Cases Of Enzootic Bovine Leukosis Research Articles

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis.

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in eight genes, TP53, KMT2D, CREBBP, KRAS, PTEN, NOTCH1, MYD88, and CARD11. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to high impact mutations affecting protein translation and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylcytosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection.IMPORTANCEEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

Seroprevalence of bovine leukemia virus infection in contrasting farming systems in Kenya

Enzootic bovine leukosis (EBL) is a worldwide disease of cattle caused by the bovine leukemia virus (BLV) and clinically characterised by occurrence of multiple lympho-sarcomas. In Kenya, cases of bovine lympho-sarcomas have been reported but limited information available on prevalence and distribution of BLV infection in the country. The objectives of this cross-sectional study were to estimate the seroprevalence of BLV infection in Kenya and how the seroprevalence is affected by different livestock farming systems. In 2016, 1383 bovine serum samples were randomly collected from 14 counties which were purposively selected to represent 3 livestock farming systems in the country. The sera were tested for the presence of antibodies against BLV using the IDEXX anti–BLV indirect ELISA test. An overall seroprevalence of 7.6% (95% CI: 6.3% - 9.1%) BLV infection was estimated. A multivariable mixed logistic regression model, with county as a random variable controlling for clustering, identified age and farming system as significant risk factors associated with BLV seropositivity. Zero-grazing (0.6%), ranching (4.4%) and pastoral systems (18.3%) differed in seroprevalence. Cattle under 1 year of age had a prevalence of 6.4%, while cattle over 1 year of age had a prevalence of 7.9%. BLV infection was present across the three farming systems but in only five of the fourteen counties assessed. This information contributes to designing effort on control programs of BLV infection in Kenya. Further research should be carried out to determine the frequency of clinical cases of EBL and the impact on the livestock industry in Kenya.Keywords: Bovine Leukemia Virus; Cattle, Seroprevalence; Kenya

The effectiveness of colostral antibodies for preventing bovine leukemia virus (BLV) infection in vitro

BackgroundBovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). The incidence of EBL in Japan is increasing annually; and the cases of EBL in cattle younger than 2 years old has been reported. Therefore, it is vital to find a method to control BLV infection, especially in young calves. In this study, to evaluate the protective ability of colostral antibodies against BLV infection, as well as the potential for BLV infection mediated by colostrum/milk, we investigated temporal fluctuations in the anti-BLV antibody titer and BLV proviral load (PVL) in colostrum/milk and peripheral blood of six infected dams during lactation. The association between PVL and antibody titer in colostrum and peripheral blood was then investigated using samples from a further twenty-seven cattle. Antibody concentrations were measured with a Syncytium-induction Inhibition Assay using colostral/milk whey and serum. PVL in peripheral blood and colostrum was measured by real-time PCR.ResultsColostral antibodies showed high inhibitory activity until day 3 of lactation. The antibody titer and PVL in peripheral blood showed lesser changes than those in colostrum/milk throughout lactation. The colostral antibody titer was significantly higher than the serum antibody titer in all samples, whereas the colostrum PVL was significantly lower than the blood PVL. The blood PVL showed a significant correlation with serum antibody titer, colostrum PVL, and colostral antibody titer. However, there were no major correlations between the serum and colostral antibody titers.ConclusionsThis is the first report investigating the temporal changes in colostral antibody titer in terms of inhibiting BLV infection in vitro. The results of antibody detection by Syncytium-induction Inhibition Assay suggested that the protective activity of the colostral antibodies against BLV infection would be conferred by anti-BLV gp51 antibody. The high antibody titer of colostral whey suggests that colostral whey could be a potential source of antibodies with a low risk of infection in neonatal calves.

Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis.

Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.

Pathological findings of Japanese Black Cattle living in the restricted area of the Fukushima Daiichi Nuclear Power Plant accident, 2013–2016

Fifty-one Japanese black cattle from four farms in the evacuation zone of the Fukushima nuclear accident were examined pathologically during the period, 2013-2016. We found no evidence of a radiation effect on pathological findings in any of these autopsy cases, although nine cases (3.7%) of enzootic bovine leukosis (EBL) and three cases (1.2%) of goiter were diagnosed. Estimated integrating dose of external exposure in EBL cases ranged from a maximum of 1200mSv to a minimum of 72mSv. Clinically, five cases showed wobble, dysstasia or paralysis. Exophthalmos was observed in three cases. Macroscopically, enlarged lymph nodes, multiple irregular masses of intra-abdominal and intrapelvic adipose tissue, diffuse thickening of the abomasal submucosa with ulceration, and numerous white nodules of myocardium were observed. Histologically, neoplastic lymphoid cells were extensively proliferated and infiltrated in the lesions. Lymphoid tumor cells showed mature or large lymphoblastic appearance. Immunohistochemically, tumor cells were positive for BLA-36, CD20 and CD5, and negative for CD3. Three cases showed diffuse enlargement of the thyroid gland with no mass or nodule lesion. Histologically, diffuse proliferation of follicular epithelium was observed with preservation of normal structures. There were no malignant findings such as cellular atypia or invasion to capsule.

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis.

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.

Evaluation of Gene Expression in Peripheral Blood Cells as a Potential Biomarker for Enzootic Bovine Leukosis

Expression of six selective genes in peripheral blood cells was evaluated as diagnostic biomarkers for enzootic bovine leukosis (EBL) by using 10 EBL and 15 clinically healthy cattle. The clinically healthy cattle generally showed lower gene expression levels. Although wide variations of gene expression were found in some clinical cases of EBL, 4 and 5 among 10 EBL cattle showed higher expression of interleukin 2 receptor gene (IL2R) and Wilms' tumor gene (WT1), respectively. Expression of IL2R in peripheral blood cells in EBL cattle was statistically increased; however, the lower sensitivity and higher variation in the gene expressions among clinical cases of EBL would be problems as diagnostic biomarkers.

Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

Determination of seric levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB). In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated), and TB-negative animals (n = 204, TB-free herds). The mean ADA seric value from the TB-positive group (4.45 +/- 2.33 U/L) was significantly lower (p = 0.008) than that observed in sera from the TB-negative group (6.12 +/- 4.47 U/L). When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 +/- 3.75 U/L) was not significantly different anymore from that of the TB-positive group (p = 0.28). There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively). In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

Immunohistochemical Analysis of Expression Patterns of Tumor Necrosis Factor Receptors on Lymphoma Cells in Enzootic Bovine Leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.

Relation between phenotype of tumor cells and clinicopathology in bovine leukosis.

Thirty-three cases of enzootic bovine leukosis (EBL) and 14 cases of sporadic bovine leukosis (SBL) were examined by immunohistochemistry using 6 monoclonal antibodies against leukocyte differentiation molecules of bovine leukocytes. There were 17 cases of B-1a cell type, 10 cases of B-1b cell type and 6 cases of B-2 cell type in EBL, and 5 cases originating from B cells (B-2 cell type) and 9 cases originating from immature T cells in SBL. The average age for the EBL cases of B-1a cell type was 8.6 years, B-1b cell type was 6.5 years, and of B-2 cell type was 4.5 years. In cases of SBL, immature T cell type patients were younger than B-2 cell type ones. The lymphoma originating from B cells differed from that originating from T cells in morphology. In T cell tumors, the nucleus of tumor cells was round, the edge of the cytoplasm obvious, and tumor cells were sporadically present and proliferated. When compared with T cells, the region among B cells was obscure. But, there was no relation between phenotype and the histologic classification of tumor cells. In EBL, beyond the lymph node, tumors of B-1a and B-1b types had developed in the heart and abomasum, and those of the B-2 type tended to occur in liver. In SBL, B-2 type and T type cells formed tumors in the liver, kidney, thymus, and one case of T-cell type tumor formed on the skin. We would like to propose a new classification of bovine leukosis as EBL, calf type B-cell lymphoma, juvenile T-cell lymphoma and skin type T-cell lymphoma.

Distribution of T-lymphocyte Subpopulation in Blood and Spleen of Normal Cattle and Cattle with Enzootic Bovine Leukosis

Immunocytochemical and flow cytometry techniques were used to examine T-lymphocyte subpopulations in peripheral blood lymphocytes (PBLs) and spleen from cases of enzootic bovine leukosis (EBL) in adult cattle, and from normal cattle (adult and young), with a panel of monoclonal antibodies against bovine leucocyte differentiation molecules. Both in PBLs and spleen, the percentages of T-lymphocyte subpopulations (CD3+, CD4+, CD8+, and WC1+γδT lymphocytes) of EBL-affected and normal adult cattle were significantly lower than those of normal young cattle. The percentages of these T-lymphocyte subpopulations in the PBLs of adult cattle with EBL were lower than those of normal adult cattle, but the converse was true in the spleen. It is suggested that tumour immunity occurred in the spleen. Histological examination revealed no follicular hyperplasia in the spleen, and the proliferation of neoplastic cells began in the red pulp. It is concluded that the spleen is not the organ initially responsible for the transformation of EBL lymphoma and that neoplastic cells migrating from peripheral blood are metastatic.

Bovine leukaemia-virus infection in Costa Rica.

The geographical distribution of bovine leukaemia-virus (BLV)-infected herds in Costa Rica and the isolation of BLV from cases of enzootic bovine leukosis are presented. It was found that BLV is prevalent throughout the country, affecting mostly dairy cattle (Bos taurus) located in the highlands around the central valley of Costa Rica. By contrast, beef cattle (Bos indicus) raised in the lowlands were affected to a lesser extent. One out of four isolates of BLV obtained from local cattle with lymphosarcomatous tumours was analysed and had similar physical, serological and biological characteristics to a reference strain of BLV isolated in the USA. However, different patterns of recognition of BLV-protein p24 were observed among naturally infected cattle using the western-blotting technique.

Properties of Nine Continuous B‐Cell Lines Established from Enzootic Bovine Leukosis Tumors

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.

Early diagnosis of enzootic bovine leukosis.

For establishment of early diagnosis of enzootic bovine leukosis (EBL), the complement-dependent antibody cytotoxicity (CDAC) test using monoclonal antibody against tumor-associated antigen (TAA) expressed on EBL tumor cells, lymphocyte counts, lactate dehydrogenase (LDH) activities and concentration of serum sialic acid (SA) were examined in clinical subclinical and EBL cases. In TAA-positive clinical and subclinical cows, lymphocyte counts, total LDH, LDH2 and LDH3 activities and concentration of SA increased, whereas LDH1 activity decreased from those of TAA-negative healthy cattle. The cytotoxic index (CI) values of the CDAC test from individual cows examined were compared with the results of other clinical examinations. Significant correlation coefficients were observed between CI values and the lymphocyte counts, total LDH, LDH1 and LDH2 activities and concentration of SA (p<0.05-0.005).

Establishment of bovine leukemia virus-producing and -nonproducing B-lymphoid cell lines and their proviral genomes

We have established four cell lines in vitro from peripheral leukemic cells of four independent enzootic bovine leukosis (EBL) cattle. All cell lines exhibited differentiated B-cell characters. Two of them produced infectious bovine leukemia virus (BLV), but the others did not. Nonproducer cell lines contained single copies of defective BLV proviral genomes with the same integration profiles as the uncultured cells. On the other hand, numerous proviral copies were detected in producer cell lines. One of the producer cell lines, BL407, whose original uncultured cell contained complete and defective proviral genomes retained the original two copies and had increased only complete genomes in different integration sites after long term culture. These findings suggest that the monoclonal leukemic cells from EBL cases are preferentially established in vitro irrespective of their proviral structures, and the producer B-lymphoid cells amplify their proviral copies by reinfection with viruses re-expressed from the cells during in vitro cultivation.

Difficulty in detecting in vivo extracellular infective virus in cattle naturally infected with bovine leukemia virus

A search was made for the presence in vivo of infective bovine leukemia virus (BLV) in secretions and excretions as well as in several tissues from five bulls, three cows and one calf. Two of the bulls and three of the cows were natural tumour cases of enzootic bovine leukosis, another three of the bulls were natural cases of persistent lymphocytosis and the calf was a natural tumour case of juvenile bovine leukosis. BLV infection was confirmed for all cattle, except the juvenile leukosis case, by detection of BLV-specific agar gel immunodiffusion (AGID) antibodies Cell-free preparations were made from homogenates of lymphocytes, lymph nodes, spleens, livers, intestine, urinary bladders, salivary glands, mammary glands, pools of prostate glands and testicles and feces as well as from plasma, urine and milk, by passing them through 5 μm membrane filters. BLV infectivity in these cell-free preparations was examined by syncytia infectivity assay using susceptible cell cultures of bovine or ovine origin. Infective BLV could not be isolated from any of these cell-free preparations of plasma, secretions, excretions and tissues, although it was isolated consistently from the in vitro cultures of viable lymphocytes obtained from BLV-infected cattle. There was no indication of BLV involvement in the case of juvenile bovine leukosis.

Studies on the sporadic and enzootic forms of bovine leukosis

The relationship between the skin form of bovine lymphosarcoma (SLS) and bovine leukemia virus (BLV) was studied by a number of immunological techniques. Neither C-type virus expression, BLV antigens in culture cells nor antibody to BLV antigens were found in the SLS case. The percentage of B and T cells in lymphoid cells of the SLS case was lower than that of normal bovine lymphoid cells. On the other hand, anti-BLV antibodies were found in 7 cases of enzootic bovine leukosis (EBL) and BLV was detected in 4 of 6 EBL cases tested. The percentage of B cells in peripheral blood lymphocytes and tumour cells from EBL was higher than that in normal cattle, but loss of B cell surface marker may occur in some cases. No BLV antibodies were found in 65 sera from 3 herds where sporadic leukosis, including the SLS case, had recently occurred. However, antibodies were found in 15 of 63 sera from herds where enzootic leukosis had recently occurred. These results suggest that SLS is not aetiologically related to EBL.