Expression of SARS-CoV-2 entry molecules ACE2, NRP1, TMPRSS2, and FURIN in the reproductive tissues of male macaques.

Coronavirus disease 2019 (COVID-19) reportedly affects male reproductive function by causing spermatogenesis dysfunction and suppressing testosterone secretion. However, the relationship between COVID-19 and impaired reproductive function, such as whether these effects on reproductive function are a direct effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in male reproductive organs or an indirect effect of high fever, is not known. Here, we examined whether the cell entry molecules of SARS-CoV-2, namely, ACE2, NRP1, TMPRSS2, and FURIN, are expressed in the male reproductive organs using the testes and accessory gonads of macaques during the breeding season. RT-PCR expression analysis showed that the testes alone expressed all four molecules. Immunohistochemical staining of testis tissue sections revealed that ACE2 is expressed in Leydig cells and the apical region of Sertoli cells, whereas NRP1 is expressed in the cell bodies surrounding the Leydig and Sertoli cell nuclei. FURIN is mainly expressed in Leydig cells, secondary spermatocytes, and spermatids. However, TMPRSS2 immunopositive cells were not observed. Therefore, it was not possible to observe cells expressing all four molecules in the gonads and accessory gonads of male primates. These results suggest that SARS-CoV-2 is unlikely to directly affect spermatogenesis in primates or proliferate in cells of the seminiferous tubules and undergo release into the semen through the previously known ACE2-mediated infection route. However, the expression of three molecules, including ACE2, was observed in Leydig cells, suggesting that testosterone synthesis and secretion may be affected when primates, including humans, are infected with SARS-CoV-2.

Sclerosing encapsulating peritonitis in a dog with pancreatic ductal adenocarcinoma.

BACKGROUND: Sclerosing encapsulating peritonitis (SEP) is a rare clinical syndrome characterised by fibrosis and thickening of the peritoneum with massive adhesions of the abdominal organs. In humans, abdominal tumours, such as pancreatic adenocarcinoma, can be underlying diseases of SEP. This report describes a case of SEP in a dog with pancreatic ductal adenocarcinoma. CASE PRESENTATION: An 11-year-old male neutered French Bulldog presented with chronic vomiting. Ultrasonography revealed a mass in the centre of the abdomen. A small amount of ascites, interpreted as modified transudate, was present in the abdominal cavity. Computed tomography (CT) revealed peritoneal effusion with a thickened peritonium. Laparoscopy revealed a large nodular lesion occupying the central portion of the abdomen, continuous with the falciform ligament. Histological examination of the biopsy specimens of the mass, abdominal wall, and gastric peritoneum revealed marked fibroplasia with mild lymphoplasmacytic infiltrates. Based on these results, a tentative diagnosis of early stage sclerosing encapsulating peritonitis (SEP) was made. Prednisolone and tamoxifen were administered with the expectation of ameliorating SEP, however, the dog died 61 days post diagnosis. At autopsy, the intestinal loop and mesentery were encased in the fibrous membrane, which is a typical finding in SEP. Histopathology and immunohistochemistry of the samples obtained at autopsy supported the diagnosis of pancreatic ductal adenocarcinoma with peritoneal dissemination and distant metastasis with desmoplasia. The unexpectedly hardened skin, where previously laparoscopic ports were inserted, histologically contained the same carcinoma cells with desmoplasia. CONCLUSIONS: To the best of our knowledge, this is the first report of canine SEP with pancreatic ductal adenocarcinoma that also caused metastasis to port insertion sites as well as distant organs.

Pathology and Proteomics-Based Diagnosis of Localized Light-Chain Amyloidosis in Dogs and Cats.

Amyloidosis is classified according to the amyloid precursor protein, and accurate diagnosis of the amyloidosis type may guide appropriate treatment. Immunohistochemistry and Congo red staining are the most frequently used methods used to distinguish types of amyloidosis, but problems with specificity and sensitivity indicate the need for an alternative diagnostic method. In this study, we evaluated laser microdissection-liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS) for the diagnosis of amyloid light-chain (AL) amyloidosis in animals. Plasmacytomas with amyloid deposits from 15 dogs and 2 cats were subjected to Congo red staining with or without potassium permanganate pretreatment, immunohistochemistry for kappa and lambda light chains, and LMD-LC-MS/MS. Congo red staining was diagnostic in 12 of 17 cases based on resistance to potassium permanganate pretreatment, but in 5 of 17 cases the pretreatment unexpectedly reduced Congo red staining or abrogated the birefringence and a definitive diagnosis could not be reached. Immunohistochemistry detected kappa or lambda light chains in 6 of 17 cases. With LMD-LC-MS/MS, immunoglobulin lambda light chain was detected in all 17 cases. The amyloid signature proteins ApoA-I, ApoA-IV, and ApoE were detected in 9, 1, and 3 of the 15 canine cases by LMD-LC-MS/MS, but not in the feline cases. In conclusion, LMD-LC-MS/MS consistently determined the amyloid type in all examined specimens, while Congo red staining after potassium permanganate treatment and immunohistochemistry were less sensitive tests.

Suspected renal interstitial cell tumor causing polycythemia in two dogs.

Here we report a case series of two dogs diagnosed as renal interstitial cell tumor (RICT) accompanied by elevated serum erythropoietin level and marked polycythemia. RICT is a rare tumor in dogs, originating from renal interstitial cells. While several renal tumors such as renal lymphoma, adenocarcinoma, carcinoma, sarcoma, fibrosarcoma and nephroblastoma may cause polycythemia, polycythemia caused by RICT has never been reported in dogs. The tumors in both dogs were solitary and lied within cortex or cortico-medullary junction. Histopathology revealed spindle-shaped cells suggesting mesenchymal origin, with no mitotic figures suggesting that the tumors in both dogs were benign. Following surgical removal of the affected kidney, serum erythropoietin level and polycythemia normalized in both dogs.

Canine Gallbladder Erosion/Ulcer and Hemocholecyst: Clinicopathological Characteristics of 14 Cases.

(1) Background: Gallbladder mucosal erosion and/or ulceration are illnesses associated with unexpected gallbladder intra-cystic bleeding (hemocholecyst), an under-reported problem in dogs. (2) Methods: Clinicopathological characteristics of 14 dogs with gallbladder erosion/ulcer were investigated in this single-center retrospective study using clinical data and archived gallbladder tissues of client-owned dogs. (3) Results: Canine gallbladder erosion/ulcer tends to occur in older, neutered dogs of various breeds. Vomiting, lethargy, and anorexia are common. Concurrent gallbladder rupture occurred in 5/14 cases (35.7%), while rupture was absent in 6/14 cases (42.8%) and undetermined in 3/14 (21.4%) cases. Histologically, the gallbladder wall was markedly thickened due to mucosal hyperplasia, inflammatory infiltrates, fibrosis, edema, hemorrhage, and smooth muscle hyperplasia/hypertrophy. Twelve out of fourteen cases (85.7%) had concurrent cholecystitis of varying severity. Bacteria were detected by Giemsa or Warthin-Starry stain in 8/14 (57.1%) cases. Bacterial rods immunoreactive to the anti-Helicobacter antibody were present in one case. Mucosal epithelial cells of the gallbladder erosion/ulcer cohort were immunopositive for the cyclooxygenases COX-1 or COX-2 in only 5/14 (35.7%) cases. In contrast, COX-1 and COX-2 were more frequently expressed in a reference pool of cases of gallbladder mucocele (n = 5) and chronic cholecystitis (n = 5). COX-1 was expressed in 9/10 cases (90.0%) of gallbladder mucocele and chronic cholecystitis and in 10/10 cases (100%) for COX-2. (4) Conclusions: Canine gallbladder erosion/ulcer is an under-reported condition which requires active clinical intervention. Based on the clinicopathological information reported in this study in addition to the COX-1 and COX-2 IHC results, we suggest that canine gallbladder erosion/ulcer may be related to decreased cytoprotection physiologically provided by arachidonic acid, but which is decreased or absent due to reduced COX expression because of yet undetermined etiologies.

Magnetic resonance imaging findings of the primitive neuroectodermal tumour in lumbosacral spinal cord in a cat.

A 5-year-old, castrated, male domestic short-haired cat presented with neurological deficits in the pelvic limbs, back pain and dysuria. Magnetic resonance imaging showed a mass lesion caudal to the L4 vertebrae. In addition, suspected haemorrhage was observed at the cranial aspect of the mass. There was no evidence to support the presence of extravertebral intrusion or vertebral body, osteolysis. Dorsal laminectomy and durotomy were performed to debulk the intraspinal mass. Histopathological and immunohistochemical assessment revealed a primitive neuroectodermal tumour (PNET). To our knowledge, this is the first report to describe the clinical and pathological features and imaging diagnosis of intraspinal PNET without extraspinal invasion in a cat.

Vascular ring anomaly with a right patent ductus arteriosus and a left aortic arch in a juvenile cat.

This paper reports the clinical findings and surgical treatment of feline right patent ductus arteriosus (RPDA) with a left aortic arch. A two-month-old female Maine Coon was referred for an investigation of regurgitation after weaning. RPDA with a left aortic arch was diagnosed based on the echocardiographic and computed tomography (CT) findings. A right-fourth intercostal thoracotomy was found to be an appropriate approach to the duct. Preoperative diagnosis is crucial and diagnostic imaging, including radiography, echocardiography, and cardiac CT examination, is essential for determining if the aortic arch is right or left.

Phenotypic and molecular characterization of novel pulmonary adenocarcinoma cell lines established from a dog.

Canine pulmonary adenocarcinoma (PAC) resembles human lung tumors in never-smokers, but it is rarer than human pulmonary adenocarcinoma. Therefore, research on canine PAC is challenging. In the present study, we successfully established various novel canine PAC cell lines from a single lesion in a dog, including two parent cell lines and fourteen cloned cell lines, and characterized their cellular properties in vitro. Several of these cell lines showed epithelial-mesenchymal transition (EMT)-like and/or cancer stem cell (CSCs)-like phenotypes. We additionally assessed the sensitivity of the cells to vinorelbine in vitro. Three clonal lines, two of which showed EMT- and CSC-like phenotypes, were resistant to vinorelbine. Furthermore, we evaluated the expression and activation status of EGFR, HER2, and Ras signaling factors. The findings indicated that the cell lines we established preserved the expression and activation of these factors to varying extents. These novel canine PAC cell lines can be utilized in future research for understanding the pathogenesis and development of treatments for canine PAC.

Concurrent lacrimal gland melanocytoma and ocular melanocytosis in a dog.

A 9-y-5-mo-old, spayed female, mixed-breed dog with buphthalmia and elevated intraocular pressure in the left eye, consistent with glaucoma, was evaluated. Black-pigmented, slightly elevated tissue with irregular margins was noted on the dorsolateral aspect of the left globe. Ultrasonography detected a mass, later identified as lacrimal gland, adjacent to the globe and the thickened uvea. The surgically removed lacrimal gland was effaced by dense sheets of melanin-laden cells. Within the enucleated globe, numerous melanin-laden cells infiltrated and expanded the rostral two-thirds thickness of the cornea, the entire anterior uvea (iris and ciliary body), and a rostral portion of the choroid. Melanin-laden cells in the left lacrimal gland and globe showed no nuclear atypia or mitotic figures, and reacted to anti-S100 and anti-melan A antibodies by immunohistochemistry. Our final diagnosis was concurrent lacrimal gland melanocytoma and ocular melanocytosis. The trabecular meshwork of the eye was obliterated by melanin-laden cells, which was the likely cause of glaucoma in this patient. To our knowledge, melanocytoma affecting the lacrimal gland has not been reported previously in a non-human mammalian species. Veterinary clinicians are encouraged to include melanocytoma in the differential list when examining an enlarged lacrimal gland.

Effects of Oxygen Gas Injection on the Subsequent Development of Chick Embryos in a Shell-Less Culture System.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.

Effects of Calcium Lactate on the Development of Chicken Embryos in a Shell-less Culture System up to Day Seventeen of Incubation.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

Expression profile of immunoregulatory factors in canine tumors.

Cancers utilize a variety of molecules to escape host immune responses. Better understanding the immune environment surrounding cancer may facilitate application of innovative cancer immunotherapies, such as immune checkpoint inhibitors, to dogs as well as humans. In this study, we screened the expression of 20 immune regulatory molecules in diverse canine tumors (n = 59). Quantitative RT-PCR (qPCR) analysis revealed that some immune regulatory molecules, such as LGALS9 (coding Galectin-9) and CD48, were expressed in most canine tumors, but other molecules, such as CD274 (coding PD-L1), IL4I1, PVR, TNFSF18, ICOSLG, and TNFSF4, were rarely expressed. NECTIN2 was highly expressed in epithelial tumors but was low in non-epithelial tumors. In contrast, VSIR and CD200 expressions were low in epithelial tumors but high in non-epithelial tumors. Interestingly, several tumors expressed distinctive immunoregulatory factors. Hepatocellular carcinomas expressed FGL1, mast cell tumors expressed PDCD1LG2 (coding PD-L2), transitional cell carcinomas expressed VTCN1 (coding B7x), and lymphomas and squamous cell carcinomas expressed CD70. Consistent with qPCR results, immunofluorescence staining confirmed that hepatocellular carcinomas expressed FGL-1 protein. Thus, this study reveals the expression profile of immunoregulatory molecules in canine tumors and opens the door to better understanding the relationship between canine tumors and host immunity.

Chronic Cholecystitis of Dogs: Clinicopathologic Features and Relationship with Liver.

(1) Background: Chronic cholecystitis of dogs has not been vigorously investigated histopathologically. In addition, the relationship between gallbladder and liver diseases is not known. (2) Methods: We aimed to provide a hallmark for canine chronic cholecystitis using clinical data, histopathology, histochemistry, immunohistochemistry, and statistical analysis. (3) Results: Our investigation of 219 ultrasonographically abnormal surgically resected canine gallbladders revealed 189 cases (86.3%) of mucosal lymphoplasmacytic infiltration (chronic cholecystitis). Sludge, a gravity-dependent or nondependent fine granular hyperechoic material, was more prevalent (105/219, 47.9%) than mucocele (51/219, 23.2%) in this cohort. Mucosal lymphoid follicles were detected in 68/219 cases (31%), suggesting the influence of long-standing antigenic stimulation. Bacteria were histochemically detected in 41/60 (68.3%) of heavily inflamed gallbladders, 18/129 (14%) of lightly inflamed, and 3/18 (16.7%) of uninflamed gallbladders, suggesting a possible relationship between bacteria and chronic cholecystitis. Simultaneous liver biopsies revealed mild or no inflammation, changes consistent with primary portal vein hypoplasia, and mild hepatocellular degeneration. (4) Conclusions: Based on the results of our statistical analysis, we conclude that canine chronic cholecystitis is a long-standing inflammatory process of unknown (but possibly bacterial) etiology and that liver pathology is unlikely the cause of chronic cholecystitis in dogs.

Anti-CD71 antibody immunohistochemistry in the diagnosis of acute myeloid leukemia, subtype acute erythroid leukemia with erythroid dominance (AML M6-Er), in a retrovirus-negative cat.

CD71 is an immunohistochemical marker used in diagnosing acute myeloid leukemia (AML) M6-Er in humans; however, to our knowledge, it has not been reportedly used for immunohistochemistry in veterinary medicine. We evaluated the pathologic features of AML M6-Er in a retrovirus-negative cat and used CD71 to support the diagnosis. A 4-y-old spayed female Scottish Fold cat was presented with lethargy, anorexia, and fever. Whole-blood PCR assay results for pro feline leukemia virus/pro feline immunodeficiency virus and feline vector-borne diseases were negative. Early erythroid precursors were observed in the peripheral blood smear. Fine-needle aspiration of the enlarged spleen and splenic lymph node showed many early erythroid precursors. Bone marrow aspirate smears revealed erythroid hyperplasia with 68.4% erythroid lineage and 3.6% rubriblasts. Dysplastic cells infiltrated other organs. The patient was diagnosed with myelodysplastic syndrome, progressing to the early phase of AML M6-Er. The patient died on day 121 despite multidrug treatments. Postmortem examination revealed neoplastic erythroblasts infiltrating the bone marrow and other organs. Neoplastic cells were immunopositive for CD71 but immunonegative for CD3, CD20, granzyme B, von Willebrand factor, CD61, myeloperoxidase, and Iba-1. Although further studies are necessary for the application of CD71, our results supported the morphologic diagnosis of AML M6-Er.

New canine parvovirus 2a infection in an imported Asian small-clawed otter (Aonyx cinereus) in Japan.

Post-import from the Republic of Indonesia to Japan in 2017, two juvenile, captive bred Asian small-clawed otters (Aonyx cinereus) exhibited gastrointestinal symptoms, including vomiting, diarrhea, and hematemesis, and died. One of them was examined postmortem. Microscopically, the small intestinal mucosa was necrotic with crypts lined by regenerating large epithelial cells. A gastric cardiac mucosal ulcerative lesion containing fungal yeasts and pseudohyphae morphologically indicated Candida spp. The lymph nodes exhibited marked lymphoid depletion. Canine parvovirus 2 (CPV-2) was isolated from an oral swab, and virus protein 2 (VP2) gene sequencing revealed new CPV-2a. To our knowledge, this is the first new CPV-2a infection report in Asian small-clawed otters. This infection should be considered in gastrointestinal symptom-related cases in this species.

Forestomach developmental failure in an 11-month-old Japanese Black steer with severely retarded growth and chronic ruminal tympany.

This study reports findings from the pathological examination of the forestomach of an 11-month-old Japanese Black steer with severely retarded growth (41% of expected weight) and chronic ruminal tympany. The ruminal papillae were weakly formed (0.3-0.5 cm long) and unevenly distributed. The cellulae and cristae reticuli were underdeveloped; the cristae were 0.4-0.7 cm in height and milky white. The keratinized layer in the stratified squamous epithelium was thickened. Ruminal pH was 5.25, and ruminal volatile fatty acid concentration was 11.7 mM. The steer's severely retarded growth was considered to be caused by malnutrition due to developmental and functional failure of the forestomach.

Dysuria Associated with Non-Neoplastic Bone Hyperplasia of the Os Penis in a Pug Dog.

A three-year-old male Pug presented with a three-year history of urolithiasis and repeated urethral obstruction. Biochemical analysis, ultrasonography, and retrograde urethrocystography revealed probable portosystemic shunt and incomplete urethral obstruction due to uric acid ammonium calculi. Enhanced computed tomography (CT) revealed portosystemic shunt and proliferation of the osseous tissue of the os penis, which was surgically removed. Histopathologically, the excised osseous tissue comprised bland lamellar bone without atypia or inflammation. Hyperplasia of the os penis was diagnosed based on the image findings and histopathology. The dysuria improved postoperatively. This is the first report of dysuria associated with non-neoplastic bone hyperplasia of the os penis in a dog. Careful evaluation of the os penis by CT is needed for accurate diagnosis in case of repeated penile urethral obstruction.

Sudden death associated with hypertrophic cardiomyopathy in an alpaca (Llama pacos).

A healthy, 3-year-old, intact male alpaca (Llama pacos) died suddenly and unexpectedly and had an unusual pattern of hypertrophic cardiomyopathy that was restricted to the interventricular septal and right ventricular myocardium. Grossly pale areas in the affected myocardium corresponded histologically to thickened and disorganized myofibers with excessive branching and marked nuclear pleomorphism. Mitoses were absent. Inflammation and fibrosis were minimal. A few thickened muscular arteries were scattered in the affected myocardium.

Lobular diameters of autopsied dog livers give clues for an appropriate liver biopsy methodology.

Hepatobiliary diseases of animals are frequently diagnosed by a combination of imaging, clinical pathology, and histopathology. A standardized surgical liver biopsy protocol, however, has not been established in veterinary medicine with regard to the selection of lobe and site of the liver to yield the most diagnostic information. To address this matter, we histologically examined 33 livers of autopsied dogs from which tissue samples of 4 different lobes as well as 4 different sites of each lobe were prepared. We measured the hepatic lobular diameter (HLD) as an objective variable to refer to the inter-lobar or inter-site difference among the biopsied samples. A measurement of 2,623 hepatic lobules resulted in 1.042 mm as the average of all the HLD values. Statistical analysis further revealed that the HLD tended to be small in a superficial 2 mm area of the liver parenchyma regardless of biopsy location, thus this area should be evaluated carefully by pathologists. The results also suggest that the HLD values of the quadrate lobe may measure smaller than those in the other lobes. Therefore, one would be able to obtain representative data of the entire liver by taking a sample from any single lobe except for the quadrate lobe. HLD measurements are needed in order to accumulate potentially useful information on the microanatomy and pathophysiology of the liver.

A laryngeal inflammatory polyp in a miniature dachshund.

A 15-year old, female miniature dachshund was presented for a 5-year history of progressive dyspnoea. A laryngeal mass was detected on cervical radiographs. Surgery was performed 5 days later to reduce the volume of the mass and to obtain biopsy samples; the dyspnoea improved. Based on the histopathological findings, the mass was diagnosed as inflammatory polyp. This case of inflammatory polyp in the larynx suggests that it should be listed in the differential diagnoses for laryngeal masses in dogs and that surgical treatment may be curative.