Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

AN OUTBREAK OF FELINE INFECTIOUS PERITONITIS IN THREE RELATED SAND CATS (FELIS MARGARITA) IN HUMAN CARE.

Feline infectious peritonitis (FIP) is a systemic disease in felid species caused by infection with mutated forms of feline coronavirus (FCoV), and outbreaks can devastate exotic felid populations in human care. Feline infectious peritonitis was diagnosed in three of four related juvenile sand cats (Felis margarita) from a single institution over a 6-wk period. Case 1 was a 7-mon-old male found deceased with no premonitory signs. Case 2, an 8-mon-old male (littermate to Case 1), and Case 3, a 6-mon-old male (from a different litter with identical parentage), were evaluated for lethargy and anorexia 1 mon after Case 1. Both exhibited transient anisocoria and progressive lethargy, anorexia, and dehydration despite antibiotic and supportive treatment. Approximately 1 wk after initial presentation, Case 2 was humanely euthanized, and Case 3 was found deceased. Necropsy findings included intrathoracic and/or intra-abdominal lymphadenopathy (3/3 cases), bicavitary effusion (2/3), multifocal tan hepatic and intestinal nodules (1/3), and multifocal yellow renal nodules (1/3). Histologically, all cats had severe pyogranulomatous vasculitis in multiple organs, and the presence of FCoV antigen was confirmed using immunohistochemical staining. Next-generation sequencing of the virus from Case 3's affected kidney demonstrated ∼93% homology to the UG-FH8 virus, a serotype 1 feline alphacoronavirus isolated from Denmark. Future research will focus on comparative viral genomic sequencing with the goals of identifying potential sources of FCoV infection and identifying features that may have contributed to the development of FIP in this species.

American Society for Veterinary Clinical Pathology-Recommended Clinical Pathology Competencies for Graduating Veterinarians.

Given the move toward competency-based veterinary education and the subsequent reevaluation of veterinary curricula, there is a need for specialties to provide guidance to veterinary college administrators and educators on the core knowledge and skills pertaining to their specialty to ensure their inclusion in revised or redesigned curricula. The American Society for Veterinary Clinical Pathology (ASVCP) Education Committee sought to create a list of competencies specific to clinical pathology expected of graduating veterinarians. The stimulus for this project was the American Veterinary Medical Association Council on Education Standards of Accreditation for Colleges of Veterinary Medicine, further driven by the 2018 publication of the Association of American Veterinary Medical Colleges Competency-Based Veterinary Education Working Group framework. The recommendations made in this document are the culmination of the 2016 ASVCP Education Forum for Discussion, multiple remote subcommittee communications, and feedback obtained from ASVCP membership. The final framework includes 8 clinical pathology-focused domains of competence with 20 clinical pathology competencies and 61 clinical pathology illustrative sub-competencies. The clinical pathology-focused domains of competence are: the pre-analytical phase of testing, laboratory medicine and instrumentation, principles of test selection and interpretation, hematology and hemostasis, chemistry, endocrinology, urinalysis, and cytology. These are not intended to replace the nine established AAVMC domains of competence with supportive competencies and illustrative sub-competencies but to guide institutions for how clinical pathology aligns within the competency-based veterinary education (CBVE) framework for the practice-ready veterinary graduate. This clinical pathology competency framework may prove useful and empowering during discussions of curriculum revisions and redesigns.

Virtual Microscopy Is More Effective Than Conventional Microscopy for Teaching Cytology to Veterinary Students: A Randomized Controlled Trial.

Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown. Our objective was to determine whether instruction using VM, compared to CM, is a successful method of training veterinary students for the application of cytology in practice (i.e., using light microscopes). Seventy-one veterinary students from Colorado State University who attended a voluntary 3-hour cytology workshop were randomized to receive the same instruction with either VM (n = 35) or CM (n = 36). We compared these students to a control group (n = 22) of students who did not attend a workshop. All students took a post-workshop assessment involving the interpretation of four cases on glass slides with CM, designed to simulate the use of cytology in general practice. Students also took an 18-question survey related to the effectiveness of the workshop, providing their opinions on cytology instruction in the curriculum and their learning preference (VM or CM). The mean assessment score of the VM group (14.18 points) was significantly higher than the control group (11.33 points, p = .003), whereas the mean of the CM group (12.77 points) was not statistically significantly different from controls (p = .170). Not only is VM an effective method of teaching cytology to veterinary students that can be translated to a real-world case scenario, but it outperformed CM instruction in this study.

Unlicensed antiviral products used for the at-home treatment of feline infectious peritonitis contain GS-441524 at significantly different amounts than advertised.

OBJECTIVE: To analyze the content of unlicensed GS-441524-like products being used as a largely successful at-home treatment for cats suspected to have FIP. The remdesivir content and pH were also measured. SAMPLE: 127 injectable and oral samples from 30 of the most popular brands of black market producers. METHODS: Unlicensed GS-441524-like products were procured through donations and tested for GS-441524 and remdesivir content by liquid chromatography with tandem mass spectrometry. A pH meter measured the pH of injectable samples. RESULTS: Of the 87 injectable formulations, 95% contained more (on average 39% more) GS-441524 than expected based on the producer's marketed concentrations. The average pH (1.30 pH) was well below the physiologic pH conditions recommended for SC injections. The oral formulations were more variable, with 43% containing more GS-441524 (on average 75% more) than expected and 58% containing less (on average 39% less) than the expected content. There was minimal variability in GS-441524 content between replicate samples in the injectables formulations (measured by coefficient of variation). One injectable and 2 oral samples additionally contained remdesivir. CLINICAL RELEVANCE: All unlicensed products used for the at-home treatment of FIP that we tested contain GS-441524. The injectables generally contain significantly more drug than advertised at a below-physiologic pH. Unlicensed oral products vary more widely in drug content and suffer from unconventional dosing and labeling. These data should highlight the need for regulation of these products and the development of legal pathways to procure GS-441524.

An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Guidelines for resident training in veterinary clinical pathology. IV: Laboratory quality management-Teaching domains, competencies, and suggested learning outcomes.

BACKGROUND: The 2019 ASVCP Education Committee Forum for Discussion, presented at the annual ASVCP/ACVP meeting, identified a need to develop recommendations for teaching laboratory quality management principles in veterinary clinical pathology residency training programs. OBJECTIVES: To present a competency-based framework for teaching laboratory quality management principles in veterinary clinical pathology residency training programs, including entrustable professional activities (EPAs), domains of competence, individual competencies, and learning outcomes. METHODS: A joint subcommittee of the ASVCP Quality Assurance and Laboratory Standards (QALS) and Education Committees executed this project. A draft guideline version was reviewed by the ASVCP membership and shared with selected ACVP committees in early 2022, and a final version was voted upon by the full QALS and Education Committees in late 2022. RESULTS: Eleven domains of competence with relevant individual competencies were identified. In addition, suggested learning outcomes and resource lists were developed. Domains and individual competencies were mapped to six EPAs. CONCLUSIONS: This guideline presents a framework for teaching principles of laboratory quality management in veterinary clinical pathology residency training programs and was designed to be comprehensive yet practical. Guidance on pedagogical terms and possible routes of implementation are included. Recommendations herein aim to improve and support resident training but may require gradual implementation, as programs phase in necessary expertise and resources. Future directions include the development of learning milestones and assessments and consideration of how recommendations intersect with the American College of Veterinary Pathologists training program accreditation and certifying examination.

Flow Cytometry in Veterinary Practice.

This article summarizes the current applications of flow cytometry in clinical veterinary medicine, which is largely restricted to the diagnosis of hematopoietic neoplasms (lymphomas and leukemias) of domestic dogs, cats, and horses. A brief background on the technique of flow cytometry and fundamentals of data interpretation are included. Major emphasis is placed on clinical indications for flow cytometry, principles of sample collection and submission, and awareness of diagnostic and prognostic utility. Expectations regarding both the benefits and limitations of flow cytometry in a clinical setting, and its complementary nature with other types of testing, are also reviewed.

Diagnostic accuracy of cytology for the detection of bacterial infection in fluid samples from veterinary patients.

BACKGROUND: Bacterial sepsis is a relatively common, life-threatening condition with a high case fatality rate. The current primary diagnostic tools for detecting bacterial infection in fluids are bacterial culture and fluid cytology. While culture is the gold standard, it can take up to several days for results to be made available to clinicians, which can delay recognition of bacterial sepsis and negatively impact patient outcomes. OBJECTIVES: The aim of this study was to evaluate the diagnostic accuracy of cytology for detecting bacterial infection in body fluids. METHODS: We retrospectively reviewed 10 years of medical records at the Ohio State University's Veterinary Medical Center for mammalian patients with both cytology and bacterial culture of fluid samples, including body cavity fluids (abdominal and thoracic effusion), blood, joint fluid, and CSF. The overall sensitivity and specificity of cytology relative to the reference method of bacterial culture was recorded, as well as among the subcategories of fluid type. RESULTS: The overall sensitivity and specificity of cytology for the diagnosis of sepsis were 42.6% and 93.0%, respectively. Individual sensitivities and specificities were also calculated for each fluid type. Thoracic fluid cytology had relatively high sensitivity and low specificity, in contrast to the other fluid types analyzed. CONCLUSIONS: Overall, cytology is poorly sensitive but highly specific for the detection of bacterial infection in fluid samples. The results from this study will allow a better comparison between the diagnostic accuracy of cytology and emerging diagnostic tests for the detection of bacterial sepsis in mammalian patients.

Unlicensed Molnupiravir is an Effective Rescue Treatment Following Failure of Unlicensed GS-441524-like Therapy for Cats with Suspected Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a complex and historically fatal disease, though recent advances in antiviral therapy have uncovered potential treatments. A newer therapeutic option, unlicensed molnupiravir, is being used as a first-line therapy for suspect FIP and as a rescue therapy to treat cats who have persistent or relapsed clinical signs of FIP after GS-441524 and/or GC376 therapy. Using owner-reported data, treatment protocols for 30 cats were documented. The 26 cats treated with unlicensed molnupiravir as a rescue therapy were treated with an average starting dosage of 12.8 mg/kg and an average ending dosage of 14.7 mg/kg twice daily for a median of 12 weeks (IQR = 10-15). In total, 24 of 26 cats were still living disease-free at the time of writing. One cat was euthanized after completing treatment due to a prolonged seizure, and the other cat underwent retreatment for relapsed clinical signs. Few adverse effects were reported, with the most notable-folded ears (1), broken whiskers (1), and severe leukopenia (1)-seen at dosages above 23 mg/kg twice daily. This study provides a proof of principle for the use of molnupiravir in cats and supports the need for future studies to further evaluate molnupiravir as a potentially safe and effective therapy for FIP.

Comparison of pathologist review protocols for cytologic detection of prostatic and urothelial carcinomas in canines: A bi-institutional retrospective study of 298 cases.

Urothelial carcinoma, also known as transitional cell carcinoma, is the most common primary bladder tumour in dogs, and can also involve the prostate gland. Cytology is a common diagnostic tool utilized for dogs with bladder or prostate gland lesions. The objectives of this retrospective study were to compare the sensitivity and specificity of cytologic evaluation for urothelial or prostatic carcinoma between two institutions with different cytology review protocols as well as determine if certain collection methods resulted in higher cytologic accuracy. A total of 298 cases met inclusion criteria. The overall sensitivity and specificity for institution 1 were 91.8% and 50%, respectively, compared to 31.1% and 97.4%, respectively, for institution 2. When the urine sample review protocol at institution 2 was matched to that of institution 1, sensitivity and specificity were more similar to institution 1 (71.2% and 88.9%, respectively). Our results show that the sensitivity and specificity of cytology are affected by screening and review protocols implemented by different institutions. The data also demonstrate that sensitivity and specificity vary by collection method. Diagnostic catheterization had the highest performance: of the 11 cases between two institutions, it had 100% sensitivity and specificity. In contrast, examination of urine sediment not collected via diagnostic catheterization had low sensitivity and specificity that varied greatly by institution. In summary, cytologic interpretation should be undertaken with consideration given to both processing and collection protocols.

Optimizing the u411 automated urinalysis instrument for veterinary use.

BACKGROUND: The Cobas u411 Analyzer (Roche Diagnostics) is an automated, reflectance photometry-based urinalysis instrument designed for use with Roche's CHEMSTRIP 10UA technology and human urine samples. OBJECTIVE: We aimed to optimize and validate the Cobas u411 Analyzer for use in canine and feline urinalysis. METHODS: Patient urine samples presenting to the Clinical Pathology Laboratory at the Colorado State University Veterinary Teaching Hospital were analyzed with the Cobas u411 and by manual readings in parallel. Initially, 223 canine and 83 feline urine samples were run using the u411 factory settings. Following comparisons with manual results, and evaluation for directional bias, adjustments to the reflectance values were made in the instrument's programming. An additional 183 canine and 95 feline samples were run using the adjusted settings. Total urine protein concentrations were measured in 48 samples and used to generate receiver operating characteristic curves for the protein test pad. RESULTS: Following adjustments in reflectance programming, concordance between u411 and manual results was increased by 17.7% for protein, 11.7% for ketones, and 4.5% for bilirubin. Concordances for pH, glucose, and blood were not substantially changed. Discordance for all analytes was ≤3%. Canine and feline samples had similar levels of discordance, though marginal concordance was higher in dogs for ketones, bilirubin, and blood. CONCLUSIONS: Adjustments to the reflectance programming of the Cobas u411 Analyzer improved concordance with manual results for canine and feline samples. This instrument has the potential to greatly increase both efficiency and consistency of urinalysis procedures in higher throughput veterinary diagnostic laboratories.

Multiplex fluorescent immunocytochemistry for the diagnosis of feline infectious peritonitis: Determining optimal storage conditions.

BACKGROUND: Feline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF-ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples. OBJECTIVE: We aimed to develop and optimize an MF-ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay. METHODS: A feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF-ICC were made at 0, 1, 2, 3, and 5 days post-collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF-ICC for the presence of mononuclear cells with co-expression of vimentin and coronaviral antigen. RESULTS: MF-ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post-harvest, 22℃ through 48 hours post-harvest, and 37℃ through 24 hours post-harvest. The MF-ICC assay was successfully performed on fixed slides through the 12-week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP. CONCLUSIONS: The MF-ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.

Formulae to correct sodium concentrations for serum water fraction in cases of hypo- and hyperproteinemia in cats.

BACKGROUND: Biochemistry analyzers in many high-throughput laboratories use indirect potentiometry to determine serum electrolyte concentrations, which involves a pre-analytical dilution step that may be associated with artifactual increases or decreases in electrolyte concentrations under circumstances of altered serum water fraction (SWF). Severe hypo- and hyperproteinemia, conditions that cause altered SWF, are recognized but under-emphasized causes of falsely measured serum sodium concentrations. OBJECTIVES: The goals of this study were to determine the average actual SWF (SWFA ) and establish formulae to correct serum sodium concentration measured by indirect potentiometry in hypo- and hyperproteinemic cats. METHODS: Serum samples from 112 feline patients were analyzed for electrolytes (measured by both indirect and direct potentiometry), total protein, albumin, triglycerides, and cholesterol. Each serum sample was also lyophilized to determine the SWFA . A feline-specific formula to estimate SWF (SWFE-FEL ) was developed and evaluated with a multivariable linear model. RESULTS: The mean SWFA in this population of cats was 91.2%, which was significantly different (P < .0001) than the mean (93.9%) calculated using the human estimated formula (SWFE-HUM ). The formula devised for the SWFE-FEL better recapitulated the SWFA than did the SWFE-HUM , and the corrected sodium concentrations calculated using the feline formula were better correlated with serum sodium measured by direct potentiometry than those determined using the human formula. CONCLUSIONS: Application of feline-specific formulae is expected to limit the misinterpretation of electrolyte data from indirect potentiometry when altered SWF occurs. To demonstrate this, a case example of a hypoproteinemic cat is provided.

Validation of an immunocytochemical assay for immunophenotyping of lymphoma in guinea pigs (Cavia porcellus).

BACKGROUND: Lymphoma is an important disease of pet guinea pigs, although validation of immunophenotyping techniques based on cytologic or hematologic samples has not been reported. OBJECTIVE: To describe an immunocytochemical method for immunophenotyping of lymphoma (as either T- or B-cell) in guinea pigs, and to validate antibodies for this purpose. METHODS: Blood and tissues were obtained at the time of necropsy from laboratory guinea pigs and a privately owned dog (control) euthanized for reasons unrelated to lymphoproliferative disease. Fine-needle aspirates of enlarged peripheral lymph nodes were obtained from a case of spontaneous lymphoma in a pet guinea pig. Anti-CD3 and anti-Pax5 antibodies were validated by a combination of western blotting performed on splenic lysates of both the dog and guinea pigs, immunohistochemical studies on normal guinea pig tissues, and immunocytochemistry on normal guinea pig peripheral blood and splenic impression smears. RESULTS: The antibodies bound to antigens of an appropriate size in both the dog and guinea pig splenic lysates by Western blot analysis. Immunohistochemistry and immunocytochemistry demonstrated the expected distribution of putative T- and B-lymphocytes in normal tissues, peripheral blood, and splenic impression smears. As a proof-of-principle for its clinical utility, this immunocytochemical assay was used to diagnose a B-cell phenotype in a spontaneous lymphoma case in a pet guinea pig. CONCLUSIONS: Here, we validated an immunocytochemical method for immunophenotyping of lymphoma in guinea pigs as either a T- or B-cell phenotype. This enables future research into the clinical attributes of these subtypes and may ultimately improve both prognostication and therapy of lymphoma in guinea pigs.

Basal cell enumeration does not predict malignancy in canine perianal gland tumor cytology.

BACKGROUND: Perianal (hepatoid) gland tumors are common in dogs, and the distinction between the benign and malignant forms is clinically important. Cytology of these tumors typically contains many large hepatoid cells and fewer small basal cells. OBJECTIVES: The objective of this study was to determine whether the proportion of the smaller basaloid reserve cells in cytologic samples from perianal tumors correlates with malignancy. METHODS: Eighty-three cases of cytologically diagnosed perianal gland tumors with corresponding histopathologic sections were identified from two separate institutions and included six (7.2%) malignant tumors and 77 (92.8%) benign tumors. The proportion of basal cells from each sample was evaluated. RESULTS: No difference between these groups was found, although the study was sufficiently powered to detect an approximately 1.5-fold change in basal cell proportion. CONCLUSIONS: This report found no evidence that the proportion of basal cells in canine perianal tumor cytology is an indication of the potential for malignancy. We, therefore, do not recommend citing this feature in cytologic reports or when communicating with clinicians.