Monoclonal proliferation of B-cells with two integration sites of bovine leukemia virus proviral DNA in cattle with enzootic bovine leukosis.

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.

Diffuse large B-cell lymphoma with DNA copy number changes in a Japanese black calf.

A 2-month-old Japanese Black calf exhibited mandibular and superficial cervical lymph node swelling. Fine needle aspiration cytology of the superficial cervical lymph node revealed large lymphoblast-like cells with mitoses. Hematological examination revealed remarkable lymphocytosis with atypical lymphocytes. Increased activities of serum total lactate dehydrogenase and thymidine kinase were detected. At necropsy, generalized swelling of lymph nodes was observed. Histopathological analysis revealed diffuse proliferation of medium-sized round centroblastic neoplastic cells that were positive for CD20, CD79α, PAX5, and BLA-36, and negative for CD3, CD5, CD10, and CD34. The calf was diagnosed with centroblastic diffuse large B-cell lymphoma (DLBCL) based on these findings. Analysis of DNA copy number variation revealed an increased copy number for the GIMAP family relative to that in healthy cattle. Moreover, decreases in copy numbers of GBP-1, MIR3141, OR5P1E, and PTPRG relative to those in healthy cattle were also observed. Because DNA copy number variation represent a major contribution to the somatic mutation landscapes in human tumors, these findings suggest that DNA copy number changes might have contributed to the onset of DLBCL in the present case.

Non-suppurative and necrotizing testicular arteritis in the male reproductive organs of a heavy draft horse.

Equine testicular arteritis commonly occurs as a consequence of the migration of nematode larvae or equine arteritis virus (EAV) infection. However, testicular arteritis without evidence of these infections has been reported, and the underlying pathogenesis remains unclear. We encountered testicular arteritis without evidence of nematode or EAV infection in a 3-year-old male heavy draft horse with scrotal enlargement. Grossly, the volume of the pampiniform plexus was markedly increased due to edema. Histologically, non-suppurative and necrotizing testicular arteritis, characterized by lymphocyte infiltration and fibrinoid necrosis of the arterial walls, was diffusely observed in the spermatic cord, pampiniform plexus (most severe), testis, and epididymis. We were unable to identify the cause of arteritis, such as a viral infection or autoimmune abnormality.

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA.

Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.

Multiple endocrine neoplasia in a sheep: insulinoma, adrenocortical carcinoma with myxoid differentiation, and thyroid C-cell carcinoma.

An ~10-y-old male sheep had anorexia and progressive weight loss for ~1 mo. The sheep was emaciated, and 20 d later, became recumbent and lethargic, and was hypoglycemic (0.33 mmol/L; RI: 2.6-4.4 mmol/L). The sheep was euthanized because of poor prognosis, and submitted for autopsy. We found no gross lesions in the pancreas; however, histologically, focal proliferations of round-to-polygonal cells were separated by connective tissue into small nests. These proliferating cells, which had abundant eosinophilic-to-amphophilic cytoplasm and hyperchromatic nuclei, were immunopositive for insulin and negative for glucagon and somatostatin; the lesion was diagnosed as an insulinoma. Insulinoma has not been reported previously in sheep, to our knowledge. In addition, autopsy and histologic examination revealed the presence of an adrenocortical carcinoma with myxoid differentiation and a thyroid C-cell carcinoma. Our case indicates that multiple endocrine neoplasms can occur in sheep, as in other animal species.

Epididymitis in mice experimentally infected with Trypanosoma equiperdum: a histopathological and immunohistochemical study.

Trypanosoma equiperdum is a causative agent of dourine in equids and is transmitted from stallions to mares by coitus. Dourine-affected stallions develop orchitis and epididymitis, and these lesions are considered to be responsible for the sexual transmission of T. equiperdum during coitus. However, the parasitic site of trypanosomes in the reproductive organs and the mechanisms underlying transmission have not yet been elucidated histopathologically. We examined the reproductive organs of male mice infected with T. equiperdum histopathologically and identified severe epididymitis with sperm granulomas. Many trypanosomes were detected in the epididymal interstitium and a few were seen within tubular lumen. Interstitial inflammatory cells mainly consisted of Iba1-, iNOS- and CD204-positive cells with a few CD3-, FOXP3- or PAX5-positive cells. There was diffuse immunolabelling of tumour necrosis factor-α (TNF-α) within these inflammatory foci. While caspase-3-positive epithelial cells in the epididymis were not observed in control mice, they were detected multifocally in infected mice and were frequently associated with loss of immunolabelling of zonula occludens-1 (ZO-1), a major protein that forms tight junctions between epididymal epithelial cells. Anti-laminin immunofluorescence revealed an indistinct basement membrane of the epididymal duct. These results suggest that trypanosomes in the epididymal interstitium induce the infiltration of TNF-α-secreting macrophages. Secreted TNF-α may impair the tight junctions of the epididymal duct by inducing apoptosis and downregulating ZO-1 expression.

Hemophagocytic syndrome in a cat with immune-mediated hemolytic anemia.

A 10-year-old spayed female domestic short-haired cat presented with depression, anorexia, and tachypnea. A complete blood count revealed moderate regenerative anemia, severe leukopenia, and mild thrombocytopenia. Antibodies against feline immunodeficiency virus (FIV) were also detected. Abdominal radiography and ultrasonography revealed severe splenomegaly. Cytologic evaluation of the spleen revealed macrophagic infiltration with hemophagocytosis. Bone marrow aspiration revealed erythroid hyperplasia with no other abnormalities. A presumptive diagnosis of hemophagocytic syndrome secondary to immune-mediated hemolytic anemia was made based on a positive direct Coombs test result. Blood transfusion, prednisolone, and immunosuppressive treatments were performed; however, the blood abnormalities did not improve. The cat was then administered prednisolone and chlorambucil, followed by splenectomy. Leukopenia immediately recovered, and packed cell volume increased slightly. However, the blood abnormalities recurred, and the cat died. To the best of our knowledge, this is the first report of hemophagocytic syndrome secondary to immune-mediated disease in an FIV-positive cat.

Cholesterol granuloma associated with degenerative neuropathy in the cauda equina of a dog.

An 8-y-old Labrador Retriever dog had mild ataxia of the hindlimbs 4 mo after lumbosacral dorsal laminectomy for intervertebral disk disease. Ataxia of the hindlimbs gradually worsened over the next 6 y. On autopsy, gross lesions were not recognized in the spinal cord. Histopathology revealed an intradural extraparenchymal cholesterol granuloma in the cauda equina associated with remnant nerve roots. Nerves associated with the cholesterol granuloma had axonal degeneration, myelin vacuolation, and edema. In those foci, macrophages were increased in number between nerve fibers. Immunohistochemistry for neurofilament protein and Luxol fast blue staining highlighted the presence of remnant axons and myelin sheaths within the granuloma. Inflammatory cell infiltrates in the granuloma were mainly macrophages and CD3- or CD20-immunopositive T or B lymphocytes, respectively. We conclude that the cholesterol granuloma likely formed subsequent to degenerative neuropathy in the cauda equina.

Enzootic bovine leukosis in a 21-month-old Japanese Black cow with high susceptibility.

A 21-mo-old Japanese Black beef cow had swollen mandibular and superficial cervical lymph nodes. Fine-needle aspiration cytology of the superficial cervical lymph node revealed large lymphoblasts with mitoses present. The bovine leukemia virus (BLV) proviral load was relatively high, and phylogenetic analysis of the whole BLV genome classified the BLV strain as one with high viral replication activity. Genotyping of bovine leukocyte antigen genes indicated that the cow was susceptible to enzootic bovine leukosis (EBL). The bone morphogenetic protein 6 (BMP6) gene promoter region was hypermethylated. Monoclonal proliferation of B cells and monoclonal integration of the BLV provirus in the bovine genome were detected by a clonality test of B cells and an inverse PCR assay, respectively. At autopsy, generalized swelling of lymph nodes and spinal canal invasion by tumor tissue at vertebrae L5-6 were observed. Histologic analysis revealed diffuse proliferation of large round neoplastic cells that were positive for BLA36 and negative for CD3. The cow was definitively diagnosed with EBL based on these findings. Infection with a highly pathogenic strain of BLV, susceptibility of the BoLA-DRB3 alleles, and hypermethylation of the BMP6 gene may have contributed to the development of EBL in our case.

Bilateral Plaque-Like White Matter Degeneration in Cerebral Septal Regions of a Chapman's Zebra (Equus quaggachapmani).

Histopathological examination of an approximately 19-year-old female Chapman's zebra (Equus quagga chapmani) revealed multifocal bilateral plaque-like lesions in rostral septal regions of the cerebrum. The centre of these lesions consisted of radiating, acicular eosinophilic structures, which were surrounded by rare glial cells. Spheroids were also seen around the lesions. Luxol fast blue and Bodian staining revealed loss of normal myelin and axonal structures within the plaque-like lesions. The glial cells surrounding the lesions were considered reactive astrocytes due to their immunoreactivity to vimentin, glial fibrillary acidic protein and alpha-smooth muscle actin. Based on these findings, the lesions were consistent with focal white matter degeneration localized in bilateral, rostral septal regions; these lesions have not been previously reported. The cause of the lesions was not determined despite the application of various histochemical stains.

Renal Dysplasia with Hydronephrosis and Congenital Ureteral Stricture in Two Holstein-Friesian Calves.

We investigated the pathological characteristics of renal dysplasia with hydronephrosis and congenital ureteral stricture in two calves. Macroscopically, the affected kidneys were enlarged and the renal calyces were dilated and associated with ureteral strictures. Histopathologically, multifocal regions of mesenchyme were observed in the renal medulla. This mesenchyme was weakly eosinophilic with haematoxylin and eosin, blue with Alcian blue and pale blue with Masson's trichrome, and was immunopositive for vimentin and smooth muscle actin, consistent with persistent mesenchyme. There was asynchronous differentiation of the renal cortex characterized by immature glomeruli, immature tubules and arteriolar proliferation. Similar persistent mesenchyme was observed in the ureteral walls with ureteral stricture, and the ureteral musculature or smooth muscle bundles had a disorganized arrangement. Congenital ureteral stricture appeared to have caused ureteral obstruction and hydronephrosis. The lesions may represent a new phenotype of renal dysplasia with concomitant congenital ureteral stricture in Holstein-Friesian calves.

Characteristic computed tomographic myelography findings in 23 Thoroughbred horses.

The purpose of this observational study was to determine the characteristic computed tomographic (CT) myelography findings of cervical vertebral stenotic myelopathy (CVSM) lesions in Thoroughbred horses. A total of 23 Thoroughbred horses (age range, 155-717 days on CT examination; mean, 410.9 days) were analyzed. All 23 Thoroughbred horses underwent unenhanced radiography, radiographic myelography, and CT myelography. Unenhanced radiographs were observed the presence of cervical vertebral malalignment and osseous lesions. Radiographic myelograms were observed for signs of cervical spinal cord compression; additionally, CT myelograms were used to detect cervical vertebral osseous lesions. Ventral compressions were frequently observed in the cranial cervical vertebrae (C2-C4), whereas dorsal compressions were frequently observed in the caudal cervical vertebrae (C5-C7). Furthermore, osseous lesions of the caudal articular process developed more frequently than those of the cranial articular process. CT myelography in Thoroughbred horses is a useful method for detecting CVSM changes.

Chronic otitis externa with heat shock protein 70-positive intranuclear inclusion bodies in the ceruminous gland epithelium of a Chihuahua dog.

A Chihuahua dog showed persistent itching in the right ear canal. Anti-inflammatory medicines and prednisolone were ineffective and total ear canal ablation was performed. Histological diagnosis was chronic otitis externa. Eosinophilic intranuclear inclusion bodies (Cowdry type A and full-type) were occasionally observed in the ceruminous gland epithelium. The inclusion bodies were negative for nucleic acid and ultrastructurally composed of fibrous structures (approximately 10 nm in width). Viral infection was initially suspected, but polymelase chain reaction tests did not detect the expected viral genes. Immunohistochemistry revealed that the inclusion bodies were positive for heat shock protein 70 (HSP70), suggesting that these bodies could be protein aggregates including HSP70. The etiology of this lesion has not been elucidated, but chronic inflammation may influence the cytoplasm-to-nuclear transportation of HSP70. To the best of our knowledge, this is the first report of canine chronic otitis externa with HSP70-positive intranuclear inclusion bodies.

Congenital cutaneous fibropapillomatosis without evidences of papillomavirus infection in a Holstein-Friesian calf.

A male Holstein-Friesian calf was born with multiple, cauliflower-like, pale pink cutaneous masses on the head and limbs. On histopathological examination, the cutaneous masses were diagnosed as congenital cutaneous fibropapillomatosis. Those lesions involved focal proliferation of sebaceous gland in the dermis. There were no histological findings to suggest bovine papillomavirus infection, such as the presence of intranuclear inclusion bodies, large keratohyalin granules, and koilocytosis. Furthermore, papillomaviral antigens and DNA were not detected by immunohistochemistry and polymerase chain reaction, respectively. These results suggested that there was no association between these cutaneous lesions and bovine papillomavirus infection, and the lesions were considered as harmartomatous changes.

Acute myelomonocytic leukemia negative for alpha-naphthyl acetate esterase stain in a Holstein cow.

A 4-year, 7-month-old Holstein cow presented with anorexia. Physical examination revealed masses in the interscapular region and vagina. Blast cells were detected in the masses and peripheral blood by fine needle aspiration cytology and hematological examination. By bone marrow aspiration, blast cells constituted up to 24.2% of all nucleated cells, and 22% and 2% of non-erythroid cells stained positive for myeloperoxidase and alpha-naphthyl acetate esterase (ANAE), respectively. Pathological examination revealed the mass lesions consisted of a proliferation of tumor cells, which were positive for monocytic markers (HLA-DR and Iba-1). The cow was diagnosed with acute myelomonocytic leukemia (AMML). Even when tumor cells are ANAE-negative, AMML cannot be completely ruled out and should be considered when diagnosing cattle with leukemia/lymphoma.

Spontaneous pulmonary adenocarcinoma in a common marmoset (Callithrix jacchus).

A seven-year-old female common marmoset (Callithrix jacchus) presented with weight loss. Imaging revealed a left thoracic mass, confirmed at necropsy. Histology and immunohistochemistry suggested a well-differentiated pulmonary adenocarcinoma. No evidence of local lymphovascular invasion or distant metastasis was observed. This is the first report of pulmonary adenocarcinoma in marmosets.

Case report: The case of a 17 kg ovarian granulosa cell tumor in a Breton draft mare.

Granulosa cell tumor (GCT) is a benign tumor which affects the mare's ovaries. In this report, a case of unilateral GCT in an ovary, which weighed 17.04 kg, of a 9-year-old Breton draft mare is described. A transrectal ultrasonography exam revealed a unilateral multi-cystic enlarged ovary. Laparoscopic ovariectomy was difficult due to enlargement of blood vessels in the ovarian broad ligament. The mare was necropsied, and the pathological changes in the GCT-affected ovary and unaffected ovary were evaluated. The ovarian mass in the GCT-affected ovary had a cribriform pattern and was positive for anti-Müllerian hormone (AMH) and its receptor (AMHR2). The contralateral ovary showed no follicular development and was negative for AMH. AMHR2 was positively expressed in stromal cells. The AMH concentration in plasma was 4,210 ng/ml. This is the first report showing the presence of AMH (2,210 ng/ml) in ascites fluid, and it also shows that laparoscopic ovariectomy might not be suitable for larger ovaries affected by a GCT. Ultrasonographic, endocrine, and histopathological analyses were helpful for making a definitive diagnosis of GCT in this mare.

Pathology of female mice experimentally infected with an in vitro cultured strain of Trypanosoma equiperdum.

Dourine, caused by infection with Trypanosoma equiperdum, is one of the trypanosomiasis in equids. The clinical course of dourine is long-term, ranging from 1-2 months to several years. Since the pathogenesis of dourine has not yet been elucidated, experimental studies using mouse infection models are needed. Although mice are not susceptible to most T. equiperdum strains, some strains can infect mice. Even in such strains, infected mice develop rapidly transient parasitemia and die within 2-8 days. Therefore, mice experimentally infected with these T. equiperdum strains are not suitable for mouse infection models to analysis the pathogenesis of dourine. A sequential method of isolating parasites from dourine-affected horses and adapting them to in vitro cultures using soft agarose media was recently developed. Various T. equiperdum strains adapted to in vitro conditions have been established using this technique. We used one of these strains, the T. equiperdum IVM-t2 strain. In the present study, T. equiperdum IVM-t2 strain inoculated mice developed periodic parasitemia during the experimental period of 60 days. Histopathologically, vaginitis and dermatitis were observed. These findings were comparable to those of dourine-affected horses. Therefore, mice infected with T. equiperdum IVM-t2 strain may be a valuable tool for pathological, immunological, and parasitological in vivo research, and will contribute to investigations on the mechanisms underlying the disease process and the host-parasite relationship.

Morphological and Histological Features of the Vomeronasal Organ in African Pygmy Hedgehog (Atelerix albiventris).

The vomeronasal organ (VNO) detects specific chemicals such as pheromones and kairomones. Hedgehogs (Eulipotyphla: Erinaceidae) have a well-developed accessory olfactory bulb that receives projections from the VNO, but little is known about the hedgehog VNO. Here, we studied the histological features of the VNO in five individual African pygmy hedgehogs by hematoxylin-eosin, periodic acid-Schiff, and Alcian blue stains. The hedgehog VNO comprises a hyaline cartilage capsule, soft tissue and epithelial lumen, and it branches from the site just before the incisive duct opening into the nasal cavity. The soft tissues contain several small mucous (or mucoserous) glands and a large serous gland, and many venous sinuses all around the lumen. The VNO lumen is round to oval throughout the hedgehog VNO, and the sensory epithelium lines almost the entire rostral part and medial wall of the middle part. These findings indicate that the VNO is functional and plays an important role in the hedgehog. Notably, the VNO apparently has a characteristic flushing mechanism with serous secretions like those of gustatory glands, which the hedgehog might frequently use to recognize the external environment.