RESUMO
The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.
Assuntos
DNA Viral/análise , Leucose Enzoótica Bovina/diagnóstico , Vírus da Leucemia Bovina/genética , Reação em Cadeia da Polimerase/veterinária , Animais , Sequência de Bases , Bovinos , Primers do DNA/análise , Primers do DNA/química , Primers do DNA/genética , DNA Viral/sangue , DNA Viral/genética , Leucose Enzoótica Bovina/epidemiologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Alemanha/epidemiologia , Imunodifusão/métodos , Imunodifusão/normas , Imunodifusão/veterinária , Incidência , Vírus da Leucemia Bovina/isolamento & purificação , Estudos Longitudinais , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Eslováquia/epidemiologiaRESUMO
Polymerase chain reaction (PCR) has been used for direct detection of bovine leukemia virus (BLV) proviral DNA in cattle, but it is still mainly used for experimental research. One bottleneck for routine diagnosis of BLV by PCR has always been the isolation and purification of DNA. We compare the use of not purificated with highly-purified DNA in the PCR-based diagnosis of BLV infection. DNA extracted from whole blood by chloroform extraction (CP-DNA) and DNA prepared only by osmotic shock, washing, heating and freezing procedures (RPoS-DNA), were utilized. Fifteen cattle well characterized serologically were investigated for BLV-provirus with PCR using this different DNA preparations. With both methods all but one investigated animal were correctly identified. It was estimated that in case of CP-DNA PCR 10 BLV-provirus copies were sufficient to obtain a positive result. The sensitivity of RPoS-DNA PCR was similar. Because of the greater practicability of the latter technique we used it in a small field study with ten cattle. All serologically positive animals were correctly identified by the PCR. In addition one seronegative animal was found to carry BLV-provirus. Therefore RPoS-DNA PCR might be a good tool for the routine diagnosis of BLV-infected cattle.
