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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.

A 38-month-old Japanese Black bull presenting with anorexia was given supportive treatment without improvement. Findings including bovine leukemia virus positivity and monoclonal B-cell proliferation strongly suggested the onset of enzootic bovine leukosis (EBL). Pathological findings confirmed the diagnosis of EBL. B-cell clonality were analyzed over time using pre-onset preserved genomic DNA at ages 6 months, 16 months, and 30 months. In the B-cell clonality analysis, two minor peaks at 140 and 220 bp were observed before onset, but another large peak at 175 bp appeared at the time of EBL diagnosis. Although the reason for the proliferation of an independent clone is unknown, detection of clonality abnormalities may lead to the detection of cattle at high risk of developing EBL.

A 7-year-old Japanese Black cow with anorexia was presented at a local veterinarian. As rectal palpation revealed an enlarged induration of the uterus, lymphoma was suspected, and the patient was introduced to the Veterinary Teaching Hospital of Obihiro University of Agriculture and Veterinary Medicine. Although lymphadenopathy and lymphocytosis were not found, a high proviral load of bovine leukemia virus (BLV) with 464 copies/10 ng DNA was recorded, and lymphocytes with constricted or flower-like nuclei were found occasionally in the peripheral blood; accordingly, enzootic bovine leukosis (EBL) was suspected. According to PCR for clonality analysis of B cells based on immunoglobulin heavy chain (IGH) gene rearrangement, monoclonal proliferation of peripheral blood B cells was evident, suggesting the presence of tumorigenic B cells in the peripheral blood and onset of EBL. Pathological examination including necropsy and histopathological examination confirmed the diagnosis of EBL. Clonality analysis for B cells in the peripheral blood is thought to be useful for diagnosing B-cell tumors such as EBL, especially in cases lacking lymphadenopathy and lymphocytosis. This is the first clinical case of EBL diagnosed by demonstrating monoclonal proliferation of peripheral blood B cells using IGH gene rearrangement-based PCR.

Peripheral B-lymphocyte clonality of 274 bovine leukemia virus-infected cattle with lymphocytosis was analyzed using clonality PCR based on sequences of the variable region of the bovine immunoglobulin H chain. None of the cattle showed monoclonal proliferation, while 10, 31, and 233 showed minor-clonal, oligoclonal, and polyclonal proliferation, respectively. A total of 163 cattle were analyzable the following year, and lymphocytosis was maintained in 157, indicating persistent lymphocytosis (PL). B-lymphocyte clonality of the 157 PL cattle was minor-clonal in 6 (3.8%), oligoclonal in 8 (5.1%), and polyclonal in 143 (91.1%). A higher rate of enzootic bovine leukosis (EBL) onset within a year was observed in PL cattle with minor-clonal (50.0% (3/6)) and oligoclonal (25.0% (2/8)) proliferation compared to those with polyclonal (5.6% (8/143)) proliferation. Minor-clonal and oligoclonal proliferation in PL cattle may be a prognosis factor for developing EBL.

Enzootic bovine leukosis (EBL) is characterized by systemic B-cell lymphoma associated with bovine leukemia virus (BLV) infection in cattle over 3 years old. This study evaluated the use of inverse polymerase chain reaction (PCR) as a diagnostic method for EBL. Inverse PCR revealed monoclonal or biclonal integration of BLV proviral DNA in the genomes of 71 of 82 cattle with EBL, and polyclonal integration of BLV proviral DNA in the genomes of 16 cattle with B-cell type sporadic bovine leukosis (SBL), 5 cattle with T-cell type SBL, 4 cattle with tumors other than lymphoma, 20 cattle with inflammatory diseases, 5 cattle with diseases other than tumors and inflammatory diseases, and 20 healthy cattle. The sensitivity, specificity, positive predictive value, and negative predictive value of inverse PCR for diagnosis of EBL were 86.6%, 100%, 100%, and 86.4%, respectively, suggesting that the risk of false positives is low. Inverse PCR can be a useful tool for definitive diagnosis of EBL in cattle with difficult diagnosis, such as young cattle.

A 2-year, 3-month-old Holstein cow presented with anorexia and enlarged superficial lymph nodes. Fine needle aspiration cytology of the superficial lymph nodes revealed large blast cells. Hematological examination revealed anemia, neutropenia, and blast cells in peripheral blood. Blast cells were the predominant cell type in bone marrow aspirates. Of the non-erythroid cells, 26%, 58%, and 18% were positive for myeloperoxidase, α-naphthyl acetate esterase, and naphthol AS-D chloroacetate esterase, respectively. Pathological examination revealed the proliferation of neoplastic cells, which were positive for monocytic markers, in the affected lymph nodes. The cow was diagnosed with acute myelomonocytic leukemia based on these findings. This report highlights the importance of performing bone marrow aspiration cytology and cytochemical staining when diagnosing bovine myeloid leukemia.

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.

Enzootic bovine leukosis (EBL) is typically observed in cattle over 3 years old. However, some cases of EBL onset in young beef cattle have been reported in Japan. The mechanism for early EBL onset is unclear. In Japan, beef cattle are given large amounts of concentrated feed with low vitamin A. Bone morphogenetic proteins (BMPs) are regulators of cell proliferation, differentiation, and apoptosis, and thought to represent one of the key players in tumor malignancy. The purpose of this study was to evaluate the differences in BMP-6 methylation status between EBL beef cattle under 3 years old and other cattle. We investigated the methylation status of the BMP-6 promoter region in 32 EBL beef cattle under 3 years old. We also compared the methylation status of EBL dairy cattle to that of healthy cattle. Median methylation rate of the BMP-6 promoter region in EBL beef cattle under 3 years old was 8.9%, which was significantly higher than that of other groups. Hypermethylation of the BMP-6 promoter region might contribute to early onset of EBL in beef cattle under 3 years old, and animal feeding management practices specific to beef cattle may affect the methylation status of the BMP-6 promoter region.

A 5-day-old male crossbred beef calf presented with a well-coordinated bilateral hopping gait of the hind limbs. Postmortem CT showed a poorly defined oval-shaped region at the L3-L4 spinal segments, which had high signal intensity on T2 weighted postmortem MRI images. On pathological examination, we identified a large cystic cavity filled with a large amount of cerebrospinal fluid on the cut surface of the spinal region. Histopathological examination revealed that the spinal cord parenchyma was compressed by the cystic structure, and the cystic cavity was lined with a thin layer of discrete ependymal cells, indicating syringohydromyelia. This is the first reported case of a Holstein-Friesian × Japanese Black crossbred calf with solitary syringohydromyelia. Our findings suggest that myelodysplasia with cystic cavities can be suspected by CT, without the need for MRI.

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.

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.

We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility as a method to diagnose bovine B-cell lymphoma. PCR for IgH gene rearrangement indicated monoclonal proliferation of B-cells in 24 of 35 cattle with B-cell lymphoma. In contrast, PCR for IgH gene rearrangement in lymph nodes and tumor tissues from 65 cattle diagnosed with tumors other than B-cell lymphoma and non-tumors revealed polyclonal population of B-cells. Sensitivity, specificity, positive predictive value, and negative predictive value for PCR for IgH gene rearrangement for bovine B-cell lymphoma were 68.6%, 100%, 100%, and 85.5%, respectively. Clonality analysis using PCR for IgH gene rearrangement may be useful for adjunctive diagnosis of bovine B-cell lymphoma.

application/pdf, Circulating cell-free DNA (cfDNA) is extracellular DNA released into the bloodstream by apoptotic or necrotic tumor cells, with cfDNA determination proposed as a noninvasive, sensitive marker for the diagnosis of human cancer. We evaluated cfDNA quantification as a diagnostic and prognostic tool in dogs with various tumors. We quantified plasma cfDNA concentration by absolute real-time PCR of long interspersed nuclear elements in 50 dogs with malignant tumors, 13 dogs with benign tumors or nodules, and 11 healthy controls. Six patients with malignant tumors were followed-up, and plasma cfDNA was quantified throughout disease progression. We found that plasma cfDNA concentrations were significantly elevated in dogs with malignant tumors compared with dogs with benign nodules or healthy controls. The DNA integrity index (the ratio between long and short cfDNA fragments) was significantly lower in dogs with malignant tumors compared to healthy controls. Significantly higher cfDNA levels and a lower DNA integrity index were observed in dogs with lymphoma or leukemia, hemangiosarcoma, and distant metastasis; cfDNA levels correlated well with clinical stage and tended to increase during or before periods of disease progression, suggesting potential efficacy of cfDNA for the detection of distant metastasis and to monitor the clinical stage of neoplasia.

In the present study, we analyzed bovine leukemia virus (BLV) integration sites in under 3 years old with enzootic bovine leukosis (EBL) cattle and compared these to 30 cattle over 3 years old with EBL. BLV proviruses were integrated near CpG islands and into long interspersed nuclear elements more frequently in EBL cattle under 3 years old than in those over 3 years old. These results suggest that cattle under 3 years old with EBL have different BLV provirus integration sites from those of cattle over 3 years old with EBL, and the BLV provirus integration site may represent one factor contributing to early onset of EBL.

A 19-mo-old Holstein heifer was inactive and dyspneic. Physical examination revealed wheezing, exophthalmos, a cervical mass, and lymphadenopathy. Cytology of the cervical mass and lymph nodes showed predominantly large atypical lymphocytes. Lactate dehydrogenase and thymidine kinase activities were elevated. Although nested PCR for bovine leukemia virus (BLV) using blood was positive, quantitative PCR showed a low number of provirus copies. Autopsy revealed enlargement of most lymph nodes examined, as well as white masses of various sizes in muscles of the left hindlimb and thoracic and abdominal organs. Histopathology revealed severe infiltration with neoplastic lymphocytes in these organs. The cervical mass was immune-positive for B-cell markers. The final diagnosis was thymic B-cell lymphoma with BLV infection.

International audience; Mycoplasma wenyonii, a hemoplasma infecting cattle, was never detected in France. In 2014, evocative inclusions were observed in erythrocytes from cattle presenting milk drops, anemia, and edema in Brittany (France). A survey was then initiated to investigate the epidemiological situation and correlate mycoplasma detection with clinical signs. For this purpose, a new PCR assay targeting polC gene was designed. Comparative results with published PCR assays place this new one as more specific, allowing a one-step diagnosis without further sequencing. A total of 181 cows were included in this study and 4.97% (n=9) were positive, resulting in the first molecular identification of M. wenyonii in France. All positive animals presented anemia, edema and milk drop. When selecting animals presenting evocative clinical signs, the prevalence of M. wenyonii in Brittany was estimated to 25.6%. Further studies are needed to evaluate the importance of the infection, the implication of arthropods and the existence of asymptomatic carriers.

We evaluated the prevalence of Anaplasma infection in 332 dogs from Ibaraki, Japan, using serological and molecular methods. An immunofluorescence antibody assay against Anaplasma phagocytophilum indicated that 7 of the 328 serum samples tested (2.1%) were positive for A. phagocytophilum. Screening by polymerase chain reaction (PCR) analysis demonstrated that 8 of the 331 peripheral blood samples tested (2.4%) were positive for Anaplasmataceae. Phylogenetic analysis of the partial 16S rRNA sequence of the PCR amplicons revealed that 6 sequences were most similar to the 16S rRNA sequence of a Wolbachia sp., and the remaining 2 to A. bovis. Further analysis by A. phagocytophilum-specific nested PCR demonstrated that 1 dog infected with A. bovis was also positive for A. phagocytophilum. This is the first study to report the dual infection of a dog in Japan with A. bovis and A. phagocytophilum.

application/pdf, This study proposed a novel radiographic positioning in order to image the cranioventral lung region using a portable X-ray unit and digital radiography system. In the novel position, calves were restrained in a chute and a unilateral forelimb was pulled cranially with the contralateral forelimb tied to the chute; the forelimbs were then spread cranio-caudally as in a scissor position (Three-legged view: TL view). In a preliminary study, we applied the TL view for imaging of 14 clinically healthy calves. In a clinical study, accuracy in detecting cranioventral lung lesions was compared between the standard standing view and the TL view for 19 calves, which were culled from herd; the results of postmortem examination were used as gold standard. Seven evaluators independently interpreted the images. The median (range) number of trials and the time for obtaining optimal position were 2 (1-7) and 263 sec (105-488), respectively in 14 healthy calves. Calves thicker than approximately 40 cm were not considered candidates for TL view in this setting because of difficulty in restraint and the low output of the portable X-ray unit. The TL view improved the detection of consolidation in the cranioventral lung region, compared with the standard view. The TL view was considered an optional view when the cranioventral lung region was an area of interest, because this view was relatively easy to perform and required a small number of personnel, even for large calves.

A 15-day-old Holstein calf with lethargy and tachypnea presented to the Veterinary Teaching Hospital at Obihiro University of Agriculture and Veterinary Medicine for evaluation of suspected congenital heart defect. A Levine grade 6 systolic murmur was noted at right apical site auscultation and phonocardiogram also recorded systolic a murmur. Electrocardiography findings include increased R and S waves, R wave split, and negative T waves without arrhythmia. Echocardiography revealed a single ventricle with a trace of the right ventricular wall, atrioventricular valve regurgitation, and turbulent in a single ventricle. Arterial blood analysis showed a marked decrease in oxygen saturation of 78% and oxygen partial pressure of 44 mmHg. Post-mortem examination confirmed the diagnosis of a single left ventricle.

A 2-day-old Japanese Black male calf that presented with opisthotonos with spastic extension of all four limbs and nystagmus was presented. Evaluation of cranial neurology revealed a horizontal slow nystagmus and absence of menace response in the left eye. Necropsy revealed a mass located between the posterior margin of the cerebrum and anterior margin of the cerebellum, and continuously with the cerebellar lesion. The brainstem was severely compressed by those lesions. Original structures of the cerebellum were mostly replaced by grayish-white and brownish tissues. Those lesions were diagnosed as presumed cerebellar medulloblastoma by histopathological and immunohistochemical examination. As neuron-specific enolase in the cerebrospinal fluid which is a biomarker for neuronal damage was increased compared with healthy calves.

Circulating tumor DNA (ctDNA), which carries tumor-specific mutations, is an emerging candidate biomarker for malignancies and for monitoring disease status in various human tumors. Recently, BRAF V595E mutation has been reported in 80% of dogs with urothelial carcinoma. This study investigates the BRAF V595E allele concentration in circulating cell-free DNA (cfDNA) and assesses the clinical significance of BRAF-mutated ctDNA levels in canines with urothelial carcinoma. A total of 15 dogs with urothelial carcinoma were included. cfDNA concentration was measured using a real-time polymerase chain reaction (PCR) of the LINE-1 gene. To measure the concentration of the mutated BRAF gene in cfDNA, allele-specific real-time PCR with a locked nucleic acid probe was performed. BRAF mutations were detected in 11 (73%) of the 15 tested tumor samples. BRAF-mutated ctDNA concentrations were significantly higher in dogs with the BRAF mutation (14.05 ± 13.51 ng/ml) than in wild-type dogs (0.21 ± 0.41 ng/ml) (p = 0.031). The amount of BRAF-mutated ctDNA in plasma increased with disease progression and responded to treatment. Our results show that BRAF-mutated ctDNA can be detected using allele-specific real-time PCR in plasma samples of canines with urothelial carcinoma with the BRAF V595E mutation. This ctDNA analysis may be a potentially useful tool for monitoring the progression of urothelial carcinoma and its response to treatment.

Here, we report a case of rhinocerebral zygomycosis due to a Lichtheimia ramosa infection in a calf. A histopathological examination revealed that a fungus had invaded the brain through the olfactory nerves. Lichtheimia ramosa was detected by polymerase chain reaction analysis of DNA extracted from formalin-fixed paraffin-embedded samples of the affected tissue. This is the first case of rhinocerebral zygomycosis to involve cattle. Also, this is the first such case to involve fungal invasion into the central nervous system through the cranial nerve itself, rather than through perineural tissue.

Anaplasma phagocytophilum DNA was detected from a dog with canine granulocytic anaplasmosis (CGA) in Japan. Phylogenetic analysis of the DNA using 16S rRNA, gltA, and groEL sequences revealed that the strain was nearly identical to A. phagocytophilum detected from Apodemus agrarius (black-striped field mouse) in China and Korea. To our knowledge, this is the first report of molecular detection and phylogenetic analysis of A. phagocytophilum from a clinical case of CGA in Japan.

application/pdf, A 4-week-old female Holstein Friesian calf presented with hindlimb paresis. Neurologic examination of spinal reflexes revealed depressed or absent reflexes of the hindlimbs. Menace responses on both sides disappeared on examination of cranial nerves. The calf was finally diagnosed with Neospora caninum infection by pathological findings including nonsuppurative inflammation associated with cysts in the cerebrum and spinal cord. High levels of antibody against recombinant surface antigen 1 of N. caninum (NcSAG1) were detected by ELISA from both serum and cerebrospinal fluid (CSF) samples. This result suggests that detection of antibodies against N. caninum by NcSAG1-ELISA in serum and CSF could be useful for the clinical diagnosis of neosporosis in calves with acquired neurological signs. © 2018 The Japanese Society of Veterinary Science.

Five calves that had shown neurological symptoms within 9 days after birth were histopathologically diagnosed as encephalomalacia. Two calves showed bilateral laminar cerebrocortical necrosis and neuronal necrosis in the corpus striatum and hippocampus. Since the distributional pattern of the lesions was consistent with that of global ischemia in other species, the lesions were probably hypoxic/ischemic encephalopathy consistent with the history of dystocia and perinatal asphyxia. One calf also showed bilateral laminar cerebrocortical necrosis. However, the lesions were chronic ones, because the calf had survived for long time and necropsied at postnatal day 118. Additionally, the lesions did not involve the corpus striatum and hippocampus. The other two calves showed multifocal necrosis with vascular lesions characterized by fibrin thrombi, perivascular edema and perivascular hyaline droplets in the cerebral cortex, corpus striatum, thalamus, brain stem and cerebellum. Considering the age of onsets and histopathological appearance, it was possible that latter three calves were also hypoxic/ischemic encephalopathy, however, exact cause of them was not revealed. In all calves, degenerated/necrotic neurons showed positive reactions for Fluoro-Jade C and degenerated axons showed immunoreactivity for Alzheimer precursor protein A4. Therefore, these markers were applicable to examination of brain injury in neonatal calves.

We collected 1,084 ticks by flagging vegetation in Tsukuba and Moriya (Ibaraki, Japan), where several cases of canine granulocytic anaplasmosis were reported. The DNA of the collected ticks was molecularly examined for infection with the family Anaplasmataceae. Twenty-six positive samples of Anaplasmataceae-specific PCR of partial 16S rRNA gene were subjected to semi-nested PCR, covering the divergent regions, the gene, and sequence analysis. Anaplasma phagocytophilum was detected in 3 pools of Haemaphysalis longicornis larvae and A. bovis from a H. flava male. Sequences of both amplicons had high homologies to those from dogs in our previous studies in Ibaraki. These results suggest that Haemaphysalis ticks are candidate vectors of A. phagocytophilum and A. bovis in Ibaraki, Japan.

Babesia odocoilei-like parasites were first reported in 2003, and their virulence and hosts remain unknown. We report three cases of dogs with canine babesiosis in Iwate Prefecture. Since Iwate Prefecture area is an area of Japan where canine babesiosis is not endemic, we suspected that these cases of canine babesiosis were caused by B. odocoilei-like parasites. In the present study, we tried to identify the Babesia species that caused these cases of canine babesiosis. To classify Babesia parasites, the heat shock protein 70 (HSP70) gene was examined. Accordingly, we cloned and analyzed the HSP70 gene sequences of B. odocoilei-like parasites from three Ixodes ovatus ticks. It was determined that the nucleotide sequence of the HSP70 gene of the B. odocoilei-like parasites was not consistent with that of B. odocoilei, which suggests that these parasites were from a different species than B. odocoilei. Second, we identified the Babesia species that infected the three dogs by using the HSP70 gene and 18S rRNA. A partial HSP70 gene of B. odocoilei-like parasites was detected in the three dogs, but that of B. gibsoni was not detected. Additionally, a partial sequence of 18S rRNA of B. odocoilei-like parasites was detected in two dogs. These results demonstrated that two dogs were certainly infected with B. odocoilei-like parasites and that one dog was probably infected with B. odocoilei-like parasites. Therefore, these dogs were diagnosed with canine babesiosis due to the presence of B. odocoilei-like parasites. As there were only three cases, additional cases are needed to confirm our findings.