Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in dairy herds in Minas Gerais state, Brazil / [Soroprevalência do Mycobacterium avium subespécie paratuberculosis e vírus da leucemia bovina e fatores de risco associados em rebanhos leiteiros de Minas Gerais, Brasil]

Mycobacterium avium subesp. paratuberculosis (MAP) e o vírus da leucemia bovina (BLV) são agentes que causam grandes perdas econômicas nos rebanhos. O objetivo deste estudo foi avaliar a situação epidemiológica da paratuberculose bovina (PTB) e leucose enzoótica bovina (EBL) em rebanhos leiteiros de Lagoa Formosa, Minas Gerais, Brasil. Foram coletadas 236 amostras de sangue de vacas, as quais foram submetidas aos testes ELISA e imunodifusão em gel de ágar para detecção de anticorpos contra MAP e BLV. A soroprevalência de anticorpos contra MAP e BVL foi de 20% para os rebanhos e 6% para os animais e de 85% para os rebanhos e 50,42% para os animais, respectivamente. A presença dessas enfermidades deve servir como um alerta para os produtores e veterinários, para que concentrem maior atenção na implementação de medidas higiênico-sanitárias, incorporando elementos de vigilância com base nos riscos identificados no estudo.(AU)

Infección experimental en ovinos con el virus de leucosis bovina: dosis mínima de células BLV-FLK y virus libre de células, y actividad neutralizante de anticuerpos naturales / Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies

Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especialmente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ovejas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV.

Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil / Avaliação do desempenho de ELISA importado no sorodiagnóstico da leucose enzoótica bovina em rebanhos do estado de Pernambuco, Brasil

Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa de distribuição cosmopolita e caráter crônico causada por um vírus da família Retroviridae, o vírus da leucemia bovina (VLB). A situação epidemiológica da LEB no Brasil vem motivando estudos para o aprimoramento do seu diagnóstico, tendo como base as técnicas sorológicas recomendadas: imunodifusão em gel de ágar (IDGA) e Enzyme-Linked Immunoabsorbent Assay (ELISA). Este estudo teve como objetivo avaliar o uso de ELISA importado para a detecção do VLB em rebanhos leiteiros criados em Pernambuco, Brasil, comparando-o ao IDGA. Amostras de soro sanguíneo de 327 bovinos leiteiros do estado de Pernambuco foram testadas para IDGA e ELISA comercial importado CHEKIT-Leucose-serum, produzido pelo laboratório IDEXX® para o diagnóstico da LEB. Descartadas 25 amostras inconclusivas de um ou ambos os testes, foram analisadas 302 amostras, sendo 24,1% positivas (73/302) na IDGA e 45% (136/302) no ELISA, que em relação à IDGA, técnica considerada padrão, apresentou sensibilidade de 98,6%, especificidade de 72% e coeficiente Kappa de 0.55. A falta de concordância entre os métodos diagnósticos deveu-se, provavelmente, à elevada sensibilidade do ELISA, que possibilita detectar anticorpos mesmo em situações com baixos teores séricos. Apesar da IDGA se mostrar até o momento um teste eficiente, em etapas mais avançadas de um programa de controle e erradicação da LEB, com baixos índices de prevalência, o ELISA apresentará melhor desempenho, por possuir maior sensibilidade, evitando-se a permanência de animais disseminadores da doença nos rebanhos.​(AU)

Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.

A novel association of BoLA DRB3 alleles in BLV infected cattle with different proviral loads / Uma nova associação de alelos de BoLA DRB3 em bovinos infectados com BLV com diferentes cargas provirais

Bovine leukemia virus (BLV) is associated with the most common neoplastic disease of cattle. BLV has a silent dissemination in the herd due to infected cell exchange, thus the concentration of BLV-infected cells in blood should play a major role in the success of viral transmission. Genes from Bovine leukocyte antigen (BoLA), the MHC system of cattle, are associated with genetic resistance and susceptibility to a wide range of diseases, and also with production traits. Some BoLA DRB3.2 allele polymorphisms in Holstein cattle have been associated with resistance or susceptibility to BLV-disease development, or with proviral load (PVL). This investigation studied 107 BLV-infected Argentinean Holstein dairy cows, all of them belonging to one herd. PVL was analysed by qPCR and animals were classified as high proviral load (HPVL, N = 88) and low proviral load (LPVL, N = 19), and BoLA DRB3.2 alleles were genotyped. Alleles BoLA DRB3.2*1501 and *1201 were significantly associated with HPVL (p = 0.0230 and p = 0.0111 respectively), while allele BoLA DRB3.2*0201 was significantly associated with LPVL (p = 0.0030). The present study aims at contributing to the knowledge of the association between BoLA polymorphism and development of a BLV infection profile. Genes that best explain the PVL in this population resulted BoLA DRB3.2*0201 (as a protection factor) and *1501 (as a risk factor). Allelic differences may play an important role in the development of effective immune responses. A better understanding of how BoLA polymorphism contributes to these responses and the establishment of a BLV status is desirable to schedule and evaluate control measures.(AU)

O vírus da leucemia bovina (BLV) está associado à doença neoplásica mais comum do gado bovino. O BLV tem uma disseminação silenciosa no rebanho devido à troca de células infectadas, assim, a concentração de células BLV infectadas no sangue deve desempenhar um papel importante no sucesso da transmissão viral. Os genes do antígeno leucocitário bovino (BoLA), sistema MHC do gado bovino, estão associados à resistência genética e à susceptibilidade a uma ampla gama de doenças, bem como às características da produção. Alguns polimorfismos de alelos de BoLA DRB3.2 em bovinos Holstein têm sido associados à resistência ou susceptibilidade ao desenvolvimento da doença BLV, ou com carga proviral (PVL). Esta investigação avaliou 107 vacas leiteiras da raça Holstein argentina infectadas com BLV e pertencentes a um único rebanho. A PVL foi analisada por qPCR, os animais foram classificados em alta carga proviral (HPVL, N = 88) e baixa carga proviral (LPVL, N = 19), e os alelos BoLA DRB3.2 foram genotipados. Os alelos BoLA DRB3.2*1501 e *1201 estavam significativamente relacionados à HPVL (p = 0,0230 e p = 0,0111, respectivamente), enquanto o alelo BoLA DRB3.2*0201, à LPVL (p = 0,0030). O objetivo deste estudo é contribuir para o conhecimento da associação entre o polimorfismo de BoLA e o desenvolvimento de infecção por BLV. Os genes que melhor explicam a PVL na população analisada resultaram em BoLA DRB3.2*0201 (como fator de proteção) e *1501 (como fator de risco). As diferenças alélicas podem desempenhar um papel importante no desenvolvimento de respostas imunitárias eficazes. Uma melhor compreensão de como o polimorfismo BoLA contribui para estas respostas e o estabelecimento de um estado BLV é desejável para agendar e avaliar as medidas de controle.(AU)

Development and evaluation of an immunochromatographic assay using a gp51 monoclonal antibody for the detection of antibodies against the bovine leukemia virus

Infection of cattle with bovine leukemia virus (BLV) has been observed and reported worldwide, including in Korea. The onsite identification of infected cattle would help decreasing and eradicating BLV infections on farms. Here, we present a new immunochromatographic assay that employs monoclonal antibodies (MAbs) for the detection of antibodies against BLV in the field. BLV envelope glycoprotein (gp)51 was expressed in E. coli, and MAbs against recombinant BLV gp51 were generated for the development of an immunochromatographic assay to detect BLV antibodies in cattle. The sensitivity and specificity of the assay were determined by comparing these results with those obtained from a standard enzyme linked immunosorbent assay (ELISA). A total of 160 bovine sera were used to evaluate the new immunochromatographic assay. Using ELISA as a reference standard, the relative specificity and sensitivity of this assay were determined to be 94.7% and 98%, respectively. Because of its high sensitivity and specificity, this BLV antibody detection assay would be suitable for the onsite identification of BLV infection in the field.

Genetic analysis of env and gag gene fragments of bovine leukemia virus identified in cattle from Korea / 대한수의학회지

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. This study was conducted to clarify the molecular characteristics of BLVs obtained from a specific region in Korea. Proviral BLVs were detected in anti-BLV antibody-positive blood samples by PCR. Env and gag fragments were sequenced and compared to previously published reference sequences. Analysis of the env gene sequence revealed that the YI strain was highly similar to genotype 1, including United States and Japanese strains. The gag gene sequence had the highest degree of similarity with a Japanese strain.

Intercorrência entre leucose enzoótica e tuberculose em bovinos leiteiros do estado de Pernambuco / Assessment of intercurrence of enzootic leucosis and bovine tuberculosis in dairy cows in the state of Pernambuco, Brazil

Objetivou-se com este estudo avaliar a intercorrência entre leucose enzoótica e tuberculose em rebanhos bovinos leiteiros em oito municípios do Estado de Pernambuco, pelo estabelecimento da prevalência de bovinos reagentes às provas diagnósticas específicas. Foram analisados sorologicamente 662 animais, pela técnica de imunodifusão dupla em gel de agarose e 612 foram avaliados imunoalergicamente, por meio do teste de tuberculinização. Nos 15 rebanhos examinados, as prevalências de bovinos que apresentaram positividade aos testes IDGA e tuberculina foram 32,2% (213/662) e 14,1% (86/612), respectivamente. Os resultados obtidos nesta pesquisa permitiram concluir que as doenças estudadas encontram-se amplamente disseminadas na população avaliada, com crescimento em níveis significativos da leucose, e as infecções pelo vírus da leucose bovina (VLB) e Mycobacterium bovis encontram-se ativas e em expansão, com risco do comprometimento da saúde dos rebanhos bovinos e, pelo caráter zoonótico da tuberculose bovina, das pessoas que lidam com os bovinos.

We carried out this study to evaluate the intercurrence of tuberculosis and enzootic leukosis in dairy cattle herds in 8 districts of the State of Pernambuco, Brazil, by establishing the prevalence of bovines reagent to specific diagnostic tests, and to also verify the correlation between enzootic leucosis, cattle tuberculosis, the leukocytes count and lymphocytes count among the cattle studied. A total of 662 animals were tested serologically and hematologically, by the techniques of double immunodiffusion in agarose gel and the white blood cell count with the total and differential leukocyte count, respectively, and 612 were evaluated immunoallergically by the tuberculin test. In the 15 herds examined, the prevalence of cattle that were positive to the AGID and tuberculin tests were 32.2% (213/662) and 14.1% (86/612), respectively. Analyzing the correlation between the variables total leukocytes and lymphocytes, we found that total leukocyte is highly related to the variable lymphocytes (72%). The results in this study indicated that the diseases studied are widespread in the population studied, with significant levels of growth in leukemia, and infections of bovine leukosis virus (VLB) and Mycobacterium bovis are active and growing, with risk of impairment of the health of cattle herds, and, due to the zoonotic nature of bovine tuberculosis, of the health of people who work with cattle.

Soroprevalência de anticorpos para o Vírus da Leucose Enzoótica em bovinos criados na região metropolitana de Curitiba, Paraná / Seroprevalence for Bovine Leukosis Virus (BLV) in dairy cattle of Curitiba and the surrounding area, Paraná, Brazil

A Leucose Enzoótica Bovina (LEB) é uma doença infecto-contagiosa causada pelo Vírus da Leucemia Bovina (VLB), um retrovírus oncogênico da família Retroviridae. O presente estudo teve como objetivo avaliar a soroprevalência e a respectiva influência de fatores etários da LEB em bovinos leiteiros das raças Holandesa Preta e Branca, Jersey, Pardo-Suíço e mestiços, criados na região metropolitana de Curitiba, PR. Foram testadas 268 amostras de soros sanguíneos pela prova de Imunodifusão em gel de Agar (IDGA), colhidas em cinco propriedades situadas nos municípios de São José dos Pinhais, Campina Grande do Sul, Pinhais e Fazenda Rio Grande. Foram encontrados 151/268 (56,34%) animais positivos e 117/268 (43,66%) negativos. Animais mais velhos mostraram um aumento estatisticamente significativo de soropositividade. Pode-se concluir que a LEB está amplamente disseminada em bovinos leiteiros da região metropolitana de Curitiba e há necessidade de adequada aplicação de medidas de controle e prevenção da LEB.

Enzootic bovine leukosis (EBL) is aninfectious and contagious disease caused by bovine leukemia virus (BLV), an oncogenic retrovirus of the family Retroviridae. The present study aimed to evaluate the seroprevalence and respective age influence of BLV in Holstein, Jersey, Brown Swiss and mixed-breed dairy cattle raised in Curitiba and the surrounding area, Paraná, Brazil. A total of 268 samples of bovine serum from five different herds in the counties of São José dos Pinhais, Campina Grande do Sul, Pinhais and Fazenda Rio Grande were tested with immunodiffusion (ID). A total of 151/268 (56.34%) testedseropositive, while 117/268 (43.66%) were considered seronegative. Older animals prsented a significant rise in seropositivity. In conclusion, BLV is widely distributed among dairy cattle of Curitiba and the surrounding area, present in all tested herds, thus requiring adequate application of measures in EBL control and prevention.

Occurrence of seropositive sheep (Ovis aries) to bovine leukemia virus in Brazil / Ocorrência de ovinos (Ovis aries) soropositivos ao vírus da leucemia bovina no Brasil

Occurrence of seropositive sheep (Ovis aries) to Bovine Leukemia Virus (BLV) by agar-gel immunodiffusion test (AGID) using the antigen gp51 was surveyed for the period 2005-2007. Samples were collected from sheep in the Brazilian states of Rio Grande do Sul, Paraná, São Paulo, Pernambuco, Maranhão, Pará, Bahia, Mato Grosso, Rondônia, and Acre. Two of 35 (5.7%) flocks were seropositive to BLV, and the rate of seropositive animals was 0.077% (two of 2,592). The two seropositive sheep were female, one 13-month old Santa Inês breed and other of unknown age and breed, both from the state of São Paulo. Distribution of BLV in the ovine population studied proved to be a rare event in Brazil.

A ocorrência de ovinos sororreagentes ao vírus da Leucemia Bovina (VLB) pelo teste de imunodifusão em gel de ágar (IDGA) utilizando o antígeno gp51 foi avaliada no período de 2005-2007, em diferentes regiões do Brasil. Amostras foram colhidas de ovinos dos Estados do Rio Grande do Sul, Paraná, São Paulo, Pernambuco, Maranhão, Pará, Bahia, Mato Grosso, Rondônia e Acre. Duas em 35 cabanhas (5,7%) e dois em 2592 ovinos (0,077%) foram soropositivos. Os únicos animais com anticorpos contra o VLB eram fêmeas, uma com 13 meses de idade e da raça Santa Inês e a outra não se conhecia a idade e raça, ambas provenientes do Estado de São Paulo. A distribuição da soropositividade na população estudada demonstrou ser rara a infecção pelo VLB em ovinos no Brasil.

Serological and genomic detection of bovine leukemia virus in human and cattle samples

Bovine leukemia virus [BLV] is a retro virus responsible for lymphoproliferative disorders in cattle. Although infections of BLV in animals are well known, little is known about its capacity to infect humans. This study investigated the presence of anti-BLV antibodies and BLV pro viruses in human and cattle samples. An indirect enzyme-linked immunosorbent assay [ELISA] was used to detect anti-BLV antibodies while nested PCR was employed to identify BLV provirus sequences. The overall prevalence of anti-BLV antibodies in human and cattle samples were 12.50% and 16.73%, respectively. When using ELISA as a reference test, sensitivity and specificity for nested PCR were 0.625 and 0.970, respectively. The predictive value of a positive test was 0.862 and the predictive value of a negative test was 0.897. The percentage of cattle correctly classified by nested PCR assay was 89.1%. Nested PCR and Southern blot analysis, using primers specific for BLV gag sequences, revealed that BLV pro viruses were detectable in cattle and human samples. Our results highlight the risk of human exposure to BLV and the need for further investigations to determine whether BLV infection poses a health hazard for humans

[ abattoir survey on prevalence of enzootic bovine leukosis virus infection and associated clinical, haematological and flow cytometric findings in holestein cattle in Tehran]

Bovine leukosis virus [BLV] is a retrovirus that primarily affects lymphoid tissue of cattle. To determine the seroprevalence of Enzootic Bovine Leukosis Virus infection and its effects on some clinical, hematological and flow cytometric indications, this study was conducted on 197 Holstein cattle slaughtered in an abattoir in Tehran province. ELISA gp5 1 test of the obtained serums of the animals showed that the mean of infection rates was 22.3%. Age arrangement of the relative frequencies of the infection showed a significant correlation between EBLV infection and age increasing [0.001

Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-)

Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.

Simultaneous presence of bovine papillomavirus and bovine leukemia virus in different bovine tissues: in situ hybridization and cytogenetic analysis

Bovine papillomavirus (BPV) DNA sequences were detected in different tissues, in addition to epithelium. Cytogenetic abnormalities were observed in blood lymphocytes. The presence of more than one virus in a single tissue is a difficult aspect to evaluate, especially when the DNA sequences are detected in tissues that are not specifically targeted by the virus. BPV and bovine leukemia virus (BLV) are clastogenic, causing chromosome aberrations in peripheral blood lymphocytes. In the present study, we investigated the simultaneous presence of DNA sequences of both viruses and the possibility of vertical transmission and compared the types of chromosome aberrations related to viral action. BPV 1, 2, and 4 DNA sequences were found in three females of the herd and in their offspring. BLV DNA sequences were not detected in their progeny. A newborn calf that was negative for BLV infection showed specific chromosome rearrangements possibly related to the effect of infection with BPV.

Low numbers of intestinal Shiga toxin-producing E. coli correlate with a poor prognosis in sheep infected with bovine leukemia virus

Healthy ruminants carry intestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stx has antiviral activities in vitro and STEC numbers correlate with reduced early viremia in sheep experimentally infected with bovine leukemia virus (BLV). This study assessed the impact of intestinal STEC on BLV-induced disease for one year post-BLV-challenge. High STEC scores (CFU/g feces x frequency of STEC-positive samples) correlated with good health, whereas poor weight gain, distress, and tumor development occurred only among animals with low STEC scores. STEC carriage was associated with increased percentages of B cells in peripheral blood.

Detecting the P24 antigen in lymph nodes of BLV infected cattle

In this study 19 BLV infected and 2 healthy lymph nodes were analysed by Western blotting to detect viral p24 antigen. Western blotting was carried out by Rabbit anti p24 serum and p24 monoclonal antibody. Rabbit serum in western blotting could detect 45, 50, 54, 58 and less than 19 kD bands of proteins and monoclonal antibody could detect 30, 32, 39, 45, 50 and 53 kD bands. Our results indicate polymorphism of proteins with p24 epitope in different BLVinfected samples. Otherwise, it could be concluded that BLVstructural proteins sharing p24 epitopes have high degree of phenotypic diversity

Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.

Establishment of a bovine leukemia virus-free dairy herd in Korea

In view of the high prevalence rate of bovine leukemia virus (BLV)infections in cattle over the entire country, a large dairy farm in Chungnam province was chosen and 'test and segregate' program was instituted. On July 1999, ELISA test was performed on 491 animals on the farm and only 163 cattle (139 adult cows, 18 female and 6 male calves)were BLV-seronegative. From February 2000 through April 2004, the seronegative group was placed in barns 1,500 to 2,000 m from seropositive group and thereafter tested at 3-to 5-month intervals by ELISA. Animals seroconverted in consecutive tests were removed from the seronegative group immediately after the detection of anti-BLV antibodies. The changes in management were aimed at preventing iatrogenic transfer of blood between cattle. Replacement heifers imported from other countries and calves born at the farm were repeatedly tested by ELISA, and only seronegative animals were introduced into the group. As of April 2004, there were 311 cattle in the BLV seronegative group of the farm. Twent y four cows of the initial 139 adult cows were seroconverted in 2000, and no seropositive animals were found since February 2001. Follow up of the group, from which all seropositive cattle were moved to a separate location, revealed no recurrence of BLV infection for three years. The approach in the present study might be valuable for Korean producers who would like to move toward a BLV-negative status.

Flow Cytometric Analysis of Lymphocyte Subpopulations of Cattle Infected with Bovine Leukemia Virus

We examined lymphocyte subpopulations of peripheral blood from BLV infected and noninfected Holstein-Friesian dairy cattle reared in Korea by flow cytometry using monoclonal antibodies specifically reactive with bovine leukocyte differentiation marker. Lymphocyte subpopulations expressing BoCD11b, B-B2, CD5, B, MHC II-DP, MHC II-DQ, and MHC II-DR antigens were significantly abundant in the BLV(+) group than the BLV(-) group (p<0.01). On double staining, subpopulation of B-1a(BoCD5+ BoCD11b+) lymphocytes was significantly increased in leukemic group. However, T-lymphocyte lineage expressing BoCD2, BoCD4, BoCD8, and WC1 antigens was significantly lower than in the BLV(+) group (p<0.01). However the absolute number of T-lymphocytes expressing BoCD2, BoCD4, BoCD8, and WC1 antigens in BLV(+) group remained with in the normal range. Furthermore mean ratio of BoCD4/BoCD8 in the BLV(+) groups was higher than that in the BLV(-) group. Taken together, cellular immune responses did not seem to significantly be decreased in the leukemic cattle.