Case report: Androgen-secreting adrenocortical tumors in eight cats.

Urine marking, aggression, and other behavioral concerns are common reasons for cat owners to seek veterinary care. Empiric treatment for lower urinary tract disease or primary behavior disorders are commonly pursued, especially in those cases with normal routine laboratory evaluations. Herein, we report the clinicopathologic findings in eight sexually altered cats that were diagnosed with androgen-secreting adrenocortical tumors. Nearly all cats (n = 7) initially were evaluated for inappropriate urination and pungent urine, with additional behavioral concerns including aggression (n = 3) and excess vocalization (n = 4) commonly reported. Penile barbs (n = 5) were identified in all five male cats, and an enlarged clitoris was observed in one female cat. Testing of serum androgen concentrations revealed abnormally high androstenedione (n = 1) or testosterone (n = 7) concentrations. In the five cases with available adrenal tissue, histopathologic evaluation identified either an adrenocortical adenoma (n = 3) or adrenocortical carcinoma (n = 2). Hormonal abnormalities resolved and clinical signs improved in the four cats that underwent surgical adrenalectomy, with each of these cats surviving >1 year. However, clinical signs were minimally impacted with medical treatments, including one cat in which trilostane treatment failed to improve clinical signs or testosterone concentrations. This collection of cases underscores the importance of a detailed physical examination as well as the consideration of endocrine disturbances in cats undergoing evaluation for inappropriate urination or aggression. Furthermore, this report adds to the growing body of evidence that sex-hormone secreting adrenal tumors in cats may be an under-recognized syndrome.

Computer-assisted mitotic count using a deep learning-based algorithm improves interobserver reproducibility and accuracy.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Equid herpesvirus-1 Distribution in Equine Lymphoid and Neural Tissues 70 Days Post Infection.

Equid herpesvirus-1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses worldwide. As member of the Alphaherpesvirinae, latency is key to EHV-1 epidemiology. EHV-1 latent infection has been detected in the trigeminal ganglion (TG), respiratory associated lymphoid tissue (RALT) and peripheral blood mononuclear cells (PBMC) but additional locations are likely. The aim of this study was to investigate the distribution of viral DNA throughout the equine body. Twenty-five horses divided into three groups were experimentally infected via intranasal instillation with one of three EHV-1 viruses and euthanized on Day 70, post infection. During necropsy, TG, various sympathetic/parasympathetic ganglia of head, neck, thorax and abdomen, spinal cord dorsal root ganglia, RALT, mesenteric lymph nodes, spleen and PBMC of each horse were collected. Genomic viral loads and L-(late) gene transcriptional activity in each tissue and PBMC were measured using qPCR. In addition, immunohistochemistry (IHC) was applied on neural parenchyma tissue sections. EHV-1 DNA was detected in many neural and lymphoid tissue sections, but not in PBMC. L-gene transcriptional activity was not detected in any sample, and translational activity was not apparent on IHC. Tissue tropism differed between the Ab4 wild type and the two mutant viruses.

Transconjunctival excision of an orbital conjunctival cyst using computer-assisted 3-D surgical planning in a dog.

Investigation of exophthalmos and blood-colored discharge from the left ventral punctum in a dog was consistent with a conjunctival cyst in the orbit. 3-D prints of the cyst and surrounding facial bones identified a successful transconjunctival approach without an orbitotomy and patency of the left lacrimal duct was reestablished.

Blood and bone marrow findings in two pups with mucopolysaccharidosis type VII.

Routine blood smear findings in two of four 11-day-old mixed-breed dog littermates were suggestive of a lysosomal storage disease (LSD) that was documented to be mucopolysaccharidosis type VII (MPS VII) by molecular testing. In this condition, a functional ß-glucuronidase deficiency results in the accumulation of glycosaminoglycans (GAGs) in cells and tissues where ß-glucuronidase is important in GAG degradation. Most neutrophils and a moderate number of lymphocytes within the blood had atypical cytoplasmic magenta inclusions. The bone marrow assessment from one of the two affected pups at 24 days of age revealed similar magenta granulation in myeloid precursor cells that was most prominent in promyelocytes and myelocytes. Moreover, atypical magenta material was present within vacuoles as well as extracellularly in some osteoblasts and macrophages. Histologic bone marrow sections revealed prominent vacuolation of osteoblasts, and some osteoclasts appeared separated from the bone by layers of osteoblasts or hematopoietic cells. At 2 months of age, the second affected dog showed moderate growth retardation and had similar but more prominent hematologic findings that extended to monocytes, eosinophils, and eosinophil precursors. It had an increased number of bone marrow macrophages with many vacuoles that could be seen cytologically to contain magenta material, and there was mild nonselective phagocytosis of hemic cells. Of the hematologic cells, inclusions were most prominent in promyelocytes, myelocytes, and macrophages, cells with relatively high ß-glucuronidase activity, and GAG exposure within lysosomes or lysosome-like primary granules of granulocyte precursors.

Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses.

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

Computerized Calculation of Mitotic Count Distribution in Canine Cutaneous Mast Cell Tumor Sections: Mitotic Count Is Area Dependent.

Mitotic count (MC) is an important element for grading canine cutaneous mast cell tumors (ccMCTs) and is determined in 10 consecutive high-power fields with the highest mitotic activity. However, there is variability in area selection between pathologists. In this study, the MC distribution and the effect of area selection on the MC were analyzed in ccMCTs. Two pathologists independently annotated all mitotic figures in whole-slide images of 28 ccMCTs (ground truth). Automated image analysis was used to examine the ground truth distribution of the MC throughout the tumor section area, which was compared with the manual MCs of 11 pathologists. Computerized analysis demonstrated high variability of the MC within different tumor areas. There were 6 MCTs with consistently low MCs (MC<7 in all tumor areas), 13 cases with mostly high MCs (MC ≥7 in ≥75% of 10 high-power field areas), and 9 borderline cases with variable MCs around 7, which is a cutoff value for ccMCT grading. There was inconsistency among pathologists in identifying the areas with the highest density of mitotic figures throughout the 3 ccMCT groups; only 51.9% of the counts were consistent with the highest 25% of the ground truth MC distribution. Regardless, there was substantial agreement between pathologists in detecting tumors with MC ≥7. Falsely low MCs below 7 mainly occurred in 4 of 9 borderline cases that had very few ground truth areas with MC ≥7. The findings of this study highlight the need to further standardize how to select the region of the tumor in which to determine the MC.

Histopathologic Findings Following Experimental Equine Herpesvirus 1 Infection of Horses.

Histopathological differences in horses infected with equine herpesvirus type 1 (EHV-1) of differing neuropathogenic potential [wild-type (Ab4), polymerase mutant (Ab4 N752), EHV-1/4 gD mutant (Ab4 gD4)] were evaluated to examine the impact of viral factors on clinical disease, tissue tropism and pathology. Three of 8 Ab4 infected horses developed Equine Herpesvirus Myeloencephalopathy (EHM) requiring euthanasia of 2 horses on day 9 post-infection. None of the other horses showed neurologic signs and all remaining animals were sacrificed 10 weeks post-infection. EHM horses had lymphohistiocytic vasculitis and lymphocytic infiltrates in the lungs, spinal cord, endometrium and eyes. EHV-1 antigen was detected within the eyes and spinal cord. In 3/6 of the remaining Ab4 infected horses, 4/9 Ab4 N752 infected horses, and 8/8 Ab4 gD4 infected horses, choroiditis was observed. All males had interstitial lymphoplasmacytic and/or histiocytic orchitis and EHV-1 antigen was detected. In conclusion, only animals sacrificed due to EHM developed overt vasculitis in the CNS and the eye. Mild choroiditis persisted in many animals and appeared to be more common in Ab4 gD4 infected animals. Finally, we report infiltrates and changes in the reproductive organs of all males associated with EHV-1 antigen. While the exact significance of these changes is unclear, these findings raise concern for long-term effects on reproduction and prolonged shedding of virus through semen.

Short communication: Survey of hepatic copper concentrations in Midwest dairy cows.

Previous research from our laboratory and others indicates that liver copper concentrations in dairy cattle are commonly well above those recognized as adequate for the nutritional needs of the animal. It has also been speculated that hepatic copper concentrations have been increasing in recent years. Unlike other species, the threshold at which elevated liver copper concentrations becomes deleterious to hepatocytes is not known for cattle. Therefore, the objectives of this study were 3-fold: (1) to delineate differences in the range and mean dry matter hepatic copper concentration for dairy cattle in a retrospective analysis (January 1, 2007, to December 31, 2015), (2) to investigate hepatic copper concentrations in Midwest cull dairy cattle, and (3) to evaluate histologic changes in hepatocellular morphology in the context of copper concentration in cull cows. Furthermore, microscopic changes in hepatocellular morphology or architecture were examined and scored for evidence of inflammation, fibrosis, necrosis, and abundance of rhodanine-stained granules using hematoxylin and eosin and rhodanine staining. The retrospective analysis found copper concentrations within a range of 3 to 1,963 µg/g, with a mean of 473 µg/g. Hepatic copper concentrations in our retrospective study did not increase with time. In our abattoir analysis, copper concentrations ranged from 15 to 978 µg/g, with a mean of 390 µg/g. This study found that the range and mean hepatic copper concentrations were comparatively less in the current abattoir study than copper concentrations in our retrospective analysis. There was no evidence for hepatocellular changes associated with increased copper burdens in this study population.

Physiological characterization of ocular melanosis-affected canine melanocytes.

OBJECTIVE: Cairn terriers with ocular melanosis (OM) accumulate large, heavily pigmented melanocytes in the anterior uvea. Darkly pigmented plaques develop within the sclera, leading us to hypothesize that OM uveal melanocytes may have an abnormal migratory capacity. ANIMALS STUDIED: Globes from OM-affected Cairn terriers and unaffected control eyes enucleated for reasons unrelated to this study were used for immunohistochemistry and to culture melanocytes for in vitro cell behavior assays. PROCEDURES: The scleral plaques of six dogs were immunolabeled for HMB-45, MelanA, PNL2, CD18, CD204, and Iba-1 and compared with the pigment cells accumulated within the irides. Cultured uveal melanocytes from OM-affected and control dogs were compared using conventional assays measuring cell proliferation, invasion capability, and melanin production. RESULTS: Melanocytes isolated from OM eyes had significantly elevated levels of per-cell melanin content and production compared to controls. The majority of pigmented cells in the scleral plaques were HMB45 positive indicating a melanocytic origin. Many were also CD18 positive. No differences were observed between cultured melanocytes from OM-affected and control uvea for standard in vitro proliferation or invasion assays. CONCLUSION: Pigmented cells which accumulate in the sclera of OM-affected Cairn terriers are predominantly melanocytes; however, in vitro assays of uveal melanocytes did not reveal differences in migratory behavior between OM and control cells. Migratory behavior of OM-melanocytes may be environment-dependent. We suggest that RNA sequencing and differential expression analysis would be a useful next step in understanding this disease.

Ocular Iridociliary Adenoma in a Congo African Grey Parrot (Psittacus erithacus).

A 19-year-old female Congo African grey parrot (Psittacus erithacus) presented for an oval, solid, pigmented, suspected intraocular mass with extrascleral extension through the inferior cornea of the left eye. The eye was nonvisual, and intraocular portions of the mass significantly altered the posterior chamber. Neoplasia was confirmed by biopsy, and enucleation was performed because of the severity of ocular disease, loss of vision, enhancement of patient comfort, and potential metastasis. Histopathologic examination of the entire globe revealed a pigmented iridociliary adenoma. Iridociliary adenomas have been rarely reported in birds, and this case report details diagnosis and treatment. Iridociliary adenomas in other species are often benign, indicating this neoplasia can be successfully treated with no reoccurrence by complete excision.

Malignant transformation of canine oral papillomavirus (CPV1)-associated papillomas in dogs: An emerging concern?

Canine oral papillomavirus (CPV1, also known as COPV), the most common cause of non-neoplastic papillomas, has not been shown to cause squamous cell carcinomas (SCC). Furthermore, malignant transformation of benign papillomas to SCC has only been reported in a single group of dogs with severe combined immunodeficiency infected with CPV2. Here, we report a series of 7 dogs with benign CPV1-associated papillomas with histologic evidence of CPV1 causing malignant transformation to carcinoma in situ and ultimately SCC. Expression of p53 and p16 proteins in CPV1-infected cells within the benign papillomas and lesions that progressed into SCC also supported an association between papillomavirus and malignant transformation. Moreover, our retrospective analysis indicated that while there have been increased numbers of viral papillomas with malignant transformation, the number of annually diagnosed canine viral papillomas has remained constant over the past decade in our laboratory. We speculate that either an altered host immunity from increased usage of immunosuppressive drugs or changing environmental factors, e.g. increase exposure to UV radiation, may cause an increased oncogenic potential of this "low-risk" virus. This study aims to raise awareness of the malignant potential of CPV1 and to encourage further investigations into the cause of this suspected change in its oncogenic potential.

Genomic characterization of Erethizon dorsatum papillomavirus 2, a new papillomavirus species marked by its exceptional genome size.

We report here the complete sequence and genome organization of a new papillomavirus, Erethizon dorsatum papillomavirus 2 (EdPV2), which was isolated from cutaneous lesions observed on the muzzle of a North American porcupine. The complete genome is 8809 nucleotides long and encodes five early (E6-E7-E1-E2-E4) and two late proteins (L2-L1). In addition to the upstream regulatory region, the EdPV2 genome contains an exceptionally large secondary non-coding region with no apparent functional relevance. EdPV2 is strongly divergent from the previously described porcupine papillomavirus EdPV1 and phylogenetic analysis shows EdPV2 clustering near members of the genus Pipapillomavirus, a group of rodent papillomaviruses. Pairwise sequence comparison based on the L1 open reading frame identifies Rattus norvegicus papillomavirus 1 as the closest related virus (59.97 % similarity). Based on its low sequence similarity to other known papillomaviruses, EdPV2 is thought to represent a new genus in the family Papillomaviridae.

Immunohistochemical Characterization of Canine Oral Papillary Squamous Cell Carcinoma.

Recently, histologic subtypes of oral squamous cell carcinoma (SCC) corresponding to the human classification scheme have been proposed for dogs. A papillary squamous cell carcinoma subtype is characterized by dominant exophytic architectural growth with limited invasion, a lower metastatic rate, and better overall survival compared with conventional SCC. Whereas most canine oral conventional SCCs are easily diagnosed by histologic examination, the diagnosis of canine oral papillary squamous cell carcinoma (COPSCC) can be challenging since the exophytic portion lacks histologic features of malignancy and appears similar to oral nonviral papillomas. In contrast, the invasive portion of COPSCC has morphologic similarities to conventional SCC and canine acanthomatous ameloblastoma. The goals of this study were to immunophenotype these 3 entities and to potentially identify discriminating markers. A panel of 17 immunohistochemical markers was investigated in tissue microarrays that included 25 COPSCCs, 10 conventional SCCs, and 10 canine acanthomatous ameloblastomas. Additionally, COPSCCs were screened for papillomavirus as a potential cause using immunohistochemistry and in situ hybridization. COPSCC had immunophenotypical similarities with conventional SCC and acanthomatous ameloblastoma, but the combined differences in immunolabeling for AE1/AE3, 34ßE12, p63, and calretinin discriminated between the entities. Papillomavirus was not detected in any COPSCC, making a viral pathogenesis unlikely. A better understanding of the immunophenotype of COPSCC will aid in a more accurate diagnosis and potentially improve therapeutic approaches.

Cardiac findings in Quarter Horses with heritable equine regional dermal asthenia.

OBJECTIVE To compare biomechanical and histologic features of heart valves and echocardiographic findings between Quarter Horses with and without heritable equine regional dermal asthenia (HERDA). DESIGN Prospective case-control study. ANIMALS 41 Quarter Horses. PROCEDURES Ultimate tensile strength (UTS) of aortic and mitral valve leaflets was assessed by biomechanical testing in 5 horses with HERDA and 5 horses without HERDA (controls). Histologic evaluation of aortic and mitral valves was performed for 6 HERDA-affected and 3 control horses. Echocardiography was performed in 14 HERDA-affected and 11 control horses. Biomechanical data and echocardiographic variables of interest were compared between groups by statistical analyses, RESULTS Mean values for mean and maximum UTS of heart valves were significantly lower in HERDA-affected horses than in controls. Blood vessels were identified in aortic valve leaflets of HERDA-affected but not control horses. Most echocardiographic data did not differ between groups. When the statistical model for echocardiographic measures was controlled for body weight, mean and maximum height and width of the aorta at the valve annulus in short-axis images were significantly associated with HERDA status and were smaller for affected horses. CONCLUSIONS AND CLINICAL RELEVANCE Lower UTS of heart valves in HERDA-affected horses, compared with those of control horses, supported that tissues other than skin with high fibrillar collagen content are abnormal in horses with HERDA. Lack of significant differences in most echocardiographic variables between affected and control horses suggested that echocardiography may not be useful to detect a substantial loss of heart valve tensile strength. Further investigation is warranted to confirm these findings. Studies in horses with HERDA may provide insight into cardiac abnormalities in people with collagen disorders.

Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection.

In view of the varied biologic behavior and the costs of treatment for canine cutaneous mast cell tumors, development of appropriate treatment plans for individual affected dogs can be difficult, but decisions regarding treatment should be made using a systematic, evidence-based approach. This manuscript reviews the current state of diagnostics and prognostication of canine cutaneous mast cell tumors, and suggests a combined approach based on clinical and pathologic assessment for decision making regarding treatment choices. The current state of histologic grading, evaluation of proliferation indices, evaluation of mutations in the c-kit gene and KIT expression, evaluation of excision and clinical staging are examined. On the basis of the current understanding of prognostication and treatment response, algorithms for selection of local and systemic therapy are presented.