The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

Bovine astrovirus and its role in lymphocytic encephalitis in cattle in Ontario, Canada, 1988-2019.

Astroviruses have been found in cattle and other species with encephalitis. Our objective was to determine the frequency of neurotropic bovine astrovirus (BoAstV) in cases of encephalitis in cattle ≥ 4-mo-old. Of 56 cases of idiopathic lymphocytic encephalitis examined retrospectively (1988-2019), fixed brain from 11 cases (19%) tested positive by semi-quantitative RT-PCR for BoAstV CH13/NeuroS1. None of the control cases tested positive, including 32 with other forms of encephalitis and 40 with no neurologic disease. Most astrovirus-positive cases were 1-2-y-old, with a range of 7 mo to 7 y, and affected both beef and dairy breeds with wide geographic distribution. BoAstV-positive cases had acute onset of neurologic signs of 12 h to 7 d before death or euthanasia. Affected cattle had lymphocytic inflammation throughout the brain including cerebrum, thalamus, midbrain, cerebellum, medulla oblongata, and spinal cord, and affecting gray and white matter. Further PCR testing identified a possible cause in 9 of the 45 (20%) remaining idiopathic cases of lymphocytic encephalitis, including eastern equine encephalitis virus, Listeria monocytogenes, bovine viral diarrhea virus, bovine alphaherpesvirus 1, and ovine gammaherpesvirus 2 (malignant catarrhal fever); we found no cases of infection by West Nile virus, rabies virus, or Chlamydia spp. No cause was identified in 36 of 56 (64%) cases of lymphocytic encephalitis. We frequently identified neurotropic BoAstV in cases of lymphocytic encephalitis that had no previously identified cause. Neurotropic BoAstV infections had gone undetected for decades, but the frequency of BoAstV infections has not increased among contemporary cases.

Comparison of the diagnostic yield of 3 and 5 mm laparoscopic liver biopsy forceps in cats.

OBJECTIVE: To determine whether 3 and 5 mm laparoscopic cup biopsy forceps provide samples of equivalent diagnostic quality in cats. STUDY DESIGN: Experimental study. ANIMALS: Twelve colony cats undergoing a concurrent nutrition study. METHODS: Two biopsy forceps (3 and 5 mm) and three biopsy techniques (twist, pull, and twist + pull) were used to collect 68 laparoscopic liver samples. Biopsies were performed consecutively with the 3 and 5 mm biopsy sites adjacent to each other. Data analyzed included the number of portal triads and hepatic lobules, tissue crush and fragmentation, overall sample area (mm2 ), sample weight, and agreement regarding morphologic diagnosis. RESULTS: The 5 mm forceps provided more hepatic lobules, portal triads, and a larger tissue weight and histologic area (mm2 ) (p < .01). The twist and pull techniques provide more hepatic lobules and portal triads compared to the twist + pull technique while the twist + pull technique resulted in greater tissue crush compared to the twist technique (p = .0097). There was good agreement for morphological diagnosis between the 3 and 5 mm samples using the twist + pull technique but not for the twist or pull techniques. CONCLUSION: Liver samples can be safely collected with 3 or 5 mm laparoscopic biopsy forceps and provide sufficient tissue for histopathology analysis in cats, with minimal artifact. The diagnostic accuracy of 3 mm samples remains unknown. CLINICAL SIGNIFICANCE: Although 3 mm laparoscopic cup biopsy forceps provided samples of sufficient diagnostic quality for histopathologic interpretation in cats, further studies are required to assess their diagnostic accuracy.

Comparing 3 mm and 5 mm laparoscopic liver biopsy samples in dogs.

OBJECTIVE: To determine whether 3 mm cup biopsy forceps (CBF) provide equivalent diagnostic samples to 5 mm CBF for histopathologic diagnosis, bacterial culture, and copper quantification. STUDY DESIGN: Clinical prospective study. ANIMALS: Ten client-owned dogs, presenting for laparoscopic liver biopsy (LLB). METHODS: Dogs underwent LLB, and paired samples were collected using 3 and 5 mm CBF. Portal triad and hepatic lobule counts, crush and fragmentation artifacts, copper concentration, bacterial culture results, and agreement on histopathologic diagnosis were compared. RESULTS: Both CBF sizes allowed for easy sample collection and resulted in minimal hemorrhage. An average of 12.13 (confidence limit (CL): 9.4-14.9) and 17.84 (CL: 15.1-20.6) portal triads were obtained using a 3 and 5 mm CBF, respectively (p = .0003). A portal triad count of 11 or more was achieved in 73.3% of the 3 mm and 93.3% of the 5 mm samples. Gwets AC1 coefficient showed a high level of agreement (0.8) for overall histopathologic diagnosis (p < .0001). The 3 mm CBF crush scores were higher (median of the differences: -1; range: -1 to 1) (p = .035). There was no difference in fragmentation scores (p = .935). CONCLUSION: The 3 mm CBF yielded smaller samples in terms of size and portal triad count compared with the 5 mm CBF. However, the portal triad count was sufficient in a majority of samples and histologic agreement with the 5 mm CBF was excellent. CLINICAL SIGNIFICANCE: In dogs, a 3 mm CBF yields adequate samples for histopathologic interpretation, copper quantification, and bacterial culture.

cDNA Cloning of Feline PIWIL1 and Evaluation of Expression in the Testis of the Domestic Cat.

The PIWI clade of Argonaute proteins is essential for spermatogenesis in all species examined to date. This protein family binds specific classes of small non-coding RNAs known as PIWI-interacting RNAs (piRNAs) which together form piRNA-induced silencing complexes (piRISCs) that are recruited to specific RNA targets through sequence complementarity. These complexes facilitate gene silencing through endonuclease activity and guided recruitment of epigenetic silencing factors. PIWI proteins and piRNAs have been found to play multiple roles in the testis including the maintenance of genomic integrity through transposon silencing and facilitating the turnover of coding RNAs during spermatogenesis. In the present study, we report the first characterization of PIWIL1 in the male domestic cat, a mammalian system predicted to express four PIWI family members. Multiple transcript variants of PIWIL1 were cloned from feline testes cDNA. One isoform shows high homology to PIWIL1 from other mammals, however, the other has characteristics of a "slicer null" isoform, lacking the domain required for endonuclease activity. Expression of PIWIL1 in the male cat appears limited to the testis and correlates with sexual maturity. RNA-immunoprecipitation revealed that feline PIWIL1 binds small RNAs with an average size of 29 nt. Together, these data suggest that the domestic cat has two PIWIL1 isoforms expressed in the mature testis, at least one of which interacts with piRNAs.

Intraocular neuroectodermal embryonal tumor in two rabbits.

Spontaneous intraocular tumors are rarely reported in rabbits, despite their widespread use as laboratory animals. We describe two cases of intraocular neuroectodermal embryonal tumors, formerly primitive neuroectodermal tumors, in young rabbits. Histologically, both tumors exhibited prominent rosette or pseudorosettes, consistent with the histomorphology seen in human tumors. The neuroectodermal subtype is supported by immunoreactivity for the neuronal markers, SRY-box transcription factor 2, microtubule-associated protein 2, neuronal nuclear protein, and neuron-specific enolase. In one of the rabbits, there was metastasis to the contralateral conjunctiva. Intraocular neoplasms can occur in young rabbits and eyes with refractory disease should be enucleated for clinical management.

Characterization of serum and tissue oxytocinase and tissue oxytocin in the pregnant and non-pregnant mare.

Oxytocin is a hormone with functions in: reproduction, maternal bonding, milk ejection, and feeding/social behavior, and is reported to be present in a variety of tissues. Our goal is to characterize oxytocin and leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase), a key regulator of oxytocin in mares. We measured serum and tissue LNPEP by ELISA from ovulation (D0) until D21-22 in non-pregnant (n = 5) and pregnant mares (n = 6); and in periparturient and postpartum mares (n = 18). Placenta (n = 7) and homogenized tissue of diestrus mares (n = 6) were evaluated using protein determinations and LNPEP ELISAs. Identification of LNPEP and OXT protein in tissues was also performed via western blot, immunohistochemistry and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). Serum LNPEP concentration were similar. Placental LNPEP U/mg protein was highest in the body and pregnant horn. The highest to lowest LNPEP U/mg protein by tissue were: myometrium > follicle wall > endometrium > kidney > CL > liver. Oxytocin was identified in the equine pituitary, CL and placenta and is likely to act in autocrine or paracrine manner, while LNPEP may act systemically and locally to regulate the availability of OXT.

CT features of subcutaneous, intermuscular, and intramuscular mast cell tumors in dogs.

Surgical removal is the treatment of choice for subcutaneous (SC), intermuscular (InterM), and intramuscular (IntraM) mast cell tumors (MCTs). Advanced imaging (CT or MRI) is frequently used for presurgical planning, but InterM and IntraM MCTs can be difficult to identify and delineate on CT. Aims of the current retrospective, diagnostic accuracy, observer agreement study were to describe the imaging features of SC, InterM, and IntraM MCTs on CT and to assess the limitation of CT to identify the full local extent of the MCT. Inclusion criteria for the study were dogs with a cytologically or histologically diagnosed MCTs determined to be SC, InterM, or IntraM MCT based on histology and/or a CT scan performed in the gross disease setting. Two board-certified veterinary radiologists reviewed the CT images and recorded location, contrast enhancement pattern, and delineation between the normal and abnormal tissue. Sensitivity and specificity of CT for determining location (SC/InterM versus IntraM) was 85.71% and 55.56%, respectively, when compared to consensus location based on surgical pathology report/CT/MRI review. There was a low inter-rater agreement for delineation (kappa: 0.150 (-0.070 to 0.370) and measurement had a low/moderate correlation (rho: 0.4667 to 0.5792). Upon review by a surgical oncologist, CT findings were deemed insufficient for curative surgical planning in 13 of 16 due to inadequate definition of tumor depth, compartment boundary (fascial plane) or MCT margins. The use of CT for presurgical planning of SC/InterM/IntraM MCT dogs has limitations, especially when differentiating MCT from the adjacent muscle.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

HISTOLOGIC LESIONS IN PLACENTAS OF NORTHERN FUR SEALS (CALLORHINUS URSINUS) FROM A POPULATION WITH HIGH PLACENTAL PREVALENCE OF COXIELLA BURNETII.

Coxiella burnetii is an intracellular bacterial pathogen that can be associated with significant reproductive disease or acute mortality in livestock and wildlife. A novel marine mammal-associated strain of C. burnetii has been identified in pinnipeds of the northwestern Pacific Ocean. Little is known about C. burnetii infection in regard to reproductive success or population status. Our objective was to characterize the severity and extent of histologic lesions in 117 opportunistically collected placentas from presumed-normal northern fur seals (Callorhinus ursinus) in July 2011 on St. Paul Island, Alaska, US, where a high placental prevalence of C. burnetii had been reported. Sections were examined by histology and immunohistochemistry and impression smears with modified acid-fast stain. The nature and frequency of histologic changes were compared with target COM1 PCR-confirmed C. burnetii positive and negative placentas. Overall, histologic changes were similar to placental lesions described in aborting ruminants; however, changes were variable within and between placentas. Vasculitis and occasional intracellular bacteria were seen only in C. burnetii PCR-positive placentas. Dystrophic mineralization, edema, and inflammation were seen in PCR-positive and negative placentas, although they were statistically more common in PCR-positive placentas. Results suggest that C. burnetti and associated pathologic changes are multifocal and variable in placentas from these presumably live-born pups. Therefore, multiple sections of tissue from different placental areas should be examined microscopically, and screened by PCR, to ensure accurate diagnosis as the genomes per gram of placenta may not necessarily represent the severity of placental disease. These limitations should inform field biologists, diagnosticians, and pathologists how best to screen and sample for pathogens and histopathology in marine mammal placental samples.

Disorders of sexual development in the cat: Current state of knowledge and diagnostic approach.

PRACTICAL RELEVANCE: Any congenital or developmental abnormality of any part of the male or female reproductive tract is a 'disorder of sexual development' (DSD). The tricolored male cat phenotype, cryptorchidism, gonadal hypoplasia and incidental abnormalities such as cystic remnants or embryonic ducts are well-known feline DSDs. CLINICAL CHALLENGES: Full characterization of DSDs requires sex chromosome determination and identification of genes related to development of the gonads, internal tubular genitalia and external genitalia. Fortunately, affected cats are seen sporadically and the clinical effects are usually minimal. CLASSIFICATION: The classification nomenclature has changed. In place of intersex, hermaphrodite, pseudohermaphrodite and sex reversal, the newer standard classification, based on sex chromosomes, designates sex chromosome DSD when there is an abnormality in the sex chromosomes, and XX (female) and XY (male) DSDs where there is not. Identification of the gonadal type (testes, ovaries, ovotestes or gonadal dysgenesis) and documentation of the internal and external genital components completes the classification. EVIDENCE BASE: The original basis of the DSD classification was a consensus reached in humans. It was quickly accepted in veterinary pathology, courtesy of its logic and ease of application, and it has subsequently begun to appear in peer-reviewed papers and clinical reviews. This article reviewing the various disorders in cats is based on application of the classification and draws on the feline peer-reviewed literature encompassing chromosome analysis and definition of reproductive abnormalities, syndromes and diseases.

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada.

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog's condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal's disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Beclin-1 is a novel predictive biomarker for canine cutaneous and subcutaneous mast cell tumors.

Mast cell tumors (MCTs) are the most common skin tumor of the dog, and accurately predicting their clinical behavior is critical in directing patient therapy, as they range from benign lesions to a fatal systemic disease. Grading is useful for prognosis, but it cannot predict the behavior of all MCTs. We hypothesized that biomarker immunolabeling in tumor tissues would correlate with patient morbidity and mortality. A clinically annotated tissue microarray (TMA) of primary, recurrent, and metastatic (to lymph node) canine dermal and subcutaneous MCTs was created. Some dogs whose MCTs were included in the TMA did not receive adjunctive treatment after surgical excision of the MCT, whereas others were treated with one or a combination of chemotherapy, radiation, or oral toceranib. Immunohistochemistry for beclin-1, an autophagy protein, was performed followed by digital image analysis. Beclin-1 immunolabeling was higher in recurrent tumors (mean H-score 110.8) than primary MCTs (mean H-score 73.5), and highest in lymph node metastases (mean H-score 138.5) with a significant difference in means (P < .001). While beclin-1 level was not prognostic, it was strongly predictive for survival after adjunctive treatment; dogs with high beclin-1-expressing tumors showed poorer survival compared to those with low beclin-1-expressing tumors (HR = 5.7, P = .02), especially in Kiupel high-grade tumors (HR = 16.3, P = .01). Beclin-1 immunolabeling was the only significant predictive factor by multivariable analysis (P = .04). These findings may improve our ability to predict the response to adjunctive therapy. Importantly, these data suggest that autophagy inhibitors may be useful in improving response to treatment for dogs with high-grade MCTs.

A retrospective comparison of first and second opinion histopathology with patient outcomes in veterinary oncology cases (2011-2019).

Mandatory second opinion histopathology is common practice in human surgical pathology. It is intended to confirm the original diagnosis or identify clinically significant discrepancies, which could alter the course of disease, cost of treatment, patient management or prognosis. This retrospective analysis aimed to evaluate agreement between first and second opinion histopathology cases, examine their correlation with natural history of disease and investigate the rationale for pursuing this test. Medical records from 2011 to 2019 were reviewed, identifying 109 cases where second opinion histopathology was sought. Reasons for seeking second opinion and clinical disease course were also reviewed to determine whether case progression favoured first or second opinion findings in cases of diagnostic disagreement. Diagnostic disagreement was found in 49.5% of cases. Complete diagnostic disagreement (a change in degree of malignancy or tumour type) occurred in 15.6% cases and partial disagreement (a change in tumour subtype, grade, margins and mitotic count) occurred in 33.9%. Major disagreement (a change in diagnosis resulting in alteration of treatment recommendations) occurred in 38.5% of cases. The most common reasons for seeking second opinion were an atypical/poorly differentiated tumour (31.2%; 34/109) or a discordant clinical picture (24.8%; 27/109). Among cases with any form of disagreement, natural history of disease favoured second opinion findings in 33.3%. The first opinion was favoured over the second in a single case. These findings reinforce previous literature supporting a role for second opinion histopathology in optimizing therapy and predicting outcomes in veterinary oncology, particularly in cases where diagnosis is in question based on the overall clinical picture.

International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Longitudinal study of the cutaneous microbiota of healthy horses.

BACKGROUND: Next-generation sequencing techniques have revealed that human and animal skin is colonised by a rich and diverse population of bacteria, and that microbial composition varies between different body sites and individuals. Very little is known about the normal microbiota of healthy equine skin. HYPOTHESIS/OBJECTIVES: To describe the taxonomic distributions of cutaneous bacterial microbiota in a population of healthy horses in Ontario, Canada, and to evaluate the effects of body site, individual and time of year on microbial diversity and community composition. ANIMALS: Samples were collected from four body sites (dorsum, ventral abdomen, pastern and groin) from 12 clinically healthy horses from the same farm. Samples were collected from all individuals at four time points (winter, spring, summer, autumn) within a calendar year. METHODS AND MATERIALS: Illumina sequencing of the V4 region of the 16S rRNA gene was performed following DNA extraction. Data were analysed using mothur software. RESULTS: Bacteria from 38 phyla and 1,665 genera were identified. Alpha diversity was higher in the winter and summer than spring and autumn although this was not statistically significant. Community membership and structure clustered more based on season than skin site. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy equine skin is inhabited by a marked diversity of microbiota. Individuals living in a similar environment share overlapping cutaneous microbial populations. These populations vary significantly over time and between body sites.

The impact of ecbolic therapy in the early postpartum period on uterine involution and reproductive health in dairy cows.

Postpartum uterine disease due to poor uterine involution continues to be a significant factor that contributes to poor reproductive efficiency in dairy cattle. Therapy that increases the frequency, duration and strength of uterine contractions in the postpartum period might enhance uterine involution, resulting in improved reproductive performance. The objective of this clinical trial was to study the effect of two uterine ecbolic therapies, oxytocin and prostaglandinF2α on uterine involution, postpartum endometritis, and reproductive performance. A randomized double-blinded clinical trial was conducted in 118 dairy cows from two research herds that had normal parturition and expulsion of the fetal membranes. Within 24 hr after calving, cows were randomly assigned to receive intramuscular injections twice a day of 50 IU of oxytocin, or 25 mg of dinoprost (PGF2α) or saline (control) for 7 days. Cows were monitored from day 3 to day 63 ± 3 days postpartum by transrectal palpation of the uterus, vaginoscopy, Metricheck® examination and by endometrial cytology. Blood samples were collected for measurement of progesterone in weeks 3 (21 ± 3 days), 5 (35 ± 3 days), 7 (49 ± 3 days), and 9 (63 ± 3 days) postpartum. Herd breeding records were obtained to determine reproductive performance in all cows. Neither oxytocin nor prostaglandin F2α therapy during the first week postpartum had any significant effect on the rate of uterine involution, prevalence of endometritis or reproductive performance, compared to untreated controls. Ecbolic drugs, as used here, are not recommended for use in clinical practice to improve involution or reproductive tract health in normal cows.