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.

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.

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.

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.

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.

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.

Acute monoblastic leukemia in a feline leukemia virus-negative cat.

A 12-year-old female domestic short-haired cat was presented due to weight loss, anorexia, and tachypnea. Complete blood count revealed severe anemia, leukocytosis with massive undifferentiated blast cells, and thrombocytopenia. Bone marrow aspiration showed acute myeloid leukemia, subclassified as monoblastic leukemia (M5a) based on the outcomes of the cytochemistry examinations. The SNAP feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) test using whole blood was negative. In addition, FeLV/FIV proviral polymerase chain reaction test using bone marrow aspirate was also negative. Although the cat was treated with doxorubicin, cytosine arabinoside, and prednisolone, anemia did not improve without blood transfusion. The owner declined further treatment after 2 months, and the cat died a few days later.

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.

Analysis of bovine leukemia virus integration sites in cattle under 3 years old with enzootic bovine leukosis.

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.

Molecular diagnosis of bovine B-cell lymphoma using polymerase chain reaction for immunoglobulin heavy chain gene.

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.

Disseminated thymic B-cell lymphoma in a Holstein heifer.

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.

Periventricular leukomalacia in a neonatal calf.

A 10-day-old, Japanese Black, female calf had shown astasia since just after birth. Focal symmetrical periventricular malacic lesions of the cerebrum and suppurative arthritis of the left hip joint were observed in macroscopic examination. Histologically, the cerebral lesions were confirmed as periventricular leukomalacia (PVL). The location and histological features of the lesions were similar to PVL in humans, caused by neonatal ischemia/hypovolemia. This is the first report of PVL in a neonatal calf.

Serological survey of Borrelia infection of dogs in Sapporo, Japan, where Borrelia garinii infection was previously detected.

A serological survey of Borrelia infection of dogs was performed in Sapporo, Japan, where Borrelia garinii infection in dogs was detected in 2011. A total of 314 serum samples were collected from dogs that visited three animal hospitals in Sapporo from 2012 to 2014. The two-step evaluation method, involving screening ELISA followed by Western blot analysis, was used to detect antibodies against Borrelia species. A total of 34 samples were positive by ELISA. Among those 34 samples, 32 were positive for Borrelia spp. by Western blot. These findings suggest that the 32 dogs (10.2%) generated antibodies against Borrelia burgdorferi sensu lato, such as B. garinii or B. afzelii. Antibody positivity was 7.6% and 13.3% for dogs living in urban and rural areas, respectively. Dogs with a history of tick infestation showed a positive rate of 16.7%, which was higher, although not significantly, than the 6.7% among dogs without a history.