identify the species involved in mange cases. These data are critical as we attempt to monitor the geographical extent of these parasites in bears, learn more about the epidemiology and pathology, and develop management decisions to mitigate the risks of pathogen transmission. # AAVLD Trainee Awardee + AAVLD/ACVP Pathology Award Applicant * Graduate Student Poster Presentation Award Applicant † Graduate Student Oral Presentation Award Applicant ◊ USAHA Paper

AAVLD Annual Conference Proceedings 107 AAVLD Virtual Conference Comparison of microscopy and multiplex real-time PCR (mRT-PCR) for the detection of intestinal protozoan parasites in cats and dogs Christian Savard1, Andre Broes2, Sandra Beaudoin2 1Molecular Diagnostic, Biovet, Saint-Hyacinthe, QC, Canada; 2Biovet, St-Hyacinthe, QC, Canada

Protozoa are single-celled microorganisms some of which can cause digestive disorders in various animal species. In dogs and cats the main pathogenic species are Cryptosporidium spp, Giardia duodenalis (also known as G. intestinalis or G. lamblia), Tritrichomonas foetus (new proposed name T. blagburni) and Toxoplasma gondii. Protozoan infections can result in acute and chronic diarrhoea, along with retarded growth. The identification of the protozoan species is pivotal for effectively preventing, treating and control them. Microscopical examination (ME) of feces is often considered as the reference test for detecting protozoa in enteric infections. However, ME has been recognized as having a low sensitivity. Recently, PCR assays have been developed to detect various protozoan species. More especially real-time PCR (RT-PCR) assays appeared to be very specific and more sensitive than traditional ME. Moreover, multiplex assays can detect several organisms in the same reaction allowing reducing the cost of PCR testing. We have developed mRT-PCR assays for detecting Cryptosporidium spp, G. duodenalis, T. foetus and T. gondii. For evaluating them, we tested 47 cat samples for Cryptosporidium spp, G. duodenalis, T. foetus and T. gondii and 40 dog samples for Cryptosporidium spp and G. duodenalis with both our mRT-PCR assays and ME after enrichment by zinc sulfate double centrifugation. In dogs, the mRT-PCR assay detected all (1/1) Cryptosporidium spp and (5/5) G. duodenalis samples positive by ME. On the other hand, the mRT-PCR assay detected four more Cryptosporidium spp positive samples (4/39 or 10.3%) and five more G. duodenalis positive samples (5/35 or 14.3%) compared to ME. In cats, the mRT-PCR assay detected 85.7% (6/7) of G. duodenalis samples positive by ME but did not detect the only (0/1) Cryptosporidium spp positive sample. In return, it detected one more Cryptosporidium spp positive sample (1/46 or 2.2%) and one more G. duodenalis positive sample (1/40 or 2.5% or 1/40) compared to ME. Moreover, it also detected 19.1 % (9/47) T. foetus and 6.4% (3/47) T. gondii positive samples that were not detected by ME. Our mRT-PCR assays thus appeared to be sensitive and specific tools to detect the main intestinal protozoa of dogs and cats. In dogs it is more sensitive than ME for detecting of Cryptosporidium spp and G. duodenalis. In cats, it is more sensitive than ME for detecting of T. foetus and T. gondii. Samples missed by the mRT-PCR assays had low parasitic load as demonstrated by ME. For several samples negative by ME but positive with the mRT-PCR assays, we demonstrated by Sanger DNA sequencing the presence of Cryptosporidium canis and Cryptosporidium felis, in dogs and cats samples, respectively, confirming the higher sensitivity and great specificity of our mRT-PCR assays.

AAVLD Annual Conference Proceedings 108 AAVLD Virtual Conference Virology Poster Live Q&A Friday, October 16, 2020

Moderators: Suresh Varma Kuchipudi and Solomon O (Wole) Odemuyiwa

2:40 PM Targeted, high-throughput sequencing using bait hybridization to diagnose co-infecting agents in poultry Joshua Parris, Henry Karithi, Dawn Williams-Coplin, David Suarez...... 111

2:45 PM Characterizing the effect of temperature on bluetongue virus reassortment in Culicoides sonorensis # * Molly J. Carpenter, Jennifer Kopanke, Case Rodgers, Justin Lee, Mark Stenglein, Christie E. Mayo...... 112

2:50 PM Isolation of canine distemper virus from domestic and exotic species Ruibin Liang, Rachel Hudson, Ailam L. Lim ...... 113

2:55 PM Detection of enzootic nasal tumor virus in goats Leyi Wang, Miranda Vieson, Richard Fredrickson ...... 114

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 109 AAVLD Virtual Conference blank page Targeted, high-throughput sequencing using bait hybridization to diagnose co-infecting agents in poultry Joshua Parris, Henry Karithi, Dawn Williams-Coplin, David Suarez Southeast Poultry Research Laboratory, USDA-ARS, Athens, GA

PCR and serology-based assays have been the benchmark for detecting respiratory and enteric pathogens in poultry and other livestock, however, these techniques are limited in their ability to detect multiple infecting agents, must be frequently optimized to maintain sensitivity and specificity, and provide limited or no genetic information on the pathogen. In contrast, high-throughput, next-generation sequencing (NGS) can rapidly detect numerous co-infecting agents in a sample while providing genomic sequence information. Due to the high abundance of host and other non-target background in sequencing libraries from bulk RNA/DNA extracts of field samples (>99% in most cases), enrichment of libraries for low abundance targets is desirable to fully characterize known pathogens. In the work presented here, we selected a custom sequence set of all major antigenic variants of avian influenza, Newcastle Disease Virus (NDV), 18 other viruses of relevance for poultry, and 16S rRNA sequences for potential bacterial pathogens. These sequences were used to design ~120mer, biotinylated oligonucleotide baits with two commercially available NGS library enrichment kits. Bait treatment was applied to pooled, Nextera Flex libraries prepared from RNA extracts of field-preserved poultry swabs. Treatment resulted in a 2-3 fold increase in target sequence with up to 80% of total reads on-target. Both unenriched and enriched libaries were dominated by potential bacterial pathogens including Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma synoviae, and Ornithobacterium rhinotracheale demonstrating the ubiquity of these bacteria in swab samples from poultry. Bait treatment allowed us to detect a larger number of viruses and obtain greater depth of gene coverage for all viruses identified. At an additional cost of ~$10 per sample (when pooled) and an extra overnight incubation step for NGS library prep, the enrichment strategy demonstrated here has the capability to rapidly diagnose all known pathogens of importance to poultry from heterogenous RNA extracts with low copy number of pathogens. The comprehensive design of the bait hybridization probes to target important pathogens is critical for success, and genetic variants and novel pathogens may be missed with this approach.

AAVLD Annual Conference Proceedings 111 AAVLD Virtual Conference Characterizing the effect of temperature on bluetongue virus reassortment in Culicoides sonorensis # * Molly J. Carpenter1, Jennifer Kopanke2, Case Rodgers1, Justin Lee3, Mark Stenglein1, Christie E. Mayo1 1MIP, Colorado State University, Fort Collins, CO; 2Washington State University, Spokane, WA; 3Centers for Disease Control and Prevention, Atlanta, GA

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in devastating disease and significant economic losses. In recent years, changes in the distribution of BTV have been associated with the incursion of new serotypes into Europe and Australia. While factors driving BTV’s expansion are poorly understood, reassortment between virus strains may enhance BTV’s ability to spread to new regions. Moreover, an understanding of the effect of temperature on reassortment is lacking. The objective of this project was to characterize the frequency of reassortment between BTV-10 and BTV-17 in C. sonorensis maintained at different temperatures (20°C, 25°C, or 30°C). To establish single-virus and co-infections, midges were fed a blood meal containing ~105TCID50/ml of BTV-10, BTV-17, or BTV-10+17. Pools of midges (n = 5) collected every other day were processed for BTV qRT-PCR to track virogenesis over time. Co-infected midges collected on days 3, 7, 11, 15, and 19 were processed for BTV plaque-isolation. The complete genotypes of isolated plaques were determined using a novel, amplicon- based sequencing approach. Preliminary results indicate that midges maintained at 30°C demonstrate productive virogenesis earlier in infection than midges held at cooler temperatures. However, midges maintained at 20°C had the longest survival time, followed by midges held at 25°C, and then 30°C. Plaques from midges collected at day 19 showed a markedly punctate phenotype compared to plaques from midges collected at earlier time points. Bluetongue virus reassortment patterns and their biological consequences will add an important dimension to the modeling of viral expansion and evolution in the context of climate change. Understanding the multiple factors that drive the emergence of viruses is critical for the development of improved control and prevention measures. # AAVLD Trainee Awardee * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 112 AAVLD Virtual Conference Isolation of canine distemper virus from domestic and exotic species Ruibin Liang1, Rachel Hudson2, Ailam L. Lim1 1WVDL, UW-Madison, Madison, WI; 2Breathitt Veterinary Center, Hopkinsville, KY

Canine distemper virus (CDV) causes severe infectious disease in multiple carnivore species. Real-time PCR has been routinely used at the Wisconsin Veterinary Diagnostic Laboratory (WVDL) as a diagnostic tool for detection of CDV in canine and wildlife species. Historically, propagation of CDV field isolates in cell culture has been rather challenging until the creation of the Canine SLAM-expressing Vero cells (VDS), which were found to be highly sensitive for isolation of wild-type CDV. Maintenance of VDS cells in media containing G418 is crucial for continuous selection of VDS over the background Vero cells. The sensitivity of the VDS for CDV isolation was compared to our real-time PCR assay and detection limits were identical for both assays, confirming the high sensitivity of the isolation protocol using VDS cells. Of the 34 samples attempted for isolation, 15 CDV isolates were successfully isolated from representative domestic and exotic animals, including dogs, raccoons, bobcats, foxes and mink. These CDV isolates will be further characterized using next generation sequencing techniques to assess the genetic diversity of the viruses in domestic and exotic species across multiple locations in United States.

AAVLD Annual Conference Proceedings 113 AAVLD Virtual Conference Detection of enzootic nasal tumor virus in goats Leyi Wang, Miranda Vieson, Richard Fredrickson Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL

Enzootic nasal tumor virus (ENTV) belongs to Ortervirales order, Retroviridae family, Betaretrovirus genus. Enzootic nasal adenocarcinoma (ENA) is an economically important disease in sheep and goats and was shown to be associated with ENTV. ENA was previously reported in sheep in the U.S. and Canada. ENTV of sheep was named ENTV-1 whereas ENTV of goats was named ENTV-2. Although clinical disease commonly occurs in adult sheep and goats, young animals have also been affected. Here we report detection of ENTV-2 in goats in the U.S. Two goat kids of unknown age were submitted for necropsy and investigation of the cause of death. Findings directed ancillary testing for enteric pathogens on pooled intestinal samples, which were negative for significant aerobic pathogens by culture, Salmonella and bovine coronavirus by PCR, and rotavirus by antigen testing. A sample of spleen was also negative for bovine viral diarrhea virus by PCR. A fecal egg count showed high levels of coccidia in both kids, which was considered the cause of death in these goat kids. Metagenomic sequencing using a sequence-independent single-primer amplification on iSeq 100 was applied to both intestine and spleen samples. Bioinformatic analysis revealed that ENTV-2 was present in both spleen and intestine samples and a nearly- complete genome of ENTV-2 strain 19-44754 was assembled. Blast search of the assembled 19-44754 sequence showed 98.1% and 98.0% identity with goat strains ENTV-2CHN2 and ENTV-2CHN3 from China, respectively. Phylogenetic tree analysis results showed that 19-44754 clustered together with ENTV-2CHN2 strain. We for the first time reported detection of ENTV-2 in goats in the U.S. The role of ENTV-2 in the cause of death in these two goat kids remains unknown.

AAVLD Annual Conference Proceedings 114 AAVLD Virtual Conference Serology Poster Live Q&A Friday, October 16, 2020

Moderators: Suresh Varma Kuchipudi and Solomon O (Wole) Odemuyiwa

3:00 PM Comparison of two ELISAs for the detection of antibodies to turkey arthritis reovirus Devi Patnayak, Sagar Goyal, Stacy Pollock, Sunil Kumar Mor, Robert Porter, Rahul Kumar, Maria Barrera...... 117

3:05 PM Monitoring of BHV-1 vaccinated calves comparing an indirect and a blocking ELISA with the neutralization assay Sandra Gascon, Oriol Boix, Massimiliano Baratelli, Ester Coma-Oliva, Mireia Blanch...... 118

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 115 AAVLD Virtual Conference blank page Comparison of two ELISAs for the detection of antibodies to turkey arthritis reovirus Devi Patnayak, Sagar Goyal, Stacy Pollock, Sunil Kumar Mor, Robert Porter, Rahul Kumar, Maria Barrera Veterinary Diagnostic Lab, University of Minnesota, St Paul, MN

Turkey arthritis reovirus (TARV) has been known for nearly ten years to cause significant lameness and economic production losses in 12-16-week-old commercial turkey flocks. Diagnosis is based on clinical signs, histological lesions in gastrocnemius tendon, and detection of virus by RT-PCR and/or virus isolation. Detecting antibody titers to TARV could be a diagnostic asset, but currently there is no validated assay available. However, many US labs test turkey sera with a non-validated commercial chicken ELISA that detects antibodies to chicken arthritis reovirus (CARV). We sought to develop a TARV-specific ELISA using anti-turkey IgY as the secondary antibody/conjugate and whole virus (TARV) as the determinant antigen. ELISA optimization was done with checkerboard titration by varying antigen, serum and conjugate dilutions. Sample to positive (S/P) ratios and TARV status were used in a receiving operating curve (ROC) analysis to determine optimum S/P ratio cut-off. The whole virus TARV ELISA was compared with commercial CARV ELISA using 105 sera from hyperimmunized turkeys that were inoculated with one of several strains of TARV, turkey enteric reovirus (TERV), or CARV. The results indicated that sensitivity and specificity of both assays was high for the detection of antibodies to CARV and TARV; neither assay differentiated between antibodies to TARV, TERV or CARV; and both assays detected anti-reovirus antibodies in the serum of 1-day-old poults originating from TARV-vaccinated breeder flocks. Field studies should be performed to determine if these assays have predictive capability for identifying increased anti-reovirus antibody titers that correlate with lameness and lesions of turkey reoviral arthritis at a later age.

AAVLD Annual Conference Proceedings 117 AAVLD Virtual Conference Monitoring of BHV-1 vaccinated calves comparing an indirect and a blocking ELISA with the neutralization assay Sandra Gascon, Oriol Boix, Massimiliano Baratelli, Ester Coma-Oliva, Mireia Blanch HIPRA, Amer, Spain

Introduction: The aim of this study was to compare antibody response in vaccinated calves using an indirect ELISA (CIVTEST® BOVIS IBR), a blocking ELISA (CIVTEST® BOVIS IBRgB) and a neutralization assay (SN). Materials and Methods: Fifty four calves between 6 and 7 months of age were selected for the study. Forty four animals were divided into 3 vaccinated groups (A, B and C), following the manufacturers’ instructions, and ten (group D) were not vaccinated. All the animals were tested at 42 d.p.v. and 178 d.p.v. Comparisons between assays were performed using Spearman’s rho statistics. Results: The results from each of the techniques for each group are shown in Figure 1. Using Spearman’s rho statistics, indirect ELISA showed a very strong correlation and it was described by a linear function. Blocking ELISA showed a less strong correlation and it was described by a polynomial function (Figure 2). Discussion and Conclusion: In Figure 2, we can see that the titer of the indirect ELISA increased proportionally to the value of SN. In the case of blocking ELISA, it saturated at low positive values of SN, always showing the same titer and not offering a differentiation between low and high positives. In conclusion, the ELISA kits tested can be divided into two categories: CIVTEST® BOVIS IBR, which is better at quantifying antibody levels; CIVTEST® BOVIS IBRgB, which is better at distinguishing positive animals. Depending on the purpose of your serological study, you should choose the better kit: indirect ELISA for quantifying antibodies and blocking ELISA for epidemiological studies.

AAVLD Annual Conference Proceedings 118 AAVLD Virtual Conference Toxicology Poster Live Q&A Friday, October 16, 2020

Moderators: Cat Barr and Stephen B. Hooser

3:10 PM Identification of aflatoxin transformation products in neat standards and in corn after high voltage atmospheric cold plasma treatment using an Orbitrap ID-X Dwayne Edward Schrunk, Laura Burns...... 121

3:15 PM Comprehensive quantitative and qualitative analysis of aflatoxins by UHPLC coupled to a quadrupole-Orbitrap MS Laura Burns, Dwayne Edward Schrunk...... 122

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 119 AAVLD Virtual Conference blank page Identification of aflatoxin transformation products in neat standards and in corn after high voltage atmospheric cold plasma treatment using an Orbitrap ID-X Dwayne Edward Schrunk, Laura Burns VDPAM, Iowa State University, Ames, IA

Introduction: Aflatoxin is a toxic metabolite produced by Aspergillus and Penicillium molds. The fungi that produce aflatoxin can grow on grains in the field or during storage and distribution of food products. Aflatoxins are both carcinogenic and toxic to humans and livestock. They need to be removed before food products are distributed and consumed. Recent research has shown that aflatoxin levels are reduced after treatment with High Voltage Atmospheric Cold Plasma (HVACP). Although aflatoxin levels decrease after HVACP treatment, it is not known what potential transformation or degradation products are formed during treatment. This work investigates the aflatoxin transformation products in neat standards and corn samples that have been treated with HVACP at various amounts of time. Methods: Neat standards containing approximately 200 µM of Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, or Aflatoxin G2 were treated with HVACP for either 2, 5, 10, or 20 minutes. Similarly, a set of corn samples confirmed to have Aflatoxin B1 were prepared for pre- and post-HVACP treatment. Samples were analyzed both before and after treatment using an Orbitrap ID-X Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). An MSn workflow was used to acquire data up to MS4 using both HCD and CID. A second method incorporating source CID prior to Selected Ion Monitoring (SIM) for the product ions was used on the corn samples. Resulting data was processed using Freestyle and structures were annotated using Mass Frontier 8.0. Prelim Data: Previous work has proposed that Aflatoxin B1 reacts to form six transformation products during HVACP treatment. The MSn workflow used here identified several product ions of interest in treated samples. This work confirmed some of the previously proposed transformation products but also identified new species. These new species are thought to be potential dimers of Aflatoxin B1 or Aflatoxin G1 based on comparative MSn product ion spectra. The reaction kinetics of this transformation will be discussed in further detail based on HVACP treatment duration. It was also found that Aflatoxin B2 and G2 do not significantly break down after treatment. These results show that HVACP treatment does decrease the overall concentration of Aflatoxin B1 and G1 in neat standards. Annotated structures of Aflatoxin B1 and G1 transformation products will be presented along with an evaluation of the changes in transformation products over the course of treatment time. After HVACP treatment one of the Aflatoxin B1 transformation products shows a similar full mass spectrum to Aflatoxin G2. Evidence discussing this transformation including proposed annotated structures based on higher order mass spectrum will be discussed.

AAVLD Annual Conference Proceedings 121 AAVLD Virtual Conference Comprehensive quantitative and qualitative analysis of aflatoxins by UHPLC coupled to a quadrupole-Orbitrap MS Laura Burns, Dwayne Edward Schrunk VDPAM, Iowa State University, Ames, IA

Introduction: Mycotoxins are toxic secondary metabolites produced by fungi. They can pose potential health risks to both humans and livestock if present in feedstuff. Aflatoxins are a class of mycotoxins for which the regulatory agencies have established safe levels in the low ppb range because of carcinogenicity. A current analytical challenge is to detect and quantify mycotoxins in complex feed matrices. Aflatoxin analysis was performed with a selective and sensitive quadrupole-Orbitrap mass spectrometer. Three different acquisition workflows were evaluated for the quantitative determination of aflatoxins in corn feed. Spectral library matching was used to improve confirmation at low concentration levels. Methods: Approximately 5 g of corn feed samples were extracted with a 20 mL mixture of methanol:water (70:30 v/v) followed by mixing for 30 minutes on a multi-tube shaker. The mixture was then centrifuged for 10 minutes at 4000 rpm. An aliquot of the extract was transferred into a 2 mL amber vial prior to analysis. Analysis of aflatoxins was performed on a new UHPLC with a quadrupole-Orbitrap mass spectrometer platform. LC separation was carried out on a reversed phase C18 column (100 × 2.1 mm, 1.9 µm) with an injection volume of 2 µL. The mobile phases consisted of A: water and B: methanol both containing 5mM ammonium formate, and 0.1% formic acid. Preliminary data: Comprehensive analysis of Aflatoxins B1, B2, G1, and G2 was performed in full scan mode with three separate workflows DIA, dd-MS/MS, and tMS2. The full MS mode with resolution of 60,000 FWHM was used to acquire data for the quantification, while the DIA, dd-MS/MS, and tMS2 modes with resolution of 30k were used for identity confirmation. Data were analyzed using a processing software set with an extraction mass tolerance of 5 ppm for both precursor and product ions. Confirmation of aflatoxins in the corn feed was also conducted using a curated high-resolution mass spectral library. Results showed good linearity over concentrations ranging from 5 – 200 ppb for all four aflatoxins. Correlation coefficients with a non-weighted linear regression were at R2 > 0.990. Limits of quantitation (LOQ) based on the results of 5 pre-spiked control replicates at 5 ppb. Excellent bias and precision (n=5) were obtained. One to three fragment ions were used to confirm the identity of each aflatoxin. The robustness of the method was determined by consistent retention times that were within ±0.1 min. All three evaluated acquisition methods showed sufficient sensitivity, robustness, and reproducibility. A certified reference material containing known concentrations of aflatoxins was used to validate the method with spectral library matching and fragment confirmation.

AAVLD Annual Conference Proceedings 122 AAVLD Virtual Conference Pathology Poster Live Q&A Friday, October 16, 2020

Moderators: I. Walther and Rosalie Ierardi

3:20 PM A protocol for isolation of platelet rich plasma for aggregation testing in dogs Amelia Frye, Marjory Brooks...... 125

3:25 PM Duodenal perforation in a nursery pig ◊ Youchan Bae, Hyunkyoung Lee...... 126

3:30 PM Peritoneal mesothelioma in a black bear (Ursus americanus) Jane Kelly, Thomas J. Baldwin, Annette Roug...... 127

3:35 PM Trypanosoma cruzi genotype I and Toxoplasma gondii coinfection in a red-necked wallaby (syn. Bennett’s wallaby; Macropus rufogriseus rufogriseus), Texas, USA Josue Diaz-Delgado, Thomas Kellerman, Lisa Auckland, Pamela J. Ferro, Katia Groch, Gabriel Gomez, Sarah Hamer...... 128

Symbols at the end of titles indicate the following designations: # AAVLD Trainee Awardee † Graduate Student Oral Presentation Award Applicant + AAVLD/ACVP Pathology Award Applicant ◊ USAHA Paper * Graduate Student Poster Presentation Award Applicant

AAVLD Annual Conference Proceedings 123 AAVLD Virtual Conference blank page A protocol for isolation of platelet rich plasma for aggregation testing in dogs Amelia Frye, Marjory Brooks Comparative Coagulation, Cornell University AHDC, Ithaca, NY

Introduction: Light transmittance aggregometry (LTA) is considered the gold standard for evaluating platelet function, however LTA is technically challenging and requires meticulous pre-analytic processing to yield valid results. The LTA assays are performed on platelet suspensions, referred to as platelet rich plasma (PRP), typically isolated via centrifugation from citrate-anticoagulated whole blood samples. In some species, such as horses and humans, generation of a consistent PRP yield is relatively simple using a single, standard g force centrifugation step. Dogs, however, demonstrate inter-breed and inter-individual variation in platelet number and platelet size such that serial centrifugation steps and/or variation in g force are required to obtain PRP of sufficient quality and quantity for LTA. We therefore aimed to develop a standardized, single-step procedure for routine isolation of PRP for LTA in dogs. Methods and results: Dilution of canine citrated whole blood in a Hepes buffer (3 volumes blood: 1 volume buffer) prior to centrifugation at 650 g x 5 minutes yielded PRP with platelet counts consistently 85-130% of starting platelet concentration. Using this method, dilute PRP was generated from clinically healthy canine donors (n=13 dogs, n= 9 breeds), and aggregated in a multi-channel aggregometer (PAP8, Biodata) in response to a panel of agonists including low, medium and high dose ADP, low and high dose collagen, gamma thrombin, arachidonic acid and a saline control. None of the samples displayed >10% aggregation in the saline control channel (indicating minimal platelet activation during processing). All samples had >45% maximal aggregation (MA) to high dose ADP, with expected dose dependent decrease in MA to the medium and low doses, and >45% MA to gamma thrombin. All but one sample had >44% MA to high dose collagen, with one dog appearing to be an outlier. As previously reported, canine aggregation response to arachidonic acid was highly variable. Results of LTA for dogs with hereditary platelet defects demonstrated expected LTA abnormalities, i.e. ADP and collagen defect (Basset hound) and isolated ADP defect (Greater Swiss mountain dog). Conclusion: A pre-dilution step with a standard g force and centrifugation time allows for consistent isolation of non-activated PRP from dogs suitable for LTA.

AAVLD Annual Conference Proceedings 125 AAVLD Virtual Conference Duodenal perforation in a nursery pig ◊ Youchan Bae, Hyunkyoung Lee Animal and Plant Quarantine Agency, Gimcheon, Korea (the Republic of)

Intestinal perforation is an uncommon condition in pigs, and can result in sudden death associated with peritonitis and consequential septicemia. A specific etiology has not been recognized, but hard plastic brush bristles have been found in a few cases. Herein, we describe the sudden death of a nursery pig caused by duodenal perforation, possibly due to stress. A 10-week-old nursery pig died suddenly, without any significant clinical history of illness. The farm had 800 nursery pigs at that time, and gastric ulcers and polyserositis caused by Streptococcus suis had been detected in the herd. The carcass was submitted for postmortem examination. Grossly, the skin contained numerous scratches all over the body, a 1.5-cm diameter ulcer in the flank, and tail necrosis. The lungs were diffusely red and rubbery in consistency. The abdominal cavity contained a large quantity of green turbid fluid. The serosa of abdominal organs was diffusely reddened, and covered with green fibrinous material. The proximal duodenum contained a perforation (3 x 4 cm). Microscopically, the duodenal perforation contained severe mucosal necrosis and severe neutrophilic infiltration along the margins of the perforation. The intestinal serosa contained diffuse severe fibrino-suppurative serositis. Severe congestion and thrombosis were found in the lungs and spleen. In the ulcerated skin, there were bacterial colonies on the ulcerated epidermal surface. Considering multiple scratches of the skin, ulcerative dermatitis, and tail necrosis, and the history of common gastric ulcer in the herd, duodenal perforation in this case might be associated with stress, and consequential septic peritonitis as the cause of death. ◊ USAHA Paper

AAVLD Annual Conference Proceedings 126 AAVLD Virtual Conference Peritoneal mesothelioma in a black bear (Ursus americanus) Jane Kelly1,2, Thomas J. Baldwin1,3, Annette Roug4 1Animal Dairy and Veterinary Sciences, Utah State University, Springville, UT; 2Spanish Fork, Utah Veterinary Diagnostic Laboratory, Spanish Fork, UT; 3Logan, Utah Veterinary Diagnostic Laboratory, Logan, UT; 4Utah Division of Wildlife Resources, Salt Lake City, UT

A free-ranging adult female black bear (Ursus americanus) was found weak and emaciated near a water source in central Utah. Due to the poor body condition that it was in, it was euthanized and necropsied at the Utah Veterinary Diagnostic Laboratory, Spanish Fork in July 2016. The bear weighed 95 kg and several liters of yellow, cloudy fluid were found in the peritoneal cavity and numerous, variably-sized, white to tan, firm nodules thickened the mesentery. Similar nodules were on the capsular or serosal surfaces of the bladder, spleen, intestine, and kidneys. Histopathologic examination showed multiple similar masses composed of cuboidal to polygonal cells arranged in tubules separated by moderate amounts of vascularized collagen. Immunohistochemically, neoplastic cells were immunoreactive to antibodies directed against cytokeratin and vimentin. Histopathologic and immunohistochemical findings led to a diagnosis of peritoneal mesothelioma. Mesotheliomas are rare neoplasms that arise from mesoderm-derived cells which line serosal surfaces such as the pericardium, pleura, and peritoneum. Neoplasia in general has only infrequently been reported in American black bears and, more specifically, mesotheliomas have not previously been reported in black bears. In other bear species, mesotheliomas have mostly been reported in captive bears of advanced age. Factors that predisposed this bear to mesothelioma are unknown and it is likely a singular, rare occurrence. Even so, mesotheliomas should be a differential diagnosis for tumors in black bears.

AAVLD Annual Conference Proceedings 127 AAVLD Virtual Conference Trypanosoma cruzi genotype I and Toxoplasma gondii coinfection in a red-necked wallaby (syn. Bennett’s wallaby; Macropus rufogriseus rufogriseus), Texas, USA Josue Diaz-Delgado1, Thomas Kellerman2, Lisa Auckland3, Pamela J. Ferro1, Katia Groch3, Gabriel Gomez1, Sarah Hamer3 1Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX; 2South Texas Veterinary Clinic, Beeville, TX; 3Texas A&M University, College Station, TX

While the health effects of trypanosomes in Australian mammals in their native range are not fully understood, there is evidence of an impact in those species introduced to other geographical regions. Here we report the pathological and molecular features of concurrent fatal trypanosomiasis and toxoplasmosis in an adult female captive red-necked wallaby (syn. Bennett’s wallaby; Macropus rufogriseus rufogriseus) from Bee County, Texas, USA. The animal exhibited no clinical signs prior to sudden death. On necropsy, the main findings were generalized organ congestion and bilateral renal petechiation. Microscopically, the main finding was lymphohistiocytic and necrotizing pancarditis with intrasarcoplasmic protozoal pseudocysts containing amastigotes and occasional intrahistiocytic amastigotes, morphologically compatible with Trypanosoma cruzi, as well as rare intrasarcoplasmic protozoal tissue cysts with zoites morphology compatible with Toxoplasma gondii. Other lesions included acute centrilobular to panlobular necrotizing hepatitis with intrahepatocellular T. gondii cysts, necrotizing splenitis, pulmonary oedema with fibrin, histiocytosis and rare fibrin microthrombi, and acute renal tubular degeneration with proteinosis and pigmented casts suggestive of haemoglobinuria or myoglobinuria. Immunohistochemical labelling confirmed intralesional T. gondii cysts and molecular analyses identified Trypanosoma cruzi genotype I and Toxoplasma gondii. This is a unique case that, to the best of our knowledge, represents the first description of T. cruzi and T. gondii co-infection, as well as the first record of naturally occurring infection T. cruzi genotype I infection in macropodids. This case adds to the epidemiological knowledge on Chagas disease in the USA, particularly in Texas where there is a high prevalence of human and canine trypanosomiasis.

AAVLD Annual Conference Proceedings 128 AAVLD Virtual Conference AAVLD Author Index Aboellail, Tawfik A...... 68 Clancy, Chad...... 75 Abou-Madi, Noha...... 39 Clavijo, Maria Jose...... 9, 10, 34, 62, 82 Agnew, Dalen W...... 80 Clawson, Michael...... 54 Alanis Gallardo, Valeria Maria...... 60 Clifford, Deana...... 71 Aldous, Wade...... 43 Clinton, Alexander Penn...... 67 Aljets, Ethan...... 47 Clothier, Kris A...... 55 Almeida, Henrique...... 9 Cole, Leslie...... 46 Altier, Craig ...... 88 Cole, Stephen ...... 58 Ambagala, Aruna ...... 31 Coma-Oliva, Ester...... 118 Anderson, Renee...... 35, 39, 88 Croce, Andeliene Vienna...... 81 Angelos, John...... 55 Cronk, Brittany D...... 35, 39 Anguiano, Maritza ...... 69 Crossley, Beate...... 29, 71, 74 Anis, Eman...... 53 Crouch, Esther...... 57 Armour, Natalie ...... 73 Cummings, Kevin J...... 39 Arruda, Bailey...... 9, 34, 82 Daniels, Josh...... 56 Asin, Javier...... 71 De Luca, Eliana...... 63 Athey, Jillian Marie ...... 84 DeBlois, Suzanne...... 44 Auckland, Lisa ...... 128 Delaconcha, Andres ...... 97 Aukes, Kelly...... 30, 103 Delaney, Martha...... 59 Austel, Michaela G...... 79 Derscheid, Rachel...... 9, 34, 62, 82 Bae, Youchan...... 126 Diaz-Campos, Dubraska...... 56 Bailey, Keith L...... 28, 46, 84 Diaz-Delgado, Josue...... 93, 128 Baldwin, Thomas J...... 127 Dickey, Aaron...... 54 Banda, Alejandro ...... 73 Diel, Diego...... 35, 48, 49 Banovic, Frane ...... 79 Doolittle, Kent...... 34 Baptista, Rodrigo ...... 63 Dubovi, Ed ...... 49 Baratelli, Massimiliano...... 118 Duhamel, Gerald E...... 49 Barrera, Maria...... 117 Ebling, Rafael...... 46 Bauermann, Fernando...... 46 Edwards, Erin...... 69, 93 Baughman, Brittany ...... 73 Egan, Christina...... 57 Baum, Dave...... 9, 10, 11, 23 El-Gazzar, Mohamed ...... 14 Baumgartner, Wes...... 28 Elbert, Jessica...... 79 Beaudoin, Sandra ...... 108 Ellis, Kristina ...... 84 Benfer, Jeffrey...... 43 Erdogan-Bamac, Ozge ...... 74 Berghaus, Roy D...... 13 Fagre, Anna...... 68 Blair, Emily R...... 74 Fano, Eduardo...... 82 Blanch, Mireia...... 118 Ferreyra, Franco...... 9, 34 Blomberg, Phillip...... 103 Ferro, Pamela J...... 97, 128 Boix, Oriol ...... 118 Figueroa, Alejandra...... 74 Borst, Luke...... 81 Filigenzi, Michael...... 17 Bradner, Laura ...... 38, 43 Fitzgerald, Scott D ...... 78 Britten, Justine E...... 75 Flory, Gary...... 46 Brockmeier, Susan ...... 62 Francisco, Charles...... 32 Broes, Andre...... 101, 108 Franklin-Guild, Rebecca J...... 56, 88 Brooks, Marjory...... 125 Fredrickson, Richard...... 27, 28, 114 Brown, Corrie...... 72 Freeman, Jamie Catherine...... 60 Brown, Justin ...... 107 Freeman, Kathleen ...... 70 Buchweitz, John Philip...... 78, 80 Fresneda, Karina...... 77 Buckley, Alexandra...... 31 Fritsche, Thomas...... 87 Burbank, Kyle...... 57 Frye, Amelia...... 125 Burns, Laura...... 16, 121, 122 Frye, Elisha...... 49, 57 Burrell, Caitlin ...... 59 Gallardo, Rodrigo...... 29, 74 Burrough, Eric...... 62, 82, 83 Garcia, Jessica...... 56 Butler, Erica ...... 49 Garcia, Juan...... 77 Cardia Caserta, Leonardo...... 48, 49 Gascon, Sandra...... 118 Carpenter, Molly J...... 112 Gatto, Igor...... 9 Chamba, Fabian ...... 32 Gauger, Phillip ...... 10, 31, 34, 38, 43, 45, 47, 82 Cheng, Ting-Yu ...... 22, 31 Gebhart, Connie ...... 36 Childs-Sanford, Sara E...... 39 Gilliam, John...... 46

AAVLD Annual Conference Proceedings 129 AAVLD Virtual Conference AAVLD Author Index Gimenez-Lirola, Luis G...... 9, 10, 11, 14, 23, 31 Liang, Ruibin ...... 113 Gioia, Gloria...... 60 Lim, Ailam L...... 28, 113 Gomez, Gabriel...... 69, 93, 128 Lindaberry, Crystal...... 81 Goncalves dos Santos, Jessica ...... 62, 82 Linder, Keith...... 81 Gonzales Viera, Omar Antonio...... 74 Lingsweiler, Sonia W...... 61 Gonzalez-Astudillo, Viviana...... 71 Livengood, Julia...... 33 Goodman, Laura B...... 35, 39, 88 Loy, John Dustin...... 54 Gorden, Patrick J...... 64 Maboni, Grazieli...... 63 Goyal, Sagar...... 117 Macedo, Nubia...... 62, 82 Gresch, Sarah ...... 30 Maddox, Carol Wolfgang...... 59, 92 Groch, Katia...... 128 Magstadt, Drew...... 47, 82 Grubaugh, Nathan D...... 35 Magtoto, Precy...... 9 Gunderson, Maureen G...... 39 Magtoto, Ronaldo...... 9, 11, 14 Habing, Gregory...... 56 Maier, Gabriele Ute ...... 55 Hamer, Sarah...... 128 Main, Rodger ...... 10, 43, 62, 82 Harmon, Karen M...... 31, 34, 38, 43, 45, 62, 82 Marx, Paul...... 84 Hartmann, Faye...... 56 Mayo, Christie E...... 112 Hau, Samantha ...... 62 McClintock, Dayle...... 79 Henao-Diaz, Alexandra...... 10, 11, 23 McCluskey, Brian J...... 21 Hengy, Miranda ...... 56 McDaniel, Aric...... 9 Hensley, Terry S...... 93 Mendonca, Fabio...... 76 Hille, Matt...... 54 Merodio, Maria...... 9 Hinds, C. Austin...... 56 Mete, Asli...... 29, 74 Hoffman, Jay...... 69 Michael, Alyona...... 82 Holliday, Nicole...... 87 Miknis, Robert ...... 46 Hooser, Stephen B...... 15 Miller, Jessica...... 34 Hsiao, Shih-Hsuan ...... 27, 92 Miller, Lori...... 46 Hu, Dapeng...... 22 Mitchell, Patrick K...... 35, 39, 88 Hudson, Rachel...... 113 Mohamed, Fawzi...... 72 Hung, Chien-Che ...... 59, 84, 92 Monday, Jessie D...... 61 Hutchinson, Michael...... 33 Monistero, Valentina...... 60 Jackson, Todd...... 46 Moore, Janet D...... 71 Ji, Ju ...... 23 Moroni, Paolo...... 60 Jones, Lee...... 13 Morris, Paul...... 22 Kalantari, Allen Aref ...... 13, 89, 90 Mosley, Yung-Yi ...... 89, 90 Kapczynski, Darrell ...... 44 Naikare, Hemant K...... 13, 89, 90 Kariithi, Henry ...... 37 Navarro, Mauricio...... 76, 77 Karithi, Henry...... 111 Nemeth, Nicole...... 63 Kattoor, Jobin...... 53 Nemser, Sarah...... 15 Kellerman, Thomas...... 128 Nichols, Megin...... 91 Kelly, Jane...... 127 Niedringhaus, Kevin...... 63, 107 Kelly, Kathleen...... 57 Nyaoke, Akinyi C...... 71 Khan, Owais Ahmed...... 61 O’Toole, Alicia Dawn...... 72 Killian, Scott...... 87 Olson, Brooke...... 87 Knapp, Cindy ...... 87 Otterson, Tracy Lene ...... 30, 103 Kopanke, Jennifer...... 112 Palena, Lauren...... 39 KORCHIA, Jeremie...... 70 Pantin-Jackwood, Mary ...... 44 Kumar Mor, Sunil...... 30, 36, 117 Parris, Joshua ...... 37, 111 Kumar, Rahul...... 117 Patnayak, Devi ...... 117 Lager, Kelly ...... 31 Paul, Narayan C...... 61, 93, 97 Langan, Jennifer...... 59 Pavlovic, Nada ...... 91 Lantz, Kristina...... 62 Pempek, Jessica...... 56 Laverack, Melissa...... 49 Pentella, Michael...... 43 Lee, Hyunkyoung...... 126 Perry, Micheal...... 57 Lee, Justin...... 112 Pike, Cory...... 87 Lee, Scott ...... 44 Pineyro, Pablo...... 82 Leiferman, Michele...... 103 Plummer, Paul...... 91 Lewis, Chris ...... 87 Poeta Silva, Ana Paula...... 9, 11, 34, 62, 82 Li, Ganwu...... 62, 64 Pollock, Stacy...... 117

AAVLD Annual Conference Proceedings 130 AAVLD Virtual Conference AAVLD Author Index Poppenga, Robert H...... 15, 17 Storino, Gabriel...... 34 Porter, Robert...... 36, 117 Stoute, Simone ...... 29 Primus, Alex...... 36 Sturos, Matthew ...... 36 Pulido-Landinez, Martha ...... 73 Suarez, David...... 37, 44, 111 Radermacher, Samantha...... 103 Swayne, David ...... 44 Rahe, Michael...... 82, 83 Swinford, Amy...... 61 Raleigh, Russell ...... 61 Talley, Justin...... 46 Ramachandran, Akhilesh ...... 46 Tallmadge, Rebecca L...... 39 Ramirez, Esteban ...... 10 Templeton, Alexis...... 69 Rankin, Shelley C...... 58 Ternent, Mark...... 107 Rawal, Gaurav...... 32, 47 Tewari, Deepanker...... 33 Reimscheussel, Renate...... 15 Thachil, Anil J...... 88 Reinl, Steve...... 55 Thirumalapura, Nagaraja ...... 33 Rejmanek, Daniel...... 29, 74 Tjalkens, Ron ...... 68 Remmenga, Marta...... 46 Torrison, Jerry...... 36 Renshaw, Randall...... 49 Tsoi, Mayra Frances...... 80 Richardson, Kellie...... 93 Turner, Justin ...... 46 Riordan, Deaglan ...... 47 Uzal, Francisco...... 77 Roady, Patrick...... 28 Uzal, Francisco A...... 17, 71, 76 Robbe-Austerman, Suelee...... 62 Van Engen, Matthew G...... 45 Robbins, Kara...... 54 Van Geelen, Albert...... 31 Rocha, Savannah...... 68 Vannucci, Fabio ...... 36 Rodgers, Case...... 112 Vieson, Miranda...... 28, 114 Rood, Kerry A...... 75 Vogels, Chantal B.F...... 35 Rose, Heidi Huffman ...... 73 Vuolo, Matthew ...... 46 Rossow, Stephanie ...... 30, 36, 103 Wang, Chong ...... 22, 31 Rotolo, Marisa...... 10 Wang, Leyi...... 27, 28, 59, 92, 114 Roug, Annette...... 127 Warang, Anushri...... 102 Rovira, Albert...... 36 Watson, Cynthia...... 90 Ruston, Chelsea...... 62 Whitcomb, Caleb ...... 46 Sahin, Orhan...... 62, 82, 91 Wiedmann, Martin ...... 39 Sakamoto, Kaori...... 79 Wieland, Matthias Josef...... 60 Samuelson, Jonathan P...... 27 Wilkes, Rebecca P...... 53 Sanchez, Susan...... 63 Williams-Coplin, Dawn ...... 37, 111 Santisteban, Carlos...... 60 Williams, Laura Artz ...... 72 Sathe, Swanand...... 91 Willian, Paim ...... 46 Sato, Yuko...... 14 Wilson, David J...... 75 Savard, Christian...... 101, 108 Witbeck, Wendy...... 34 Schountz, Tony...... 68 Woods, Leslie...... 29 Schrunk, Dwayne Edward ...... 16, 121, 122 Wu, Zuowei...... 91 Schumacher, Loni L...... 47 Yabsley, Michael ...... 107 Schwartz, Kent...... 82 Yang, Tzushan Sharon...... 73 Scott, Jeffrey...... 60 Yim-im, Wannarat ...... 47 Seiger, Jeremy...... 46 Yin, Yue...... 91 Seller, Holly S...... 29 Yoon, Kyoung-Jin...... 104 Shen, Zhenyu ...... 102 Yuan, Jian...... 104 Siepker, Christopher...... 82 Zhang, Jian...... 72 Sievert, Dawn...... 91 Zhang, Jianfeng...... 47 Siler, Julie...... 39 Zhang, Jianqiang...... 32, 43, 45, 47, 48 Singh, Vikash...... 36 Zhang, Michael...... 102 Sipka, Anja...... 60 Zhang, Min...... 10 Sitthicharoenchai, Panchan...... 62, 82, 83 Zhang, Qijing ...... 91 Smith, Chad...... 32 Zhang, Shuping...... 102 Smith, Mary ...... 49 Zheng, Zhiyi...... 64 Spackman, Erica...... 44 Zhu, Jinhui ...... 47 Staats, Dylan...... 87 Zimmerman, Jeffrey...... 9, 10, 11, 22, 23, 31, 34 Stein, Leah ...... 78 Zimmerman, Silvia...... 34 Stenglein, Mark...... 112 Zylich, Nancy...... 49 Stevenson, Amy K...... 84

AAVLD Annual Conference Proceedings 131 AAVLD Virtual Conference blank page AAVLD Keyword Index accurate mass high resolution...... 16 Detection...... 27, 114 Actinobacillus...... 88 Diagnostic Specificity...... 10 Aflatoxins...... 121, 122 Diagnostic transition stages ...... 23 African swine fever...... 46 Diagnostis...... 111 AMR...... 87 Digital Pathology ...... 69 Analytical sensitivity and specificity...... 32 Disease transition stages...... 23 Analytics...... 21 disease transmission models...... 22 anaplasmosis...... 13 DNA extraction...... 34 antelope...... 84 Dog and Cat ...... 61 Antibiotic sensitivity test ...... 61 dogs...... 91 antimicrobial resistance ...... 39, 91 dry treatment...... 75 Antimicrobial resistance...... 90 duodenal perforation...... 126 Assay Validation...... 70 ELISA...... 11, 14 avian...... 117 Endocrinology...... 70 backyard chickens...... 74 Enteritis...... 76 Bacteremia ...... 56 Enzootic Nasal Tumor Virus ...... 114 Bacteria...... 15 Equine...... 78 bacteria...... 88 experimental challenge...... 44 Bacterial ...... 93 Experimental inoculation...... 62 Bait hybridization...... 111 facility...... 67 bears ...... 107 Feed...... 15 Bioassay ...... 47 Feline...... 81 bioinformatics...... 36 Fly bait ...... 80 biomarker model...... 54 foreign animal disease...... 46 black bear...... 127 frog...... 59 Blood Culture...... 56 gastrointestinal ...... 93 Bluetongue virus...... 112 GCMS...... 80 Border Disease Virus ...... 49 genomic island ...... 64 bovine...... 13 Genotyping...... 48 Bovine...... 101 Glaesserella parasuis...... 82 Bovine Kobuvirus...... 27, 28 Goats...... 17, 114 bovine mastitis ...... 64 Heartland Virus, Bourbon Virus, Bovine respiratory disease ...... 89, 90 West Nile Virus, Trypanosoma cruzi...... 102 BRD ...... 89 Horse...... 76 Brucella...... 59 Horses...... 77 California ...... 71, 74 host depletion...... 37 Campylobacter ...... 91 Houseflies...... 60 Canine distemper ...... 113 IBR...... 118 Canine parainfluenza virus...... 97 IDEXX ...... 103 Carbepenem Resistant Enterobacteriaceae...... 58 inclusion body hepatitis ...... 29 casein hydrolysate...... 75 infectious bovine keratoconjunctivitis ...... 54 Cattle...... 27, 57 infectious bronchitis virus...... 74 chloroform-based extraction...... 104 klebsiella pneumoniae...... 64 clamp...... 38 Lagomorphs ...... 71 Clostridium botulinum type A ...... 57 Light Transmittance Aggregation...... 125 Clostridium piliforme...... 77 LIMS...... 21 Coagulation...... 125 Listeria ...... 92 Colitis...... 76 Lymphoma ...... 81 comparative...... 118 MALDI-TOF MS...... 54 construction...... 67 mammary involution...... 75 Cotton-Top Tamarin...... 92 management ...... 13 Covid-19...... 68 mange...... 107 cross-reactivity...... 9 mass mortality...... 46 Culicoides...... 112 Matrix-assisted laser desorption-ionization cyanobacteria ...... 104 mass spectrometry...... 57 Dairy Farms ...... 60 mesothelioma ...... 127 Data...... 21 misclassification error rate...... 9 dermatitis...... 79 Moraxella ...... 53 design ...... 67 Multiple states...... 28

AAVLD Annual Conference Proceedings 133 AAVLD Virtual Conference AAVLD Keyword Index Multiplex assay...... 33 Robustness ...... 11 Multiplex Real-time PCR...... 101, 108 S1 protein...... 14 Mycoplasma...... 55 Salmonella serovars ...... 61 Mycoplasma hyopneumoniae...... 103 sars Cov-2...... 44 Mycoplasma hyorhinis...... 82 SARS-CoV-2 ...... 30, 43 Nanopore...... 29 screening...... 16, 122 Neonatal diarrhea...... 101 seabirds...... 63 Neurologic death...... 80 Sensititre...... 87 Neuropathology...... 68, 73 Sepsityper...... 56 Neutralisation...... 118 sequencing...... 35, 38 Next Generation Sequencing ...... 111 Sequencing...... 48 Next generation sequencing...... 36 Sheep...... 49 next generation sequencing...... 37 Shelter Dogs...... 97 oomycete...... 79 spatially balanced sampling ...... 22 oral fluid...... 31 Streptoccocus suis...... 82 oral fluid samples...... 34 Streptococcus equi subspecies zooepidemicas. . . . 97 oral fluids ...... 9 Streptococcus gallolyticus ...... 83 Oral fluids...... 10, 11 Streptococcus zooepidemicus...... 62 Orbitrap...... 121, 122 stress...... 126 Pandemic...... 43 surveillance...... 22 Paralagenidium...... 79 Susceptibility Testing...... 87 Parasitology...... 108 Susceptibility testing...... 90 Pasteurellaceae...... 63 Swine...... 62 Pathogenesis...... 72 swine...... 83 Pathogens ...... 33, 60 Taqman real-time PCR...... 102 PCR...... 34, 45 targeted NGS...... 53 PCR inhibition ...... 104 Temperature ...... 112 PEDV ...... 47 temperature...... 45 peritoneal...... 127 Thevetia...... 17 Pestivirus...... 49 Tick-borne diseases...... 33 pig...... 126 Total Error...... 70 pinkeye...... 53 Toxicants...... 15 Pinkeye...... 55 Toxicity...... 78 Platelet Function...... 125 Toxicosis...... 17 Porcine Reproductive and Respiratory Toxoplasma gondii...... 128 Syndrome Virus...... 48 Treatment ...... 68 Pork...... 47 Trypanosoma cruzi...... 128 poultry...... 44 Turkey arthritis reo virus ...... 117 Poultry...... 73 Turkey coronavirus...... 14 PrimalSeq...... 35 Tyzzer’s Disease...... 77 Protozoa ...... 108 US...... 28 PRRSV...... 23, 38 validation...... 30 PRRSV ELISA...... 10 Vector borne disease...... 102 PRRSV MLV...... 32 Vegetative valvular endocarditis...... 83 pseudorabies virus...... 31 veterinary drugs ...... 16 Rabbit Hemorrhagic Disease ...... 72 viral isolation ...... 113 real time polymerase chain reaction...... 103 virulence factors...... 63 real-time PCR...... 30, 31, 43 virus isolation...... 45 Renal...... 78 vWGS...... 35 reo virus ...... 117 Wallaby...... 128 Reovirus ...... 73 whole genome sequences...... 36 resistome...... 39 Whole Genome Sequencing...... 88 respiratory...... 93 wildlife ...... 39 Retrospective...... 89 wildlife health...... 107 Reverse transcription real-time PCR ...... 32 Yersiniosis...... 84 RHDV2...... 71 zoonotic disease ...... 59

AAVLD Annual Conference Proceedings 134 AAVLD Virtual Conference