SARS-COV-2 INFECTION AND LONGITUDINAL FECAL SCREENING IN MALAYAN TIGERS (PANTHERA TIGRIS JACKSONI), AMUR TIGERS (PANTHERA TIGRIS ALTAICA ), AND AFRICAN LIONS (PANTHERA LEO KRUGERI) AT THE BRONX ZOO, NEW YORK, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (Panthera tigris altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids.

Hematology Red Flags: The Value of Blood Smear Examination in Horses.

This article uses a case-based approach, complemented with diagnostic algorithms and images, to highlight hematologic changes of pathologic relevance in horses, namely, marked erythrocytosis, anemia or leukocytosis, inflammatory leukograms, lymphocytosis in adult horses, thrombocytopenia, and pancytopenia. These hematologic abnormalities occur with certain diseases and their identification can help clinicians narrow to down differential diagnostic lists. This article highlights the importance of blood smear examination, particularly, but not only, when numerical red flags are identified on automated blood counts.

Common bile duct obstruction palliated with common bile duct re-implantation (choledochoduodenostomy) in a cat.

A cat was presented with complete biliary obstruction at the level of the distal common bile duct (CBD), with loss of normal architecture. The area was excised and submitted for histopathology. Concurrent cholecystitis and gall bladder necrosis necessitated cholecystectomy. The proximal CBD was preserved and re-implanted adjacent to the original duodenal papilla. The cat recovered and remained asymptomatic for 6 months. At clinical relapse a carcinoma of suspected biliary origin was confirmed, and the cat was euthanized. In situations in which the CBD lumen cannot be re-established, the pathology is limited to the distal CBD, and the gall bladder is not available for cholecystoenterostomy, CBD re-implantation may be an option to salvage and retain a functional biliary tree.

Remédiation d'une obstruction du canal cholédoque par réimplantation du canal cholédoque (cholédochoduodenostomie) chez un chat. Un chat fut présenté avec une obstruction biliaire complète au niveau du canal cholédoque distal (CBD), avec perte de l'architecture normale. La région a été excisée et soumise pour examen histopathologique. Une cholécystite concomitante et une nécrose de la vésicule biliaire ont nécessité une cholécystectomie. Le CBD proximal fut préservé et réimplanté de manière adjacente à la papille duodénale originale. Le chat a récupéré et est demeuré asymptomatique pendant 6 mois. Lors d'une rechute clinique une suspicion de carcinome d'origine biliaire fut confirmée, et le chat euthanasié. Dans des situations où la lumière du CBD ne peut être ré-établie, que la pathologie est limitée au CBD distal, et que la vésicule biliaire n'est pas disponible pour une cholécystoenterostomie, la réimplantation du CBD peut être une option pour sauvegarder et maintenir un système biliaire fonctionnel.(Traduit par Dr Serge Messier).

Functional Peptide Nanofibers with Unique Tumor Targeting and Enzyme-Induced Local Retention Properties.

An effective tumoral delivery system should show minimal removal by the reticuloendothelial system (RES), promote tumor uptake and penetration, and minimize on-site clearance. This study reports the design and synthesis of advanced self-assembling peptide nanofiber precursor (NFP) analogues. The peptidic nature of NFP offers the design flexibility for on-demand customization with imaging agents and surface charges while maintaining a set size, allowing for real-time monitoring of kinetic and dynamic tumoral delivery by multimodal fluorescence/positron emission tomography/computed tomography (fluo/PET/CT) imaging, for formulation optimization. The optimized glutathione (GSH)-NFP displays a reduced capture by the RES as well as excellent tumor targeting and tissue invasion properties compared to naive NFP. Inside a tumor, GSH-NFP can structurally transform into ten times larger interfibril networks, serving as in situ depot that promotes weeks-long local retention. This nanofiber, which can further be designed to release the active pharmacophores within a tumor microenvironment, displays a superior therapeutic efficacy for inhibiting disease progression and improving the survival of animals bearing triple-negative breast cancer tumors compared to free drug and liposome formulation of the drug, in addition to a favorable toxicity profile.

Dogs with acute myeloid leukemia or lymphoid neoplasms (large cell lymphoma or acute lymphoblastic leukemia) may have indistinguishable mediastinal masses on radiographs.

Acute myeloid leukemia is an uncommon hematopoietic neoplasm of dogs that should be differentiated from lymphoid neoplasms, such as lymphoma, because of different treatment protocols and a worse prognosis. Thoracic radiography is performed frequently in dogs with suspected hematopoietic neoplasia, and detecting a mediastinal mass often prioritizes lymphoma as the most likely diagnosis. However, we have observed a mediastinal mass in several dogs with acute myeloid leukemia and hypothesized that (1) the frequency of a mediastinal mass was higher and (2) the size of the mass was larger in dogs with acute myeloid leukemia compared to dogs with lymphoid neoplasms. In this analytical study (observational, retrospective, and cross-sectional), the sample population included 238 dogs with hematopoietic neoplasia. These dogs were divided into lymphoid (large cell lymphoma, acute lymphoblastic leukemia) and myeloid groups based on standard phenotyping tests. A mediastinal mass was detected during thoracic radiography in 73/218 (33%) and nine of 20 (45%) dogs in the lymphoid and myeloid groups (P = 0.21), respectively. The median size ratio of mediastinal mass to cardiac silhouette was 0.20 and 0.23 in the lymphoid and myeloid groups (P = 0.96), respectively. Additionally, we observed normal thoracic radiographs in 111/218 (51%) dogs in the lymphoid group and nine of 20 (45%) dogs in the myeloid group. In conclusion, acute myeloid leukemia should be considered when a mediastinal mass is detected during radiography in dogs with suspected hematopoietic neoplasia-but the presence or size of a mediastinal mass does not differentiate between myeloid and lymphoid neoplasms.

Body-on-a-chip systems for animal-free toxicity testing.

Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.

A mystery revealed: an update on eosinophil and other blood cell morphology of the Argentine black and white tegu (Salvator merianae).

Reptile white blood cell (WBC) morphological features are strikingly variable across species. In the Argentine black and white tegu (Salvator merianae), red tegu (Salvator rufescens), and Savannah monitor (Varanus exanthematicus), previous reports described a WBC type with a single distinct, clear, linear- to ovoid- to crescent-shaped inclusion of presumptive monocytic origin. The objective of this study was to further investigate the origin of this unique WBC type with crescent-shaped inclusions. Blood samples from two Argentine black and white tegus, tegu 1, a 4-year-old female, and tegu 2, a 2-year-old presumed male, were submitted for routine hematological evaluation. Additional blood films were prepared and stained with these cytochemical stains: alkaline phosphatase (ALP; naphthol AS-MX phosphate substrate), alpha-naphthyl butyrate esterase, alpha-chloroacetate esterase, myeloperoxidase, Periodic acid-Schiff, and Sudan black B. Blood films from tegu 1 were also stained with a second ALP stain (5-bromo-4-chloro-3-indoxyl-phosphate and nitroblue tetrazolium substrate), Luna, luxol fast blue, and toluidine blue. The blood from tegu 1 was cytocentrifuged to isolate and fix the buffy coat in glutaraldehyde 2.5% aqueous solution for transmission electron microscopy. Six morphologically distinct WBC types were identified from tegu 1, including heterophils, basophils, monocytes, azurophils, lymphocytes, and the unique WBC type, which were identified as eosinophils with inclusions. WBC types in tegu 2 were similar; however, eosinophils lacked a discernable inclusion. Proper WBC identification will be useful in obtaining accurate hemogram data for this species.

Clinical findings and outcome predictors for multinodular pulmonary fibrosis in horses: 46 cases (2009-2019).

BACKGROUND: Prognostic indicators for equine multinodular pulmonary fibrosis (EMPF), an interstitial fibrosing lung disease, are poorly described. HYPOTHESIS/OBJECTIVES: Describe diagnostic findings and outcome predictors for EMPF. ANIMALS: Forty-six adult horses with EMPF. METHODS: Retrospective multicenter case series from 2009 to 2019. Radiographic (n = 27) and ultrasonographic studies (n = 19) from EMPF horses and bronchoalveolar lavage fluid (BALF) cytology from 6 EMPF and 13 asthma cases were independently reviewed and blinded to diagnosis and outcome. Associations between predictor variables and survival were assessed by predictor screening followed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Primary clinical findings were weight loss (36/46, 78%), increased respiratory effort (33/46, 72%), tachypnea (32/46, 70%), and fever (18/46, 39%). Macrophage atypia was seen in more EMPF than asthmatic horse BALF (67% vs. 8%; P = .02). Equine herpesvirus 5 (EHV-5) was detected in 24 of 30 (80%) and hyperfibrinogenemia in 25 of 28 (89%) cases. Twenty-seven of 46 horses (59%) and 11 of 45 (24%) survived to discharge and to 3 months, respectively. Three-month survival was associated with lower median (range) respiratory rates (30 [24-36] vs. 41 [30-60] breaths per minute; P = .04), and higher BALF lymphocyte:neutrophil ratios (4.7 [1.4-22] vs. 0.47 [0.11-1.9]; P = .01) and blood lymphocyte counts (1.25 [0.93-2.55] vs. 0.90 [0.70-1.24] × 109/L; P = .03). Imaging findings, EHV-5 detection, and corticosteroid treatment were not associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Fever is not a sensitive clinical sign of EMPF. Diagnostic testing should be pursued for horses with increased respiratory rate and effort and weight loss. The prognosis for EMPF horses is poor. Corticosteroid treatment does not improve 3-month survival.

Equine herpesvirus type 1 infection induces procoagulant activity in equine monocytes.

The alphaherpesvirus, equine herpesvirus type 1 (EHV-1), is a highly prevalent cause of equine infectious abortion and encephalomyelopathy. These syndromes have been attributed to ischemic necrosis from thrombosis in placental and neural vessels, although the mechanisms underlying thrombosis are unknown. After inhalation, EHV-1 establishes a peripheral blood mononuclear cell-associated viremia, with monocytes being a target of infection. Monocytes are also the main source of tissue factor (TF) in diseased states. Since TF is the primary activator of coagulation, increased monocyte TF expression could be involved in EHV-1-associated thrombosis. We hypothesized that EHV-1 infection would induce TF-dependent procoagulant activity in equine monocytes. Monocyte-enriched fractions of blood were infected with abortigenic (RacL11, NY03) and neuropathogenic (Ab4) EHV-1 strains. All strains induced procoagulant activity, to variable degrees, within 1 to 4 h, with maximal activity at 24 h, after infection. Virus-induced procoagulant activity was similar to that seen with lipopolysaccharide, a known stimulant of TF-mediated procoagulant responses. Virus-induced procoagulant activity was factor VIIa-dependent and temporally associated with TF gene transcription, implicating TF as the main driver of the activity. Procoagulant activity was mildly decreased (30-40%) when virus was inactivated by ultraviolet light or when infected cells were treated with aphidicolin, a virus DNA polymerase inhibitor, suggesting early events of virus infection (attachment, entry or intracellular trafficking) are the primary stimulus of procoagulant activity. Our results indicate that EHV-1 rapidly stimulates procoagulant activity in equine monocytes in vitro. The EHV-1-induced procoagulant activity in monocytes may contribute to clinical thrombosis in horses with EHV-1 infection.

Transient increases in glutamate dehydrogenase (GLDH) activity occur in neonatal foals.

BACKGROUND: Liver analyte measurement is important in the evaluation of sick animals. Liver injury in horses is recognized by increased glutamate dehydrogenase (GLDH), sorbitol dehydrogenase (SDH), and aspartate aminotransferase (AST) activities, whereas biliary pathology is identified by increased alkaline phosphatase and γ-glutamyl transferase (GGT) activities or bilirubin concentrations. We have observed high GLDH, but not SDH, activities in neonatal foals admitted for conditions other than liver disease. Only one previous study have evaluated GLDH activity over time in healthy neonatal foals; however, SDH activity was not measured. OBJECTIVE: We aimed to evaluate changes in liver analytes in neonatal foals over time. METHODS: We measured serum liver analytes (GLDH, SDH, GGT, AST, total, direct, and indirect bilirubin) and creatine kinase activity of 11 clinically healthy foals before and at various times after suckling until 46 days of age. Analytes were also measured in colostrum and mare serum. RESULTS: Median GLDH activities increased after birth to peak at 3-4 days of age (106 U/L, reference interval, 0-8 U/L). Median SDH activities had a lower peak at 3-4 days (15 U/L, reference interval, 0-11 U/L) and were frequently discordant with GLDH. There was no association between foal and mare serum or colostral enzyme activities. AST activity plateaued at 5-6 days, whereas GGT activity and total and indirect bilirubin concentrations peaked at 14 and 3-4 days of age, respectively. CONCLUSIONS: Transient increases in GLDH, SDH, and GGT activities and total and indirect bilirubin concentrations occur in clinically healthy neonatal foals and do not necessarily indicate relevant liver disease.

Granular variant of a histiocytic tumor on the toe of a cat: Case report and literature review.

A 16-year-old female spayed domestic shorthaired cat was examined for lameness and a mass on the fourth digit of the right hindlimb. Cytologic examination of an aspirate of the mass revealed large discrete cells admixed with low numbers of well-granulated mast cells. The discrete cells contained single to many variably sized light pink to purple granules in their cytoplasm and had pleomorphic nuclei, with intranuclear cytoplasmic inclusions. Karyomegalic, binucleated and multinucleated cells were seen. Histologic examination of formalin-fixed sections of the excised mass showed a mildly infiltrative, unencapsulated, multinodular dermal mass that extended into the subcutis and consisted of similar discrete cells. On immunohistochemical staining, the tumor cells expressed ionized calcium-binding adapter molecule 1 (Iba1) and CD18. The tumor cells did not express CD3, CD20, CD117, pancytokeratin (AE1/AE3), melanoma antigen (Melan-A), multiple myeloma oncogene-1 (MUM1), melanoma-associated antigen (PNL-2), and S-100. Low numbers of tumor cells expressed CD204 and protein gene product 9.5 (PGP9.5). Granules were variably positive for Periodic-acid Schiff (PAS) and Alcian blue. On transmission electron microscopy, the cells contained filopodia, abundant endoplasmic reticulum, and moderate numbers of low-density membrane-bound granules. This case documents a previously undescribed granular variant of a histiocytic tumor in a cat.

Diagnosis, management and genetic analysis of a cat with primary copper hepatopathy.

Case summary: A 2-year-old spayed female domestic longhair cat was presented for evaluation of chronic ocular discharge and occasional vomiting. While physical examination findings were consistent with an upper respiratory infection (URI), serum chemistry results revealed increased liver enzyme activities. Histopathologic examination of a liver biopsy identified substantial centrilobular accumulation of copper in hepatocytes - strongly suggestive of primary copper hepatopathy (PCH). Retrospective cytologic examination of a liver aspirate also identified copper aggregates in hepatocytes. After transitioning to a low-copper diet, 1 year of chelation therapy with D-penicillamine achieved normalization of liver enzyme activities and resolution of persistent ocular signs. Subsequently, a long-term regimen of zinc gluconate has been successfully managing the cat's PCH for almost 3 years. Sanger sequencing of the cat's ATP7B gene, which encodes a copper-transporting protein, revealed a novel, 'likely pathogenic', single nucleotide variation (c.3670t/a [p.Trp1224Arg]), for which the cat is heterozygous. Relevance and novel information: Recommendations are described for the long-term clinical management of feline PCH - a previously attainable but unreported outcome - with considerations for mitigating the speculated oxidation-exacerbated ocular risks of concurrent URI. This report is the first to include identification of copper aggregates in a liver aspirate from a cat - evidence that liver aspirates from cats could be routinely examined for copper as is standard practice for those from dogs. The cat is also the first reported with PCH and a 'likely pathogenic' heterozygous ATP7B genotype, which suggests that normal ATP7B alleles could be recessive to or incompletely/co- dominant with deleterious ATP7B alleles in cats, as has been reported in other species.

Case-control exercise challenge study on the pathogenesis of high serum gamma-glutamyl transferase activity in racehorses.

BACKGROUND: High serum γ-glutamyl-transferase (GGT) activity syndrome in racehorses has been associated with maladaption to exercise. Investigation of affected horses before and immediately after standard exercise may provide critical insight into the syndrome's pathophysiology. OBJECTIVES: To investigate blood biomarker changes in actively competing racehorses with high GGT activity associated with an exercise challenge. STUDY DESIGN: Case-control study. METHODS: High GGT case (age: 2-3 years) and normal GGT control (age: 2-7 years) pairs (3 Thoroughbred, 4 Standardbred pairs) at least 3 months into their training/racing season were included. Horses with a recent history of high GGT activity (≥50 IU/L) without additional biochemical evidence of liver disease were identified by veterinarians. Horses were tested again in the week prior to a planned exercise challenge to confirm persistent increases in GGT activity. Controls from the same stable with similar training/racing intensity and serum GGT activity ≤36 IU/L were matched with each case. Blood samples were obtained immediately before, 15 and 120 min after exercise. Pre-exercise serum samples were analysed for baseline select serum chemistries, selenium and vitamin E concentrations. Cortisol concentration and markers of oxidative status were measured in serum or plasma for all time points. Individual serum bile acid and coenzyme Q10 concentrations, plasma lipid mediator (fatty acids, oxylipids, isoprostanes) concentrations and targeted metabolomics analyses were performed using liquid chromatography-mass spectrometry. Serum viral PCR for equine hepaci- and parvovirus was performed in each animal. RESULTS: Cases had higher baseline concentrations of total glutathione, taurocholic acid, cortisol and cholesterol concentrations and higher or lower concentrations of specific oxylipid and isoprostane mediators, but there were no case-dependent changes after exercise. MAIN LIMITATIONS: Small sample size. CONCLUSIONS: Results indicated that glutathione metabolism was altered in high GGT horses. Enhanced glutathione recycling and mild cholestasis are possible explanations for the observed differences.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

Hemostatic Testing in Companion Exotic Mammals.

The mammalian hemostatic system is highly conserved, and companion exotic mammals are commonly used as biomedical models for normal and disordered hemostasis. Challenges associated with sample collection, test validation, and test interpretation have limited the use of these tests in clinical exotic animal practice. However, evaluation of platelet counts, coagulation screening times, and fibrin(ogen) degradation products can be valuable for monitoring exotic patients with a range of disease presentations including intoxications, anemia, systemic viral disease, hepatopathy, and endocrinopathy.

A Urinary Drug-Disposing Approach as an Alternative to Intravesical Chemotherapy for Treating Nonmuscle Invasive Bladder Cancer.

The standard treatment of nonmuscle invasive bladder cancer (NMIBC) is transurethral resection of the tumors, followed by intravesical therapy (IT), which comprises a direct instillation of a solution of Bacillus Calmette-Guérin vaccine or chemotherapy into the bladder. However, the recurrence rate in this disease remains unacceptably high. IT is a local treatment that fails to reach tumors developed in the upper urinary tract (ureter and renal pelvis). The catheterization procedure required for IT is invasive, painful, and poses an increased infection risk, resulting in poor patient quality of life and compliance. There is an unmet need for a potent, comprehensive, and noninvasive option. Without chemical modifications, peptides are rapidly removed by renal clearance. This "shortcoming" can be advantageous when used as a drug carrier for directing therapy to NMIBC. Here we develop a urinary drug-disposing (UDD) approach to improve NMIBC treatment. A 12-amino acid bio-inert peptide (Bdd) that can be exclusively eliminated via renal filtration was generated for delivering the microtubule inhibitor DM1 to NMIBC with minimal nonspecific accumulation in other organs. The UDD approach prolonged survival of mice bearing human bladder tumors. Unlike IT, the treatment was given noninvasively (intravenously). Furthermore, it was more effective at suppressing tumor growth than clinically used IT (mitomycin) and safer than free DM1. The application of this UDD approach to treat kidney tumors and deliver other drugs such as doxorubicin was also demonstrated. Overall, the rapid renal clearance of peptides can be exploited to direct cancer therapies to the urinary system. SIGNIFICANCE: A noninvasive drug delivery approach that targets the urinary system overcomes the current barriers facing effective treatment of bladder cancer.

Investigating the pathogenesis of high-serum gamma-glutamyl transferase activity in Thoroughbred racehorses: A series of case-control studies.

BACKGROUND: High-serum γ-Glutamyl Transferase (GGT) activity has been associated with and thought to be a marker of maladaptation to training and possibly poor performance in racehorses, but the cause is unknown. OBJECTIVES: To investigate possible metabolic and infectious causes for the high GGT syndrome. STUDY DESIGN: Pilot case-control study and nested case-control study. METHODS: The case-control study in 2017 included 16 horses (8 cases and 8 controls with median [range] serum GGT 82 [74-148] and 22 [19-28] IU/L, respectively) from the same stable. In 2018, similar testing was performed in a nested case-control study that identified 27 case (serum GGT 50 ≥ IU/L)-control pairs from three stables for further testing. Serum liver chemistries, selenium measurements, viral PCR and metabolomics were performed. RESULTS: No differences were found in frequency of detection of viral RNA/DNA or copy numbers for equine hepacivirus (EqHV) and parvovirus-hepatitis (EqPV-H) between cases and controls. Mild increases in hepatocellular injury and cholestatic markers in case vs control horses suggested a degree of liver disease in a subset of cases. Metabolomic and individual bile acid testing showed differences in cases compared with controls, including increased abundance of pyroglutamic acid and taurine-conjugated bile acids, and reduced abundance of Vitamin B6. Selenium concentrations, although within or above the reference intervals, were also lower in case horses in both studies. MAIN LIMITATIONS: Observational study design did not allow us to make causal inferences. CONCLUSIONS: We conclude that high GGT syndrome is likely a complex metabolic disorder and that viral hepatitis was not identified as a cause for this syndrome in this cohort of racehorses. Our results support a contribution of oxidative stress and cholestasis in its pathophysiology.

Assay variability and storage stability of the myeloperoxidase index of the ADVIA 2120i hematology analyzer in canine and equine whole blood samples.

BACKGROUND: The myeloperoxidase index (MPXI), on ADVIA hematology analyzers, reflects the mean neutrophil myeloperoxidase staining. It is used as a marker of inflammation in animals and people, but assay variability and storage stability are unknown. OBJECTIVE: We aimed to determine MPXI precision and stability with refrigerated storage of canine and equine EDTA-anticoagulated blood and compared MPXI results between two analyzers. METHODS: Inter-assay coefficients of variations (CVs) were determined from three human-based controls assayed before and after a 20- or 21-day calibration. Blood from 14-16 dogs and 26 horses was assayed 4-10 times within 1 day for intra-assay CV measurements. Median control and single run results from 18 canine and 35 equine samples were compared between analyzers. Blood from 10-12 dogs and 10-11 horses was analyzed after collection, and 24, 48, and 72 hours of refrigerated storage. RESULTS: Inter-assay CVs of controls were 10.7%-15.9% and 6.4%-9.6% before and 4.3%-7.7% and 2.8%-17.5% after calibration, for ADVIA 1 and 2, respectively. Calibration altered peroxidase gain settings and improved precision. Intra-assay CVs were 0.6%-64% and 3%-350% for canine and equine samples, respectively. Median MPXI results differed significantly between the analyzers, likely from calibration-associated changes in gains. MPXI decreased with storage, and with variable changes between animals and analyzers. Platelet clumps and lipid contributed to the variability in replicate MPXI measurements. CONCLUSION: MPXI has a higher variability in equine samples than in canine samples. Equivalent results might not be obtained between analyzers. Results change unpredictably with repeated analyses over 72 hours. MPXI measurements might only be useful in controlled research settings.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.