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.

Rhinocerebral Zygomycosis Due to a Lichtheimia ramosa Infection in a Calf: Neural Spread Through the Olfactory Nerves.

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.

Polysplenia syndrome with duodenal and pancreatic dysplasia in a Holstein calf: a case report.

BACKGROUND: Laterality disorders of the abdominal organs include situs inversus totalis that mirrors the arrangements of all internal organs and heterotaxy syndrome (situs ambiguus) in which the thoracic or abdominal organs are abnormally arranged. Heterotaxy is often accompanied by multiple congenital malformations, and it generally comprises asplenia and polysplenia syndromes. To our knowledge, polysplenia syndrome has been reported in only three cattle, and computerized tomographic (CT) images of these animals were not obtained. CASE PRESENTATION: A six-month-old Holstein heifer had ruminal tympani and right abdominal distension. CT imaging showed that the rumen occupied the right side of the abdominal cavity, the omasum and abomasum occupied the left ventral side and the liver was positioned on the left. The colon and cecum were located at the left dorsum of the cavity, and the left kidney was located more cranially than the right. Postmortem findings revealed two spleens attached to the rumen. Significantly, the duodenum was too short to be divided into segments, except the cranial and descending parts, or flexures, except the cranial flexure, and the pancreas, which lacked a left lobe, was covered with mesojejunum. The liver comprised a relatively large right lobe and a small left lobe without quadrate and caudate lobes. The caudal vena cava that connected to the left azygous vein passed irregularly through the aortic hiatus of the diaphragm, and the common hepatic vein without the caudal vena cava passed through the caval foramen. Although the lungs and heart were morphologically normal, the right atrium received three major systemic veins. Polysplenia syndrome was diagnosed based on the CT and postmortem findings. CONCLUSION: We defined the positions of the abdominal organs and morphological abnormalities in various organs of a calf with polysplenia syndrome based on CT and postmortem findings. These findings will improve understanding of the malpositioning and malformations that can occur in the organs of cattle with polysplenia syndrome.

Higher sika deer density is associated with higher local abundance of Haemaphysalis longicornis nymphs and adults but not larvae in central Japan.

Haemaphysalis longicornis (Acari: Ixodidae) is one of the most common and important arthropod disease vectors in Japan, carrying Japanese spotted fever and bovine theileriosis. The recent expansion of sika deer (Cervus nippon, Artiodactyla: Cervidae) populations, the most common wild host of H. longicornis, has also caused concern about increasing the risk of vector-borne diseases in Japan. We used generalized linear mixed model analysis to determine the relative contribution of deer density and other biological and abiotic factors on the abundance of H. longicornis ticks questing at each developmental stage. A total of 6223 H. longicornis adults, nymphs, and larvae were collected from 70 sites in three regions of central Japan. The abundance of questing adult and nymphal ticks was associated with deer density and other biotic and abiotic factors. However, the abundance of questing larvae showed no association with deer density but did show an association with other biotic and abiotic factors. These findings show that a high density of deer along with other biotic and abiotic factors is associated with increased risk of vector-borne diseases through amplified local abundance of questing nymphal and adult H. longicornis. Further, questing larvae abundance is likely regulated by environmental conditions and is likely correlated with survival potential or the distribution of other host species.

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.

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.

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.

PCR detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis.

We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.

Mechanistic study on liver tumor promoting effects of flutamide in rats.

Flutamide (FLU), a nonsteroidal anti-androgen, is used for the treatment of prostate cancer but is also a cytochrome P450 (CYP) 1A inducer. Some CYP1A inducers are known to exert hepatocellular tumor-promoting activities in rodents, and reactive oxygen species (ROS) produced by CYP1A1 induction via a metabolism of FLU is probably involved in the liver tumor promotion. In the present study, to clarify the possible liver tumor promoting effect of FLU, a two-stage liver carcinogenesis assay was performed using male F344 rats. Rats received an intraperitoneal (ip) injection of 200 mg/kg body weight of N-diethylnitrosamine (DEN) and fed a diet containing 0, 0.1 or 0.2% FLU for 6 weeks. After 2 weeks of DEN treatment, all rats were subjected to two-thirds partial hepatectomy. Animals were killed 8 weeks after ip injection of DEN. Immunohistochemically, the number and area of glutathione S-transferase placental form (GST-P)-positive foci significantly increased in the liver of rats given 0.2% FLU as compared with the control. Ki-67-positive cell ratio also increased in rats given FLU at both concentrations. ROS generation in the microsomal fraction and production of thiobarbituric acid-reactive substance [TBARS] and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in the liver did not increase in any of the FLU-treated groups. The results of microarray and real-time RT-PCR revealed that phase 1 drug-metabolizing enzymes such as CYP1A1, Ugt1a61 and Nqo1 and phase II drug-metabolizing enzymes such as Yc2, Akr1b7, Akr1b8, Akr1b10, Aldh1a1, Gpx2 and Me1 were up-regulated in rats treated with FLU. In addition, the MAPK pathway family-related genes such as Prkcα, Mek1, Rafb, Myc, Mek2, Raf1 and Egfr were also up-regulated in FLU-treated groups. The results of the present study indicate that FLU is a CYP1A inducer but does not cause any production of microsomal ROS in the liver and suggest that microsomal ROS is not involved in the liver tumor promoting effect of FLU.

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.

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.

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.

A mechanism of excessive accumulation of abomasal gas in vagotomized cattle determined using fluoroscopy.

To better understand the mechanism of excessive gas accumulation in the abomasum in bovine abomasal displacement, we performed gastric fluoroscopy in vagotomized cattle. Fifteen 6-month-old Holstein steers were divided into three groups: a non-vagotomized control group (Group C; n=5), a ventral thoraco-vagotomized group (Group V; n=5), and a dorsal and ventral thoraco-vagotomized group (Group DV; n=5). These groups were examined by fluoroscopy before and during a 5-week observation period after surgery. In Group C, no change was observed throughout the observation period. In Group DV, immediately after surgery, reticuloruminal motility was completely absent and ruminal distention was seen. Two weeks after surgery, abnormal reticulum motility and increased gas accumulation in the abomasal body were noted. Abomasal dilatation was also observed. In Group V, 1 week after surgery, gas inflow into the abomasum and relatively normal reticulum motility were observed along with a rapid increase in abomasal gas. Abomasal dilatation was also observed. In addition, left-displaced abomasum occurred in one of the steers in this group. From these results, we concluded that one of the mechanisms of excessive gas accumulation in the abomasum is reticulum-mediated gas inflow from the rumen combined with vagotomy-induced hypomotility.

Genotypic diversity of Theileria orientalis detected from cattle grazing in Kumamoto and Okinawa prefectures of Japan.

Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.

Use of contrast-enhanced CT in the diagnosis of abscesses in cattle.

We report here the non-contrast and contrast-enhanced computed tomography performed in two calves with brain abscess and multiple pulmonary abscesses with pharyngeal abscess, respectively. Contrast-enhanced computed tomography was useful in the diagnosis of these diseases in both calves. The diseases were confirmed by histopathological examination.

Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam.

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).

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.