Shayla Bajric¹, Sandra Godden², Evan Brenner¹, Rinosh Mani³, Srinand Sreevatsan¹ 1) Pathobiology and Diagnostic Investigation, Michigan State University 2) Veterinary Population Medicine, University of Minnesota 3) Veterinary Diagnostic Laboratory, Michigan State University

Staphylococcus aureus is a gram-positive bacterium that colonizes the skin and nasal tract of animals and humans. Methicillin-resistant S. aureus (MRSA) is an antimicrobial-resistant form of S. aureus that causes mild to invasive infections. It was first identified in a hospital (HAMRSA) and has since been isolated from communities (CA-MRSA), companion animals, and livestock (LA-MRSA). Resistance to β-lactam antibiotics poses a public health risk as these antimicrobials are largely used in the treatment of infections. It was hypothesized that MRSA isolates from companion animals share ancestry and cluster with HA- or CA-MRSA of human origin and a distant relatedness with LA-MRSA. Bacterial DNA was extracted from MRSA isolates of companion animal and bulk tank milk origin and genome sequences were obtained for genomic analysis to define their lineages and the extent of evolutionary relatedness as well as strain sharing across animals and humans. The isolates were confirmed using MALDI-TOF, coagulase testing, and an antibiogram generated using disk diffusion with cefoxitin. The staphylococcal cassette chromosome mec (SCCmec) type was determined using a multiplex PCR assay to evaluate the strain types of the isolates. This study is expected to develop an understanding of interspecies transmission pathways and provide a baseline for prospective studies in the future.

Miguel Catala¹, Chelsea Yob^2,3, Dr. Dalen Agnew ^2,3 , Dr. Anneke Moresco³ , 1) College of Veterinary Medicine 2) Pathobiology and Diagnostic Investigations, CVM, Michigan State University 3) Reproductive Health and Surveillance Program

Many lesser apes (Hylobates spp.) species are critically endangered or vulnerable according to the International Union for Conservation of Nature (IUCN). The need to protect these species is recognized by many institutions and currently there are three subspecies being managed by zoos which includes white-handed gibbons, white cheeked gibbons, and siamangs. In captivity contraception is one tool used to maintain genetic diversity in these species. Common types of contraception used in lesser apes are Melengestrol acetate (MGA) and Deslorelin implants. The current literature is sparse regarding the overall effects of contraception use in Hylobates sp. This study will examine how contraception impacts the reproductive health of lesser apes. We hypothesize that contraception use increases the risk of reproductive diseases. Samples were taken from the Reproductive Health Surveillance Program (RHSP); 24 female reproductive tracts were examined. Specimens were examined grossly focusing on size, presence of abnormal growths, and overall appearance. Tissue from the vagina, cervix, uterus, and each ovary were processed and stained routinely. Samples were examined microscopically for evidence of disease including pyometra, endometrial hyperplasia, leiomyomas, and other neoplasms. Fisher's exact test was used to determine the frequency of disease in contraceptive animals versus non-contracepted individuals and identify any correlations These findings will allow more effective management of fertility and population health for lesser apes in captivity.

Josephine Daniel¹ and Valerie Johnson² 1. College of Veterinary Medicine, Michigan State University, East Lansing, MI2. Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI

This study aimed to validate giraffe cross-reactivity with commercially available assay kits to measure cytokines in giraffes with osteoarthritis (OA). Cytokines mediate the pathogenesis of OA and, through their effects and actions, are responsible for the disease progression, pain, and lameness associated with the disease. Cytokine concentrations of TNFα, IL-1β, IL-6, and IL-8 were be compared in healthy giraffes without OA to giraffes with OA and also be measure in giraffes with OA before and after mesenchymal stem cell (MSC) therapy, a novel therapeutic alternative for OA management. The purpose of this study is to provide objective evidence of efficacy of MSC therapy used in giraffes with OA by measuring cytokines as indicators of a reduction of inflammation. Commercially available ELISA kits validated for use in sheep exhibited cross-reactivity with giraffe (Giraffa camelopardalis) serum proteins, except IL-6. Banked serum samples previously collected from giraffes (n=9) from multiple institutions were tested. The clinical history of each animal was considered and placed into one of the two sample groups, healthy (n=3) or osteoarthritic (n=6). Fresh whole blood samples from multiple giraffes were also collected and will be used to validate the expression and detection of cytokines in the supernatant of Concanavalin A- and LPS-stimulated peripheral blood mononuclear cells. Cross-reactivity has been observed, however, successful quantification of cytokines in this species with this protocol is still to be determined and will be a focus of investigation moving forward.

Cheyenne N. DeQuattro¹, Emily L. Halsmer², Christine D. Harman¹, Amanda L. Anderson¹, András M. Komáromy¹ , 1: Department of Small Animal Clinical Sciences, Michigan State University College of Veterinary Medicine, East Lansing, MI, 2: Lincoln Memorial University College of Veterinary Medicine, Harrogate, TN

Purpose: Glaucoma is the leading cause of irreversible vision loss due to increasing intraocular pressure (IOP) affecting retinal ganglion cells (RGCs). Currently, there is no cure. Treatment is limited to lowering IOP but RGCs still lose function and die. We hypothesize that the use of the nutraceutical blend Ocu-GLO™ will recover function of damaged RGCs in canine glaucoma. Ocu-GLO™ consists of 12 antioxidants such as grape seed extract (GSE), lutein, vitamins, and omega-3 fatty acids. Methods: In this study, we utilized 10 Beagles with ADAMTS10-OAG to test the neuroprotective capabilities of Ocu-GLO™. Five dogs were supplemented with Ocu-GLO™ and 5 dogs were given sham treatments using a masked study design. Electroretinograms were recorded at baseline, 5-weeks, and 7-weeks of supplementation to evaluate retinal function. Ophthalmic examinations and weekly diurnal IOPs were used as additional outcome measures. For both dog groups, amplitudes and implicit times of retinal responses were compared between baseline and endpoint times to determine if the inner retina showed improvement with Ocu-GLO™. Results: The study was ongoing at the time of abstract submission. Preliminary qualitative analyses did not indicate a positive effect of Ocu-GLO™ supplementation at 5- and 7-weeks. No other clinical outcome measures were affected by supplementation. Conclusions: We did not detect a short-term neuroprotective effect of Ocu-GLO™ supplementation in canine glaucoma. Longer supplementation may be needed to restore retinal function and/or prevent progressive loss of RGCs.

Hunter Ferchaw¹, Ronan Eustace^1,2, Kimberly Thompson^1,3 , (1) Michigan State University College of Veterinary Medicine, East Lansing, MI, (2) Potter Park Zoo, Lansing, MI, (3) Binder Park Zoo, Battle Creek, MI

Species-specific reference intervals are necessary for the proper interpretation of laboratory results in zoo-housed species. Reference intervals may vary between neonates and adults. Thus, the aim of this study is to provide age-specific biochemistry reference intervals for neonatal zoo-housed giraffe (Giraffa camelopardalis). Laboratory results were obtained by performing a retrospective survey of zoological institutions throughout the United States. Inclusion criteria included: giraffe calves born between January 2016 and May 2021 which were deemed healthy by the attending veterinarian, had samples collected via jugular vein and placed into serum separator or lithium heparin tubes, taken at the time of routine neonatal health exams, and performed under manual restraint when calves were less than 72 hours old. Reference intervals were calculated using the American Society for Veterinary Clinical Pathology’s (ASVCP) consensus guidelines for determination of de novo reference intervals. It is anticipated that the results of this study will be a valuable tool for clinicians treating neonatal giraffe calves and allow for more precise interpretation of laboratory results.

Robyn Hawley¹, Peter Fowler², Meghan Milbrath³; 1) College of Veterinary Medicine, 2) Comparative Medicine and Integrative Biology, 3) Department of Entomology; Michigan State University

European foulbrood (EFB) is a significant bacterial disease affecting honey bee larvae. The causative agent, Melissococcus plutonius, causes severe infections which weaken the colony and may lead to collapse. M. plutonius can survive for over three years on microscope slide preparations, however, little is understood about its survival within the hive. Current treatment recommendations, focused on disease control and preventing transmission to other hives, are limited by a lack of information regarding the viability of M. plutonius in the environment. Oxytetracycline hydrochloride (OTC), a bacteriostatic antibiotic, has been used for the treatment of EFB since the 1950’s, and the development of resistance is a concern. Severe outbreaks lead to significant economic loss associated with treatment, labor, and decreased productivity. The aim of this study is to evaluate the viability of M. plutonius on materials involved in beekeeping, including wax foundation, wood, steel, honey, and cloth. These materials are commonly transferred between hives, and the associated risk of EFB transmission is not well understood. These substrates were inoculated with two known strains of M. plutonius: a regional “atypical” strain isolated from a sick hive, and a “typical” ATCC 35311 strain. To evaluate viability over time, cultures were performed at five time points, up to two weeks. M. plutonius was found to survive on all materials for two weeks. Compared to the atypical strain, the typical strain of M. plutonius had poor survival in honey. Understanding the viability of M. plutonius on these surfaces will inform biosecurity, treatment, and prevention recommendations to reduce mortality by EFB.

Cyrus Victor Lacuesta¹, Anna Skedel, Lauren Heine, Ryan Lewandowski, James Wagner, and Jack Harkema^1,2, 1) College of Veterinary Medicine, 2) Pathobiology and Diagnostic Investigation, Michigan State University

Epidemiological studies suggest that people with metabolic diseases are particularly prone to adverse health effects of air pollution. We recently reported that diabetic KKAy mice have more severe lung injury than nondiabetic C57BL/6 mice after repeated exposures to ozone, a common gaseous air pollutant. In the present study we further elucidated the pathogenesis of ozone-induced lung injury and resolution in KKAy mice as compared to C57BL/6 mice. We tested the hypothesis that ozone-induced lung injury is both progressive and persistent in diabetic mice, but not in nondiabetic mice. Both strains were exposed to 0 or 1 ppm ozone for 4, 8 or 12 consecutive weekdays, 4 h/day, and euthanized 1-day postexposure (PE). Another group of these mouse strains were exposed for 12 weekdays but sacrificed 26 d PE (recovery group). Lung tissue was prepared for light microscopic, immunohistochemical and morphometric analysis. Minimal lung lesions were present in ozone exposed C57BL/6 mice. Lesions were restricted to centriacinar regions and resolved by 26 d PE. In contrast, ozone exposed KKAy mice had multifocal, necrotizing alveolitis that expanded beyond centriacini to more distal alveolar parenchyma. There was a time dependent increase in severity of alveolar histopathology that included necrosis and loss of alveolar type I epithelial cells, influx of eosinophils, fibrin accumulation, hemorrhage, proliferation of alveolar type II epithelial cells and interstitial fibrosis. At 26 d PE, KKAy mice had resolving yet persistent epithelial, inflammatory, and interstitial lung lesions. These findings give biological plausibility to the epidemiologic suggestion that people with diabetes are particularly susceptible to respiratory health effects of air pollution.

Kylee Lindsey, Dr. Anneke Moresco, Chelsey Yob, Dr. Dalen Agnew Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, East Lansing, MI (Lindsey, Yob, Agnew), Reproductive Health Surveillance Program (Moresco, Agnew)

Servals are small felids native to African savannas and are considered of Least Concern (IUCN). Servals are common in private and zoo collections. Both populations have breeding individuals; however, the management and handling are drastically different. Reproduction in servals has not been extensively studied, particularly naturally occurring reproductive disease as well as that associated with contraception such as cystic endometrial hyperplasia, pyometra, infertility and cancer potentially. Because serval- specific information is lacking, reproductive care has been extrapolated from domestic cats. To address these questions, we examined reproductive tracts gathered over the last 31 years as part of the Association of Zoo and Aquaria Reproductive Health Surveillance Program (RHSP), grossly and microscopically identifying the characteristics of normal reproductive cycle and the presence of reproductive lesions. We will also use historical data to identify possible risk factors for disease including age, and contraceptive method. We will use Spearman’s correlation test to look at the effect of risk factors on the occurrence and type of lesions present within serval reproductive tracts. We hypothesize that reproductive pathology increases with age and the use of contraceptives in female servals. We expect these results to inform serval owners and population managers on the best reproductive management techniques for this species.

Courtlandt Lyons¹ and Mayra Tsoi^1,2, 1) Michigan State University Veterinary Diagnostic Laboratory, 2) College of Veterinary Medicine, Michigan State University, East Lansing, MI.

Raising backyard poultry has become increasingly popular as people are becoming more aware of their food sources. However, many of these individuals do not have the experience or resources to identify common diseases and obtain timely care. The goal of this retrospective study is to investigate the primary reasons for backyard poultry producers to submit bird(s) to the Michigan State University Veterinary Diagnostic Laboratory (MSU VDL) for necropsy and to determine the most commonly diagnosed diseases affecting these birds. Based on similar data collected in other states across the US, we hypothesized that a large proportion of backyard poultry in Michigan would present with diseases that can easily be prevented by vaccination or preventative care. A total of 436 necropsy cases, based on the USDA definition of backyard chicken flocks, were collected from the archives at the MSU VDL between January 2010-June 2022 and information was collected, including demographics, the presenting complaint, and the most significant disease finding(s). Infectious diseases were identified in 69.9% (305/436) of cases. Of these, 53.1% (162/305) were viral diseases, followed by bacterial infections in 33.1% (101/305). The most common diagnosis was Marek’s disease in 23.1% (101/436) of total cases, followed by Mycoplasma spp. in 17.8% (78/436). Nutritional deficiencies accounted for only 9.8% (43/439) of the total cases but represented the largest proportion of noninfectious cases 31.1% (43/138). Results from this study provide strong evidence that efforts and resources in Michigan should focus on educating individuals and directing veterinary care toward more preventative measures, such as vaccination and nutrition.

Sierra McClain, Margaret Kruger, Annabelle Honet, Carine Holz, Brian Petroff, Jennifer Thomas, Srinand Sreevatsan Affiliation: Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI.

SARS-CoV2, the virus causing the COVID-19 pandemic, continues to spread globally. Despite development of effective vaccines, the virus has endured via the emergence of new variants globally. The emergence of new variants are primarily due to mutations located in the viral spike (S) protein. At least five successful variants of the S protein are now well-established in human populations. This, combined with evidence of documented interspecies transmission to mink, dogs, cats, ferrets, tigers, hamsters, macaques etc., establish that this virus has a broad host range. Transmission to diverse hosts provide an opportunity for the emergence of new mutations and therefore strains. With the heavy burden of infection in human populations, the magnitude SARS-CoV2 infection of pet dogs in households are not well-understood. We hypothesize that pets are exposed to the virus via contact with their owners. An enzyme-linked immunosorbent assay (ELISA) was undertaken to evaluate exposure in pet dogs using five SARS-CoV2 spike variants (as test antigens) and Nobivac Canine 1-Cv vaccine (as control antigen). Dog sera (n =106) collected over the course of the pandemic (December 2019 – May 2021) and control sera (n = 46) collected prior to the pandemic era (2018) will be screened for evidence of SARS-CoV2 exposure in early, mid and late-pandemic era as compared with pre-pandemic times. The modulation of seroprevalence of antibodies against the five reported spike variants over the pandemic period will be evaluated.

Jacqueline Nunnelley¹ and Cheryl L. Swenson.^2,3 1) College of Veterinary Medicine, 2) Department of Pathobiology and Diagnostic Investigation, and 3) Veterinary Diagnostic Laboratory, East Lansing, MI 48824

A 6-year-old, neutered male, Australian Shepherd dog presented to a veterinary practice with a four-day history of lethargy, nonproductive cough, and exercise intolerance. Physical examination revealed a pleural effusion, and a sample was collected for evaluation at the Clinical Pathology Section of the Veterinary Diagnostic Laboratory. The pleural fluid had cell and total protein concentrations of 2,773/μL and 3.0 g/dL, respectively. Large cells with abundant, moderately blue cytoplasm that commonly contained Mott cell inclusions predominated on cytospin concentrated, modified Wright-stained slides. Nuclei of these large cells were eccentrically located and round to pleomorphic with dispersed to condensed chromatin. Anisocytosis and anisokaryosis were moderate to marked with a small proportion of bi to tri-nucleated cells. There also were smaller numbers of nondegenerate neutrophils and few small lymphocytes, macrophages, and eosinophils. Concentrated fresh cell preparations were generated for immunocytochemical (ICC) staining; the large cells expressed CD79a (B cell marker) and lacked expression of CD3 (T cell marker). In addition, sections of a formalin-fixed, paraffin-embedded cell pellet were generated for immunohistochemical (IHC) staining; the large cells exhibited strong expression of multiple myeloma oncogene 1 (MUM1), IgG, and lambda light chains, but lacked expression of IgA or IgM above background. A B cell PARR (PCR for Antigen Receptor Rearrangements) clonality assay, using shavings of the formalin-fixed cell block, was positive. Diagnostic test results support an IgG-lambda plasma (Mott) cell clonal neoplastic effusion. This is a rare diagnosis with a poor prognosis in humans and has not to our knowledge, previously been reported in animals.

Brianna Parkinson¹, Danielle R Ferguson^1,2, Jacquelyn M Del Valle², 1) CVM, 2) Campus Animal Resources, MSU, East Lansing, Michigan

Mice are routinely used as preclinical models in biomedical research; however standard laboratory housing conditions may be associated with cold stress in this species, as ambient housing temperatures range from 20-24^oC, which is below the mouse thermoneutral zone (TNZ) of 29-34^oC. Behavioral discrepancies have been documented between mice housed in standard laboratory conditions versus their TNZ, which has the potential to alter scientific outcomes. We evaluated behavior and nest building in mice provided with a heated floor plate and an elevated platform cradle enrichment device, either individually or in combination. Paired male mice (n=14; CDIIc WT) were housed in individually ventilated cages on a rack equipped with heat plates below one quadrant of each cage. Each cage cycled through four treatment conditions in a randomized order: heat plate off and no cradle (Control), heat plate on and no cradle, heat plate off and cradle, and heat plate on and cradle provided. Cage side observations and videography were utilized to monitor behavior, mouse location in the cage, and nest location and quality. Preliminary results indicate that mice provided with supplemental heat display fewer nesting behaviors, and build poorer quality nests, indicating that heated plates below the cage floor provide thermoregulatory support. Further observation showed a preference to build nests directly above the heat source in mice given supplemental heating. Mice were observed to utilize the cradle enrichment platform for resting, grooming, and urination/defecation; mice did not build nests on the platform. Presence of a cradle did not appear to increase antagonistic behaviors between mouse pairs, compared to when a cradle was not provided.

Sonia Rafique¹, Azam Ali Sher ², Julia A. Bell ³, Linda S. Mansfield ³, 1) College of Veterinary Medicine, 2) Comparative Medicine and Integrative Biology, 3) Microbiology and Molecular Genetics, Michigan State University

The GI tract is rich in bacteria that can transfer antibiotic resistance genes to each other via circular DNA molecules known as plasmids through a mating process termed “conjugation.” This can happen at high rates within the gut, even in healthy individuals. Several studies evaluating the spread of antibiotic resistance genes through plasmid transfers from lab E. coli strains to human gut microbiota isolates have been documented in vitro. However, plasmid transfers have not been examined in vivo which is more complex. Our study tracks the transfer of antibiotic resistance genes in isolates from fecal samples from mice carrying human-derived gut microbiotas to serve as an in vivo model rather than using lab strains of recipient and donor bacteria. Bacterial populations from mouse fecal samples of 3 microbiota types were grown and isolated to act as recipients, while a commensal E. coli strain carrying a fluorescently labeled RP4 plasmid was used as a donor. Filter mating was performed between donor and each microbiota, and transconjugants were isolated using selective media and confirmed by fluorescent microscopy. Two transconjugant colonies were isolated from a fecal sample grown on MacConkey agar selective for enteric gram-negative recipients. Two potential transconjugant colonies may have been isolated from fecal samples grown on Mueller Hinton agar containing nalidixic acid to select for gram-positive recipients, but further testing is needed. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) was conducted to identify the confirmed bacterial conjugants as E.coli/Shigella. We plan to confirm the presence of the RP4 plasmid in these transconjugants using Sanger sequencing of the 16S gene.

Heather Sayles and Dalen Agnew, Michigan State University College of Veterinary Medicine- Pathobiology and Diagnostic Investigation

Meerkat behavior is unique in that they belong to the <1% of mammals that participate in cooperative breeding. A dominant pair breeds and produces offspring, while the subordinate members provide altruistic care for the pups. The reproductive health in this species has not been comprehensively assessed, particularly in light of the effects of dominance and subordinance among females. Previous research has suggested that nonbreeding females have higher rates of reproductive senescence and lower potential fertility than their dominant counterparts. We hypothesize that there is more uterine and ovarian degeneration and reproductive senescence among subordinate females than dominant females. Initial review of archived uterine and ovarian specimens identified multiple morphological variations between animals. Among these, variations in both the quality and quantity of ovarian follicles as well as the presence of uterine lesions were identified for more detailed investigation. Histological examination of uterine and ovarian tissues was conducted on 17 females varying in age from 0.5 - 17 years. Age, contraceptive status, stage of the reproductive cycle, and social status information at the time of sampling was also collected Significant lesions included hyperplasia, endometrial cysts, hemorrhage, and follicular atresia. Statistical analysis is pending to determine the correlation of lesions with social dominance and other potential risk factors. By understanding the role of reproductive suppression on the overall reproductive health of captive meerkats, we can enhance conservation efforts both in zoological institutions and in the wild.

Kathryn E. Simmons, Julie M. White, Katherine R. West, Aimee C. Colbath, & Harold C. Schott II Department of Large Animal Clinical Sciences, Michigan State University

Exercise-Induced Pulmonary Hemorrhage (EIPH) is common problem in racehorses. In the United States, furosemide is administered 4 h prior to racing to attenuate EIPH. Non-steroidal anti-inflammatory drugs (NSAIDs) are also commonly administered to racehorses. Current regulations for Quarter Horse racehorses allow administration of either phenylbutazone, flunixin meglumine, or ketoprofen 48 h before competition. Previous research has demonstrated that NSAID administration, an hour prior to furosemide administration, can limit the furosemide-induced diuresis by 25-30%. To determine whether current regulations for administration of NSAIDs to Quarter Horses affect furosemide-induced diuresis, we measured urine production (volume) during the 4 h after furosemide administration after pre-treatment with these NSAIDs. We hypothesized that the 48 h withdrawal period after NSAID administration would have no impact on subsequent furosemide-induced diuresis. To test this hypothesis we studied eight healthy mares in a replicated 4 x 4 Latin Square design. Horses were administered a single dose of each NSAID or saline as a control treatment 48 h prior to furosemide administration. In the hour before furosemide administration, mares were instrumented with bilateral ureteral catheters. Following a baseline urine collection period, furosemide (1 mg/kg, IV) was administered and total urine produced in the subsequent 4 h was collected. We found no difference (mean±SD) in urine production between the four treatments: control 20±4 mL/kg; phenylbutazone 18±3 mL/kg, flunixin 20±4 mL/kg, and ketoprofen 20±2 mL/kg (p=0.19). In conclusion, administration of NSAIDs 4 h prior to furosemide administration had no effect on furosemide-induced diuresis.

Abigail Smith¹, Jared Jaffey², Sylvia Ferguson², Victoria Watson¹ 1) College of Veterinary Medicine, Michigan State University, 2) College of Veterinary Medicine, Midwestern University.

Feline Gastrointestinal Eosinophilic Sclerosing Fibroplasia (FGESF) is an entity found in the alimentary tract and lymph nodes of cats. Common clinical findings include eosinophilia and masses at the ileocecocolic junction or pyloric sphincter. The cause and pathogenesis of FGESF is undetermined, but the role of eosinophils suggests genetic predisposition to eosinophilic inflammation or potential bacterial or parasitic infection. Previous studies have demonstrated mixed, intralesional bacteria in FGESF, but was thought to be secondary to the lesion and not the cause. Nematode migration has been associated with sclerotic and eosinophilic gastritis in non-domestic felids. We hypothesize that a common nematode of cats, Toxocara cati, plays a role in the pathogenesis of FGESF. In this study, we determined common clinical and histopathologic features of FGESF in 40 cases of suspected FGESF. Clinical data was available for 34 cases; all cats presented with gastrointestinal signs of varying duration. Complete blood counts were performed on 22 cats and 10/22 (45%) cats presented with eosinophilia. Hematoxylin and eosin-stained sections of affected tissues were scored for the presence of collagen, necrosis, and inflammation. Expansion of the submucosa and muscularis was measured for each case. Histopathologic features of FGESF included expansion of the submucosa by trabeculae of collagen, eosinophils, plump fibroblasts, and mixed mononuclear inflammatory cells. The average number of eosinophils within lesions of FGESF was 40.75 eosinophils per 10x10 grid at 40x. In this study, we will also have determined the role of T. cati in the pathogenesis of FGESF via investigation of T. cati DNA within 14 cases of FGESF.

Eric Spilker ¹, Hinako Terauchi ² and Linda Mansfield ^1,2, 1) College of Veterinary Medicine, Michigan State University, 2) Department of Microbiology and Molecular Genetics

Chemotherapy-induced gastrointestinal disease (CIGD) has been associated with chemotherapy treatment resulting in symptoms of clinical nausea, vomiting and diarrhea. CIGD can decrease quality of life and is a common reason for discontinuation of chemotherapy treatment. We hypothesized that CHOP protocol chemotherapy agents either individually or synergistically decrease the diversity of the canine fecal microbiome and trigger dysbiosis of the gut microbial community gastrointestinal bacteria in dogs with lymphoma. Twenty-two canine lymphoma patients undergoing cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy had fecal samples collected weekly before and throughout treatment. 16S rRNA gene sequencing analysis was performed on DNA from fecal samples to determine the composition and relative abundance of bacterial taxa. For each dog, 16S sequencing data from longitudinal samples were analyzed using QIME2 and bacterial taxa assignments made using the SILVA database. At specific intervals during CHOP protocol, changes in the relative abundance of bacterial taxa were observed that provided evidence that CHOP protocol administration caused dysbiosis within the canine gastrointestinal tract. We found that during periods of diagnosed CIGD acute changes in relative abundance of specific bacterial taxa occurred. Acute changes in bacterial diversity were observed to correlate with diagnosed CIGD. Additional analysis of canines with clinical signs of CIGD that have an increased relative abundance of pathogenic bacteria will be assessed for significance. Early results confirm CHOP chemotherapy was associated with dysbiosis and changes in bacterial diversity correlating with CIGD in dogs with lymphoma.

Luke VanBlois, Juan-Ting Liu, Bartolomeo Gorgoglione Fish Pathobiology and Immunology Laboratory, Dept. Pathobiology and Diagnostic Investigation / Dept. Fisheries and Wildlife, Michigan State University

A massive acute mortality event of farmed fish was reported in a fish farm operating in Southeastern Michigan. Over 40,000 lbs (18.15 mt) of a mixed stock of Largemouth Buffalo ( Ictiobus cyprinellus) and Common Carp (Cyprinus carpio) were lost within a span of two days. A new batch of Largemouth Buffalo captured in another midwestern state was introduced into the earthen pond a few days ago. During the inspection, carcasses of fish of various sizes were seen on the pond’s surface. Some of the remaining fish of different species were gasping for air at the pond’s surface and lost their escape response. Some newly dead or survival fish including six largemouth buffalo, five common carp, and one bluegill (Lepomis macrochirus) were submitted for necropsy and no clear evidence of acute infection was noted except few fish lice were found on the gills and skin of the largemouth buffalo. Due to the rapid onset of mortality in bigmouth buffalo and common carp and the history of introducing new fish within the last week, some most suspected pathogens included Cyprinid herpesvirus 3 ( K o i herpesvirus, KHV), carp edema virus, and spring viremia of carp virus were suspected and fish organs including gill, spleen and kidney were sampled for biomolecular examination. Conventional PCR were applied to detect the Cyprinid herpesvirus 3, carp edema virus, and spring viremia of carp virus following OIE suggested protocol and all of them showed negative result. Concluded from the complaint, field investigation, necropsy, and biomolecular examination result, a lack of oxygen was diagnosed and increasing the amount of oxygenation sources to all ponds were recommended.

U. Abou-Rjeileh, M. Chirivi, J. Parales, A. L. Lock, and G. A. Contreras. Michigan State University

During the periparturient period, adipose tissue (AT) lipolysis is driven by insulin resistance (IS) and limited energy intake. Excessive lipolysis in periparturient cows is associated with poor lactation performance and increased incidence of metabolic diseases. Chromium (Cr) supplementation improves systemic IS, while palmitic acid (PA) enhances energy density of the diet and increases milk fat yield. However, the effect of feeding Cr alone or in combination with PA on AT metabolism is unknown. Thirty-two multiparous Holstein cows were selected at -21d before parturition. After calving, cows were randomly assigned to one of four diets; control (CON, no supplementation), Cr (Crpropionate at 0.45 ppm Cr/kg DM), PA (1.5 % DM), or Cr+PA that were fed 1-21 d postpartum. Plasma and subcutaneous AT samples were collected at -13±5.1 d prepartum (PreP) and 14±1.9 d (PP1) and 21±1.9 d (PP2) postpartum. Demand lipolysis was determined in the presence of β-adrenergic receptor agonist isoproterenol (ISO, 1μM). The antilipolytic effect of insulin (IN, 1μL/L) was evaluated during ISO stimulation (IN+ISO). Lipolysis was quantified by glycerol release in the media (nmol glycerol/mg AT). Compared with Cr+PA, Cr cows tended to have larger adipocytes at PP2 ( P=0.07). Basal lipolysis postpartum, reflection of AT capacity to store lipids, was higher in Cr compared with PA and Cr+PA (P<0.01). In Cr cows, ISO induced higher lipolysis compared to Cr+PA ( P<0.01). In Cr cows, IN+ISO reduced lipolysis by 25% compared to ISO (P<0.01). No effect of IN+ISO was observed in CON, PA, and Cr+PA. Compared with Cr+PA, Cr tended to reduce plasma NEFA (P=0.07). Compared with all diets, Cr reduced plasma glucose at PP1 ( P<0.05). Our results demonstrate that Cr supplementation, during the early postpartum period, enhances AT insulin sensitivity promoting higher lipid accumulation in AT.

Laura M. Ford¹, Laurence M.Occelli², Paige A. Winkler², Kelian Sun², Nathanial Pasmanter², Janice Querubin², Leslie A. Lyons³, 99 Lives Consortium, Simon M. Petersen-Jones^1,2 1)Genetics and Genome Sciences, 2)SACS, MSU, 3)University of Missouri

We identified a cat with a phenotype comparable to macular degeneration (MD) in humans. The area centralis (AC; region similar to the human macula) progressively degenerated and the visual streak showed abnormal hyporeflectivity. Segregation of the phenotype was supportive of an autosomal recessive mode of inheritance. Under the 99lives cat genome sequencing initiative, two clinically affected and one obligate carrier cats were submitted for whole genome sequencing. Three candidate variants exclusive to the two homozygous affected and heterozygous cats were identified using a variant calling analysis. On genotyping further affected cats in the colony a missense variant in the Retinol Dehydrogenase 5 (RDH5) gene (p.G181V) was the only one to segregate with the disease phenotype. RDH5 catalyzes the final oxidation in the visual cycle, producing 11-cis-retinal which is needed for regeneration of the photoreceptor photopigments for continued vision. Western blotting and immunohistochemistry of retinal samples from cats homozygous for the p.G181V mutation had no detectable RDH5. Humans with RDH5 mutations develop fundus albipunctatus (FA) characterized by night blindness and white-yellow flecks on the fundus with a subset experiencing MD. Similarly to RDH5-mutant humans, RDH5-/- cats show variable age of onset and severity of AC degeneration. As the cats are identically housed, environmental influences on phenotype variation are less likely, implicating a possible modifier locus. This large animal model and potential modifier locus will provide further understanding of MD and allow the development of treatments for human RDH5 retinopathies.

Carmen-Maria Garcia, Kyan Thelen, Dr. Adam Moeser 1)College of Veterinary Medicine 2)Comparative Medicine and Integrative Biology

Across species, sex differences are often found in disease risk and mortality. In pig production, castrated male piglets generally have a higher mortality rate of about 1.4 to 1.8 times that of females. Preliminary transcriptome data produced by our laboratory, indicates that current early weaning practices led to a long-term downregulation of extracellular matrix genes in male castrates. Although it has been established that there are sex differences in disease risk and immune activation, nothing is known about the effects of biological sex and castration status on the immune system of the piglet. Now, little is known about the effect of androgens on the early development of the immune system in piglets. The goal of this research is to investigate effects of castration and sex differences on the GI inflammatory response. The mucosa from the jejunum and ileum was harvested from female (F), male castrate (MC) and intact male (MI) piglets at 0hr (unweaned) and 24hr post-weaning. RNA was extracted to determine the expression level of inflammatory and extracellular matrix genes. We hypothesize that due to sex and castration status there will be differences in activation of the immune system within the gastrointestinal (GI) tract of piglets. This knowledge is not only important for furthering our understanding the impact of sex differences and the effect of castration, but it is also could be applied to the management of livestock at the level of the farms to improve overall herd health.

Ivon A. Moya Uribe^1,2, Hinako Terauchi^1,3, Susan L. Ewart³, Julia A. Bell¹, Linda S. Mansfield^1,3 1Comparative Enteric Diseases Laboratory, 2Comparative Medicine and Integrative Biology 3College of Veterinary Medicine, Michigan State University

Recent literature suggests that gut microbiota impacts lung function during allergic airway disease (AAD). We transplanted three groups of germ-free mice with a human fecal microbiota from either eczemic infants (risk), non-eczemic infants (protective), or healthy young adults. After transplantation, microbiotas remained stable over two generations. Offpsring from each microbiota group were sensitized with house dust mite (HDM) antigen or vehicle for 12 days to induce AAD and lung function was tested at 14 days. Mice with each transplanted human microbiota showed decreased lung function at baseline compared to mouse microbiota in both allergic and non-allergic conditions. In addition, the differences in lung function at baseline did not correlate with the amount of inflammation deposited around the airways, or with the amount of specific anti-HDM-IgE measured. However, mice carrying any of the human microbiotas showed increased serum baseline levels of IgE compared to those with mouse microbiota. To further examine this finding, we measured baseline serum IgE levels using ELISA in mice carrying the 3 different gut microbiotas, from different generations and in different age ranges. We also performed a correlation analysis between the lung function parameter values at baseline and baseline IgE concentration. Results showed mice carrying any of the human microbiotas had higher baseline IgE levels compared to the control mouse microbiota. This difference was maintained throughout generations and age ranges and older mice expressed higher levels of baseline IgE than younger mice. Our results also suggest that the observed decrease in lung function may be mediated by a mechanism associated with high levels of systemic baseline IgE. Our study thus suggests a major effect of gut microbiota on shaping immune system development and respiratory mechanics. Furthermore, early life microbiota composition appears to have a direct effect on lung function

Sajani Thapa¹, Raul G. Barletta², Denise K. Zinniel², John Bannantine³ and Srinand Sreevatsan¹ , 1) Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, 2) School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, 3) National Animal Disease Center, Ames, IA

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in cattle that is associated with significant impact on agricultural economy and animal health. A greater understanding of the virulence and survival mechanisms of MAP is required to control this insidious infection. Upon phagocytosis, MAP faces severe iron restriction in the phagosomes followed by an oxidative stress when the lysosome fusion occurs. Thus, intrabacterial iron regulation is critical to the in-vivo survival of MAP. To overcome the iron restriction, bacteria modulate iron acquisition from the host by iron uptake systems. MAP carries a fur-like gene which is absent from other mycobacteria. Ferric Uptake Regulator (Fur) is well studied in other bacteria and has been shown to be involved in global regulation of iron homeostasis. Very little is known about this novel regulator in MAP. The current study was performed to characterize the Fur regulon in MAP under iron stress conditions through transcriptional analysis. An in-frame deletion of Fur in MAP strain K10 was created through homologous recombination. Parent strain, mutant and a complemented strain were exposed to iron restricted conditions for 5, 35, 65, and 95 minutes. Total RNA was extracted, and quality and integrity of RNA was determined before RNA-Seq was performed. RNA-Seq analysis will be performed to elucidate the metabolic pathways that MAP employs to overcome the iron stress caused by the host providing a window into a key intracellular survival mechanism.

Michelle Volk¹, Vishvapali Kobbekaduwa², Jean Tsao^1,3 1) Department of Fisheries and Wildlife, 2) Comparative Medicine and Integrative Biology, 3) Department of Large Animal and Clinical Sciences, Michigan State University, East Lansing, Michigan, U.S.A.

Lyme disease is the most prevalent vector-borne disease in North America with an estimated 300,000 human cases each year. Over the past few decades, blacklegged ticks (Ixodes scapularis), the primary vector of Borrelia burgdorferi sensu stricto in North America, have been invading the state of Michigan, USA. Blacklegged ticks have been detected in more than half of the counties, several of which are endemic for Lyme disease. Currently, blacklegged ticks are spreading northward and inland across the state but there are still several counties in the north central Lower Peninsula where they have yet to be detected. To update our understanding of the spread of blacklegged ticks and B. burgdorferi prevalence, we conducted state-wide surveillance in 2019 and 2021. From May-July in 2019 and 2021 we sampled 96 sites and 171 sites, respectively, with suitable habitat for blacklegged ticks. In 2019, we collected blacklegged ticks from 73/96 sites and from 109/171 sites in 2021. In 2019, 46 sites were assayed for nymphal B. burgdorferi prevalence which ranged from 0-28.6%. In 2021, 17 sites were assayed, and B. burgdorferi prevalence ranged from 0-40%. We found that blacklegged tick densities and B. burgdorferi prevalence were highest in the southwestern Lower Peninsula and western Upper Peninsula, which corresponds to areas in Michigan with the greatest Lyme disease incidence. In both years, our sampling yielded few or no blacklegged ticks in the interior northern Lower Peninsula and eastern Upper Peninsula. In 2021, blacklegged ticks were detected in four new counties, and 11 new counties reported established populations, indicating increased nymphal densities and disease risk.

Charles Whitehead-Tillery¹, Lixin Zhang¹, Linda S. Mansfield¹ 1) Microbiology & Molecular Genetics, , Michigan State University

Antibiotic resistance (AR) is a major challenge for the health care system with extended-spectrum beta-lactamase (ESBL) producing bacteria being the main threat. Horizontal gene transfer tends to be the mechanism responsible for this circumstance. Infections cause by ESBL producing bacteria occur mainly in hospital settings, yet community-acquired infections are emerging. Many studies have identified CTX-M- 1 subgroup as the most prevalent enzyme disseminated worldwide. Thus, we hypothesized our collection will harbor ESBL enzymes under the CTX-M-1 subgroup and will be able to transfer ESBL genes to recipient bacteria. A panel of CTX-resistant Escherichia coli carrying unknown ESBL enzyme bearing plasmids were isolated from hospitalized patients. 14 E.coli ESBL plasmids were tested for cefotaximeresistance and put through an in vitro conjugation system to determine transfer of ESBL genes to modified E.coli strain MG1655. ESBL plasmids were categorized into different subgroups of CTX-M and incompatibility groups by PCR. Additionally, 16 ESBL plasmids were sequenced using Nanopore technology with bioinformatic tools allowing for gene annotation and identification of ARGs. Annotations of 16 isolates identified ESBL genes CTX-M-14, CTX-M-15, CMY, incompatibility groups (IncF, IncI1, and IncQ), and insertion sequences upstream/ downstream from ESBL gene among these plasmids. Lastly, 14 ESBL bearing E. coli isolates were resistant to cefotaxime and conjugation occurred with MG1655 E.coli to produce transconjugant colonies resistant to cefotaxime and rifampicin. With this information we expect to find similar genetic elements in companion animal isolates to provide insight for if these hosts are a source for the emergence of ESBL transfer in the community.

Margaret Krueger¹, Carine Holz¹, Annabelle Honet¹, Joseph Darish¹, Steven Bolin¹, Justin Greenlee² and Srinand Sreevatsan¹ 1) Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, 2) National Animal Disease Center, United States Department of Agriculture

The increasing identification of chronic wasting disease (CWD) throughout the United States and the world, emphasizes the need for quick and reliable screening tests. The current gold standard for CWD testing involves post-mortem immunohistochemistry (IHC) on retropharyngeal lymph nodes. We hypothesized that CWD -associated immunopathology and /or neuropathology results in leaky proteins in the serum that would serve as disease biomarkers. Enzyme linked immunosorbent assay (ELISA) was performed on sera from experimentally infected deer, for specific peptides defined either previously in Creutzfeld Jakob disease (S-100b, 14-3-3γ) or discovered in our laboratory using proteomics (Fetuin, Haptoglobin). First, we compared levels of 14-3-3γ levels in white-tailed (WT) deer sera before CWD infection and one-year post-infection. The post-infection samples tend to have increased reactivity and luminescence values. However the differences were not statistically significant, possibly because of the small sample sizes used. Increasing sample sizes also did not define 14-3-3γ as a reliable biomarker. Thus, a new biomarker discovery protocol with multidimensional proteomics was initiated comparing the preand post-infection samples. Data showed that haptoglobin was the only protein elevated post infection in two sets of proteomics. Future testing will involve ELISA analysis with WT deer specific antibodies for haptoglobin. This new biomarker assay will likely serve as a screening tool to determine possible CWD cases for further testing while eliminating samples from animals without neurological damage.

Swati Sharma¹, Kennedy Baldwin³, Glorián Berríos-Vázquez¹, Roger Maes^{1, 2}, Gisela Soboll Hussey¹ College of Veterinary Medicine, Department of Pathobiology and Diagnostic Investigation¹, Veterinary Diagnostic Laboratory ², College of Natural Science, Department of Microbiology and Molecular Genetics³, Michigan State University, East Lansing, MI 48824

Canine infectious respiratory disease complex (CIRDC), commonly known as “kennel cough,” is a disease caused by different etiologic agents and is characterized by contagious respiratory disease in dogs. Viral pathogens associated with CIRDC include canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine herpesvirus-1 (CHV-1) and canine influenza virus (CIV). Vaccine availability and efficacy varies and is negatively impacted by viral pathogen variance and poor immunogenicity. Our hypothesis is that canine respiratory epithelial cells cultured at air-liquid interface (ALI-CRECs) can be used to evaluate viral replication and the immune response generated after infection with viruses involved in CIRDC and test antiviral agents. To test this hypothesis, we collected epithelial cells from tracheas of 5 respiratory healthy dogs that were euthanized for unrelated reasons. Cells were isolated, cultured and characterized morphologically and immunologically before infecting them with CDV, CHV-1, CIV & CAV-2. Immune markers compared included expression of TLRs, interferons, cytokines and chemokines in tracheas of dogs, freshly isolated CRECs and ALI-CRECs. We found that ALI-CRECs resembled the natural airway morphologically and immunologically and supported infection and replication with CDV, CHV-1, CIV & CAV-2. In conclusion, ALI-CRECs maybe ideal systems to study the molecular pathogenesis and induction of innate immune responses upon infection with viruses involved in kennel cough. Furthermore, they can be used to test efficacy of antivirals aimed to prevent viral replication and spread.

Winkler PA¹, Bortolini M², Moreno J², Sato M³, Guareschi B², Montiani-Ferreira F², Petersen-Jones SM¹ 1. Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University 2. Department of Veterinary Medicine, Federal University of Paraná 3. Department of Ophthalmology, Federal University of Paraná

A family group of German Spitz dogs with early-onset progressive retinal atrophy (PRA) was characterized using full ophthalmic examination, electroretinogram and optical coherence tomography. Twelve candidate genes were screened for association with four affected German Spitz dogs. Marker variability was assessed using six unaffected dogs on the unexcluded genes. DNA samples from two affected siblings, one unaffected half sibling and one obligate carrier parent were sent for whole genome sequencing (WGS). Sequences were processed and filtered for variants unique to the affected dogs. Sanger sequencing was used to confirm segregation of the variant with additional samples from the pedigree (N=12). PRA-affected German Spitz dogs had undetectable rod and decreased cone responses at three months of age. Cone responses were unmeasurable by nine months of age. However, retinal thinning was not apparent until three years of age. Autosomal recessive inheritance was indicated by the pedigree. Nine of twelve candidate genes were excluded for association with the disease by microsatellite markers. Using WGS, a 1 bp insertion (c.1598_1599insT, p.Ser534GlufsTer20) was detected in one of the unexcluded candidate genes (GUCY2D) which segregated with the early-onset PRA phenotype within the pedigree. The combination approach of 1) well-phenotyped dogs; 2) initial candidate gene screening using microsatellites and 3) WGS variant filtering from a close family group, allowed for the quick identification of a frameshift variant within an unexcluded candidate gene.

Joseph Darish¹, Rinosh Mani^1,2, Scott Fitzgerald^1,2 and Srinand Sreevatsan¹,1)Department of Pathobiology and Diagnostic Investigation, 2) Veterinary Diagnostic laboratory Michigan State University

Bovine Tuberculosis (bTB), caused by Mycobacterium tuberculosis variant bovis (MBO) is a contagious cattle disease that accounts for $3 billion in agricultural economic losses worldwide. Bovine tuberculosis is generally screened for with a Caudal Fold Test (CFT) and confirmed with mycobacterial culture. However, a CFT test lacks specificity and can generate false positive results, while confirmatory bacterial isolation tests are slow, delaying timely diagnosis of bTB in cattle herds. We evaluated Actiphage, a novel phaged-based assay, that has been shown to detect Mycobacterium bovis from whole blood. Actiphage is based on the principle of mycobacteremia which, if present, will be lysed by bacteriophage D29 and detected by IS6110 Polymerase Chain Reaction (PCR). Eighty-Six blood samples from Michigan (n=42) and New Mexico (n=44) cattle herds were evaluated by Actiphage. None of the samples were positive by the Actiphage procedure. Twelve of these 86 samples were from CFT positive animals, and one animal was confirmed positive by culturing MBO from lymph node lesions. The 12 CFT positive samples were further analyzed with pathogen specific biomarkers (Mb2515c, Mb1895c, and pks5). Four of these 12 samples were reactive to Mb2515c (a LuxR family transcription regulator) and Mb1895c (molybdenum binding protein). The Actiphage technology did not increase MBO diagnostic sensitivity when compared to current diagnostic strategies. Evaluation of pathogen specific protein biomarkers and antigen-specific cytokine transcriptional profiles and/or protein arrays, in conjunction with bacterial and Interferon Gamma Release Assays (IGRA), offer a promising avenue for early detection of bTB.

Giuseppe Cavaliere,¹ Anneke Moresco, DVM, PhD,² and Dalen Agnew, DVM, PhD, Dipl ACVP ³,1) Microbiology and Molecular Genetics, Michigan State University; 2) Reproductive Health Surveillance Program; 3) Pathobiology and Diagnostic Investigation, Michigan State University

Gazelles are threatened or endangered and captive populations are kept as insurance populations. However, reproductive disease can impact the viability of these populations. Tools to evaluate the female reproductive tract are needed to understand these threats to fertility. This investigation focused on comparing the relative sizes of the myometrium, endometrium, and lumen as percentages of the cross-sectional area of the uterus in gazelles during follicular and luteal phases to identify and quantify hydrometra. Gazelles in this study were classified as being in the genera of Gazella, Nanger, and Eudorcas. Previous research is limited in gazelles; however, studies in rats and ruminants suggest the overall area for each of the uterine components, primarily the endometrium and lumen, change significantly throughout the estrous cycle and in disease states. Microscopic images of uterine cross sections of 19 gazelles were taken and processed using imaging software to determine the relative percentage of each compartment. The estrous phase of each gazelle was determined based on the provided history and ovarian findings. Two Mann Whitney tests and a Pearson correlation were performed. Lumen size was positively correlated with the identified estrous cycle phase (p-value=0.008), lumen size was not correlated to known contraception status (pvalue= 0.1324), and the correlation of parity was not correlated with lumen volume (p-value=0.8112). These results are significant as understanding gazelle’s reproductive cycle is important to ensure proper management of captive populations.

Taylor Rabanus, Dawn Kuszynski, Barbara Christian, and Adam Lauver, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA

Arterial thrombosis remains the most common cause of death in the developed world. Clopidogrel, a P2Y ₁₂ antagonist, is often prescribed to inhibit platelet activation and aggregation; however, patients who take clopidogrel are known to have cerebral bleeding events. The purpose of this study was to determine if the clopidogrel bleeding effect is dependent on P2Y₁₂ receptor inhibition. Previous research from our laboratory suggests that clopidogrel possesses inhibitory effects on hemostasis independent of platelet P2Y₁₂ inhibition. We hypothesized that P2Y₁₂-deficient mice treated with clopidogrel will bleed significantly more than the P2Y₁₂-deficient mice treated with the vehicle. A tailbleeding assay was used to determine clopidogrel's effect on hemostasis. P2Y₁₂ deficient mice were adminstered clopidogrel (10 mg/kg) or vehicle daily for five days before the procedure was performed. Bleeding was evaluated using a tail amputation assay. After tip amputation, the tail was quickly placed in a tube containing Drabkin's reagent. Blood loss was determined by measuring the concnetration of hemoglobin in the tube using a standard curve. Our data demonstrate that clopidogrel induced more bleeding in the P2Y₁₂-deficient mice. These results suggest that clopidogrel's bleeding effect is not completely dependent on the P2Y₁₂ receptor inhibition. Other elemnets in the hemostatic system, such as the vessel-related factors, could be responsible for the observed clopidogrel bleeding effects.

Caleb Ragsdale¹, C.J. Rendon², G. Andres Contreras^{2 1}Undergraduate at Michigan State University ²Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI

Perivascular adipose tissue (PVAT) modulates vascular function by supplying support and secreting vasoactive factors. PVAT function differs across anatomical location. Brown adipocytes predominate thoracic aortic (APVAT) but most adipocytes in abdominal aortic (ABPVAT) are white. Adipocyte populations are maintained by adipogenesis of adipocyte progenitors (AP) into adipocytes. Changes in AP populations alter PVAT function, increasing the risk for hypertension. AP expressing platelet-derived growth factor receptor alpha (PDGFRa) may help with regulating PVAT expansion and vascular smooth muscle cell (vSMC) function. Although dimorphism in PDGFRa abundance has been identified in rats, tracer models that track AP localization allows for better characterization of PVAT conditions. Our aim was to characterize AP localization and phenotypic differences across sex and APVAT and ABPVAT. Tissue slides collected from AP tracing double transgenic mice [PDGFRa-CreERT2/R26-LSL-tdTomato] were stained. Resulting in tdTomato, a traceable protein that depicts PDGFRa₊ cells upon tamoxifen treatment, and vSMCs being marked. PDGFRa ₊ abundance and proximity to vSMCs were analyzed using ImageJ® and Python®. In both sexes, PDGFRa₊/vSMC proximity was lower in APVAT compared to ABPVAT. PDGFRa ₊ abundance and vSMC proximity was enhanced in females’ APVAT and ABPVAT. We hypothesize that sex influences AP abundance and vSMC proximity. AP tracing allows for the identification of the effects of sex and anatomical location on the PVAT microenvironment and a better understanding of the PVAT/vSMC relationship.

Kimberley Sebastian¹, Rebecca Smedley¹, Alexander Bartel², Matti Kiupel¹ 1) Michigan State University, East Lansing, MI, USA 2) Freie Universität Berlin, Berlin, Germany

Hepatic lymphoma is poorly characterized in cats and differentiating between inflammation and lymphoma is often difficult. Human hepatic lymphoma diagnosis relies on certain patterns of lymphocytic infiltrates and clonality because other features have not been proven to be predicative. Herein, we define patterns of lymphocytic infiltrates in hepatic biopsies of cats and correlate them with clonality to determine which patterns were predictive of lymphoma. A retrospective study was performed using surgical biopsies from 44 cats of which all had been tested by PCR for T-cell receptor gamma gene rearrangements and 24 cats tested by PCR for immunoglobulin heavy chain gene rearrangements. Four patterns of lymphocytic infiltrates were determined: (1) tightly periportal, (2) periportal and centrilobular, (3) nodular, and (4) periportal with sinusoidal extension. A diagnosis of lymphoma was based on microscopic examination, immunophenotyping, and clonality. Various other histomorphologic features were evaluated. The sensitivity and specificity of the lymphocytic patterns in the diagnosis of lymphoma were determined using Bayesian Hui-Walter analysis against clonality results. Bayesian analysis demonstrated that the different lymphocytic patterns accurately diagnosed hepatic lymphoma with a sensitivity and specificity of 82% (CI 95%: 0.65, 0.96) and 77% (CI 95%: 0.54, 1.00), respectively. None of the other microscopic features evaluated were predictive of lymphoma or inflammation. Our study identified specific patterns of lymphocytic infiltration that help in differentiating feline hepatic lymphoma from inflammation. We highlight the lack of predictability of some histologic features previously thought to be associated with lymphocytic hepatitis, raising the importance of clonality testing.

Chelsea Stecklein¹, Cynthia Lucidi¹, and Michael Scott¹, 1) Pathobiology and Diagnostic Investigation, Michigan State University

Accurate detection of canine erythroid precursors (nRBCs) is necessary for the clinical diagnosis of precursor-targeted immune-mediated anemia (PIMA) and hematopoietic proliferative diseases, such as erythroleukemia. Early erythroid precursors are impossible to identify reliably via routine hematoxylin & eosin (H&E) staining. Additional diagnostic methods, such as immunohistochemistry (IHC) or flow cytometry, would be useful to aid in their identification. However, a commercially-available antibody to detect canine nRBCs has not been reported. The aims of this study were to identify such an antibody using formalin-fixed spleen and formalin- and B5-fixed bone marrow tissues and to develop and validate an IHC assay to detect canine nRBCs. To date, we have successfully detected canine nRBCs using an antibody that targets the transferrin receptor (CD71). The antibody showed strong immunolabeling of nRBCs and lack of labeling of other cells (e.g., myeloid precursors, megakaryocytes, and mature RBCs) in spleen and bone marrow. We are currently optimizing the IHC protocol by performing antibody dilutions to determine the optimal signal-to-noise ratio. Following optimization, we will explore the clinical application of the working antibody by confirming consistent immunolabeling of canine nRBCs yet negative immunolabeling for neoplastic nonerythroid hemic cells (lymphoma, acute myeloid leukemia, histiocytic sarcoma). Completion of this project will yield a novel IHC assay for canine nRBCs that will facilitate the diagnosis of certain erythroid disturbances in dogs and provide direction for future development of flow cytometric assays.

R.E. Egbert, A.M. Laclair, J.M. Brudvig, K.R. Refsal, B.K. Petroff. Veterinary Diagnostic Laboratory, Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University.

Canine hypothyroidism is the most commonly diagnosed veterinary endocrinopathy. Evaluation of circulating thyroid hormone concentrations is done in comparison to reference intervals (RIs) calculated from healthy dogs of many breeds. Deviation from the established reference intervals can provide evidence of thyroid disease. However, previous research suggests that healthy dogs of certain breeds can have thyroid hormone concentrations below the lower reference limits of the all-breed reference intervals. This research aims to develop breed-specific reference intervals for Borzoi dogs. Thyroglobulin autoantibody negative samples submitted to the Endocrinology section of MSU’s Veterinary Diagnostic Laboratory for a pre-breeding thyroid certification were used (n = 259). Reference intervals were calculated using Reference Value Advisor (V2.1). Breed-specific reference intervals for free thyroxine (FT4), total thyroxine (TT4), total triiodothyronine (TT3), and thyroid stimulation hormone (TSH) were calculated, as well as the current all-breed RIs. Calculated RIs for FT4D, TT4 and TT3 trended lower than the current all-breed reference intervals while TSH ran higher. This research suggests that healthy Borzoi dogs can have lower concentrations of circulating thyroid hormones, as previously observed in other sight hound breeds.