Bovine leukaemia virus: Rapid detection of proviral DNA by nested PCR in blood and organs of experimentally infected calves

Klintevall, K.; Ballagi-Pordány, A.; Näslund, Katarina; Belák, Sándór

doi:10.1016/0378-1135(94)90018-3

Cited by 68 publications

(54 citation statements)

References 23 publications

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“…These results would be supported by previous studies directed at defining PCR sensitivity for detecting BLV provirus in the peripheral blood of infected animals; limits of detection were at 1 pg of viral DNA, 10 less than 8 proviral copies (2-3 BLV-infected FLK cells) in 2 ϫ 10 6 negative lymphocytes, 1 and 1 BLV-positive cell in 2,000 noninfected cells in experimentally infected calves. 5 These latter studies would support the use of PCR for diagnostic purposes.…”

Section: Discussionmentioning

confidence: 91%

Absence of Bovine Leukosis Virus in Semen of Seropositive Bulls

2002

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Abstract.A polymerase chain reaction (PCR)-based detection system was established to identify the presence of bovine leukosis virus (BLV) DNA in bovine semen. Seventy-nine bulls were included in the study. Serum, peripheral blood leukocytes, and semen were collected from each of the 79 bulls. The BLV-specific antibody was detected in serum by agar gel immunodiffusion and viral DNA in blood and semen by PCR. Serologically, 29 of the 79 bulls were BLV positive. Twenty-seven of the 29 seropositive bulls and 1 of the seronegative bulls had BLV DNA in peripheral blood leukocytes. All 79 bulls tested PCR negative for the presence of BLV in semen. This data is strong evidence that properly collected semen from BLV seropositive bulls will not contribute to dissemination of this viral infection.Bovine leukosis virus (BLV) is an exogenous oncogenic retrovirus associated with enzootic bovine leukosis. The infection can result in 3 different clinical states, with many animals remaining asymptomatic in an aleukemic state (AL). However, 30-70% develop a persistent lymphocytosis (PL) characterized by a polyclonal expansion of B-lymphocytes and a much smaller percent, 0.1-10%, eventually develop lymphoid tumors. 3,6,11,12 A previous survey published in 1985 suggested up to 30% of dairy cows in the United States were infected with BLV. 12 Due to the relatively short life of production cattle in the United States, the greatest economic impact of BLV infection would appear to be that associated with interference in the international movement of cattle and their germ plasm rather than clinical disease. Thus, application of molecular biology techniques, such as polymerase chain reaction (PCR), to identify the presence of BLV in semen should facilitate certification of germ plasm as being free of contaminating virus.Previous studies by numerous investigators suggested that semen from BLV-infected bulls was noninfectious for recipient cows. 4,8,9 Sheep inoculated with entire ejaculates from BLV-infected bulls remained seronegative. Cows inseminated with ejaculates from seropositive bulls have also not seroconverted nor have their progeny. In addition, a 5-year study demonstrated that use of semen derived from both BLV seropositive and seronegative donor bulls in a BLV-free closed dairy did not result in any seroconversion. Donor bulls were from multiple artificial insemination (AI) centers, with 48% being seropositive. 9 Despite the above-mentioned studies that argued strongly against the possibility of recipient cows becoming infected by using semen from BLV-positive bulls, international movement of germ plasm from BLV-infected animals is still problematic. The most common method of testing for BLV in animals is the detection of antibodies to gp51 by agar gel immunodiffusion (AGID). However, the application of PCR for identification of viral DNA has provided a sensitive test for detecting BLV in tissues such as blood. 1,2,10 This report describes the application of PCR to the detection of BLV DNA in peripheral blood leukocytes and se...

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Section: Discussionmentioning

confidence: 91%

Absence of Bovine Leukosis Virus in Semen of Seropositive Bulls

2002

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“…The composition of the reaction mixture and the thermal profile of double amplification of the viral env gene were consistent with the description given by Markiewicz et al (2003). Primers with the sequence described by Klintevall et al (1994) (Sigma, USA) were used.…”

Section: Nested Pcr Testmentioning

confidence: 99%

Tumor necrosis factor-alpha (TNFα) gene polymorphism and expression of membrane-bound TNFα protein on CD11b+ and IgM+ cells in cows naturally infected with bovine leukemia virus

Bojarojć-Nosowicz¹,

Kaczmarczyk²,

Stachura³

et al. 2015

Polish Journal of Veterinary Sciences

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The aim of this study was to determine whether SNP at position -824 (promoter region) of the TNFα gene significantly differentiates the size of IgM+, CD5+ and CD11b+ cell subpopulations and affects the expression of membrane-bound TNFα protein (mTNFα) on these cells and their susceptibility to BLV infections.In this study, significant differences were determined for the first time between TNFα genotypes and the percentage of cells with the CD11b+TNFα+p24+ immunophenotype. Furthermore, greater expansion of lymphocytes with the IgM+TNFα+p24+ immunophenotype was reported in cows with the G/G genotype than in A/A homozygotes. Cells with the above immunophenotype were more frequently observed in cows with persistent leukocytosis than in aleukemic cattle.Our results suggest that polymorphism of the TNFα -824 A>G gene and mTNFα protein expression play an important role in the pathogenesis of enzootic bovine leukosis.

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“…A não identificação pela PCR em animais soropositivos foi constatada também por KUCKLEBURG et al (2003), CAMARGOS et al (2007) e RAMA et al (2011. Esse fato pode ser explicado pela existência de um pequeno número de linfócitos infectados (EAVES et al, 1994;REICHEL et al, 1998), devido à presença destas células apenas nos órgãos (KLINTEVALL et al, 1994) ou devido à supressão da multiplicação viral em animais com altos títulos de anticorpos (JIMBA et al, 2011). A não detecção de anticorpos pela IDGA em animais positivos na PCR pode ser causada por infecção recente, sendo a colheita das amostras realizada antes da soroconversão (DE BÔER et al, 1987) ou por imunossupressão no periparto (EBERTUS et al, 1987).…”

Section: Resultsunclassified

“…NAIF et al (1992), comparando IDGA, ELISA e PCR, demonstraram que a PCR diagnostica mais precocemente a infecção do que o ELISA e a IDGA. KLINTEVALL et al (1994) verificaram que a PCR detectou o DNA proviral em linfócitos sanguíneos no sétimo dia pós-infecção e os anticorpos foram detectados primeiro pelo ELISA a partir do 26 o dia após inoculação, enquanto que, pela IDGA, a soroconversão ocorreu em média a partir do 28 o dia pós inoculação. Considerando-se os riscos de contaminação de reagentes e amostras pela manipulação dos produtos da PCR externa, a necessidade de realização de eletroforese para visualizar resultados e tempo maior gasto na nPCR para finalização do método, além dos resultados obtidos neste trabalho, conclui-se que a qPCR pode ser utilizada oficialmente no diagnóstico da LEB.…”

Section: Resultsunclassified

“…A IDGA foi considerada por muitos anos a prova de eleição pela sua alta especificidade, sensibilidade e por ser de baixo custo (REICHEL et al, 1998). Em algumas situações, as provas sorológicas falham na identificação dos animais infectados, por exemplo, no periparto (GENTILE et al, 1985;EBERTUS et al, 1987), em animais infectados recentemente, visto que a detecção de anticorpos ocorre no mínimo três semanas após a infecção (DE BÔER et al, 1987), em bovinos permanentemente soronegativos ou com baixos títulos de anticorpos e em animais persistentemente infectados pelo BVDV que apresentam resposta imune deprimida a infecções pelo BLV (EAVES et al, 1994;KLINTEVALL et al, 1994).…”

Section: Introductionunclassified

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PCR em tempo real para diagnóstico da leucose enzoótica bovina

et al. 2012

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O objetivo deste trabalho foi realizar a validação de uma reação em cadeia da polimerase em tempo real com o sistema Plexor® (qPCR) para o diagnóstico da Leucose Enzoótica Bovina (LEB), por meio da comparação com testes de diagnóstico recomendados pela Organização Mundial de Saúde Animal (OIE). A qPCR foi comparada com duas outras técnicas: a PCR nested (nPCR) e a imunodifusão em gel de ágar (IDGA). Das 82 amostras analisadas pela qPCR e nPCR, 79 apresentaram resultados concordantes, sendo a concordância, classificada pelo Índice Kappa, como alta. Entre as PCRs e a IDGA, o número de resultados concordantes foi de 71 e 69, respectivamente, para qPCR e nPCR, sendo a concordância classificada como considerável. A qPCR apresentou altos valores de sensibilidade e especificidade. Os valores preditivos da qPCR observados demonstraram a alta capacidade de classificação dos casos positivos e negativos. A qPCR não foi capaz de detectar três amostras positivas e tem custo ligeiramente superior que a nPCR. Entretanto, a qPCR é uma técnica mais rápida, menos susceptível a contaminações, tem alta sensibilidade, não utiliza e não gera resíduos carcinogênicos. Concluímos que a qPCR pode substituir a nPCR recomendada pela OIE no diagnóstico de rotina em áreas em que a LEB é endêmica, como no Brasil.

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Bovine leukaemia virus: Rapid detection of proviral DNA by nested PCR in blood and organs of experimentally infected calves

Cited by 68 publications

References 23 publications

Absence of Bovine Leukosis Virus in Semen of Seropositive Bulls

Absence of Bovine Leukosis Virus in Semen of Seropositive Bulls

Tumor necrosis factor-alpha (TNFα) gene polymorphism and expression of membrane-bound TNFα protein on CD11b+ and IgM+ cells in cows naturally infected with bovine leukemia virus

PCR em tempo real para diagnóstico da leucose enzoótica bovina

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