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.

Enzootic bovine leukosis caused by bovine leukemia virus classified as Group C based on viral whole genome sequencing in a 23-month-old Holstein-Friesian heifer.

A 23-month-old Holstein-Friesian heifer presented with inactivity and diarrhea. On physical examination, no enlargement of superficial lymph nodes was observed. Hematological examination revealed lymphocytosis. The bovine leukemia virus (BLV) proviral load was 2,122 copies/10 ng DNA, and BLV was classified as Group C based on whole genome phylogenetic analysis. 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 inverse PCR, respectively. Although lymph nodes were not swollen at necropsy, histopathological examination revealed neoplastic lymphocyte proliferation in lymph nodes, which were immune positive for CD5 and CD20, and negative for CD3. The heifer was diagnosed with EBL caused by BLV classified as Group C.

BoLA-DRB3*15:01 allele is associated with susceptibility to early enzootic bovine leukosis onset in Holstein-Friesian and Japanese Black cattle.

Enzootic bovine leukosis (EBL) is typically observed in cattle older than 3 years, but some cases of onset in cattle younger than 3 years have been reported in Japan. BoLA-DRB3 polymorphisms are associated with susceptibility to EBL onset. However, little is known about the relationship between the polymorphisms and EBL onset in young cattle. In the present study, we performed BoLA-DRB3 genotyping in 59 EBL cattle younger than 3 years (25 Holstein-Friesian and 34 Japanese Black) and compared the results with those of 69 EBL cattle older than 3 years (38 Holstein-Friesian and 31 Japanese Black). The BoLA-DRB3*15:01 allele was detected at a frequency of 37.3 % (48.0 % and 29.4 % in Holstein-Friesian and Japanese Black, respectively) and was identified as an early EBL onset susceptibility allele. Nine EBL cattle younger than 3 years (5 Holstein-Friesian and 4 Japanese Black), but only 1 EBL cattle older than 3 years (1 Holstein-Friesian), had a BoLA-DRB3*15:01/*15:01 homozygous genotype. The frequency of the BoLA-DRB3*15:01 allele occurring with a different allele (BoLA-DRB3*015:01/other) in cattle younger than 3 years was 44.1 % (56.0 % Holstein-Friesian and 35.3 % Japanese Black) and significantly higher than that in cattle older than 3 years (28.9 % Holstein-Friesian and 9.7 % Japanese Black) (P = 0.0013). These results suggest that BoLA-DRB3*15:01/*15:01 and BoLA-DRB3*15:01/other genotypes are early EBL onset susceptibility genotypes. The present findings may contribute to cattle breeding selection.

Phylogenetic analysis based on whole genome sequence of bovine leukemia virus in cattle under 3 years old with enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is one of bovine neoplasms caused by bovine leukemia virus (BLV). Although EBL is typically observed in cattle over 3 years old, several cases of EBL onset in cattle under 3 years old have been reported in Japan. The mechanism for EBL onset in young cattle remains unclear. Although genetic variation of BLV is limited, the variations could affect viral properties relating to BLV pathogenesis. The purpose of this study was to clarify relationship between early onset of EBL and BLV groups. Moreover, we also aimed to characterize BLV that cause early onset of EBL. Whole genome sequences of BLV in 72 EBL cattle under 3 years old and 50 EBL cattle over 3 years old were identified. Phylogenetic analysis showed that BLV was divided into 4 groups (A, B-1, B-2 and Other). The BLV from EBL cattle under 3 years old were mainly classified as group A and B-1, while those from EBL cattle over 3 years old were mainly included in group B-2. Common sequence of group A and B-1 was compared with those of group B-2. Specific sequences in LTRs, gag-pro-pol, env and tax gene regions were identified in these groups. Amino acid substitutions of Pro and Tax protein were predicted in those nucleotide sequences. Those genetic variations might contribute to the early onset of EBL.

Time course changes in peripheral B-cell clonality in a Japanese Black bull with enzootic bovine leukosis.

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.

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.

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.

Congenital syringohydromyelia in a crossbred (Holstein-Friesian × Japanese Black) beef calf.

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.

Abnormal clonalities of B-lymphocytes in bovine leukemia virus-infected cattle with persistent lymphocytosis.

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.

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.

Three cases of canine babesiosis caused by Babesia odocoilei-like parasites in 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.

Analysis of the bone morphogenetic protein 6 gene promoter region in young beef cattle affected by enzootic bovine leukosis.

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 clinical case of acute myelomonocytic leukemia in a Holstein cow.

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 clinical case of single left ventricle in a Holstein calf.

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 clinical case of presumed cerebellar medulloblastoma in a Japanese Black calf with increased neuron-specific enolase in cerebrospinal fluid.

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.

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.

Quantitative analysis of the BRAF V595E mutation in plasma cell-free DNA from dogs with urothelial carcinoma.

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.

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis.

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.