Evaluation of an indirect ELISA for the detection of antibodies to bovine leukaemia virus in milk and serum.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to BLV in milk and serum (Juntti et al., 1989). The conjugate consists of a monoclonal anti-bovine IgG1 and IgG2 labelled with horseradish peroxidase (HRP). The indirect ELISA was calibrated with EEC reference serum E 4. Standard serum E 4 was scored positive when diluted 8192 times in negative milk and between 12,800 and 25,600 times in negative serum. The sensitivity and specificity of the indirect ELISA relative to the agar gel immunodiffusion test (AGID) were 100% and 99.8%, respectively. ELISA results for milk and sera from 614 dairy cows agreed to 100%. The absorbance value in bulk milk could be used to roughly predict the rate of BLV infection among lactating cows in a herd. An infection rate of 4 to 5% in a herd could be detected in the ELISA. Results were applied in a nation-wide screening of more than 24,000 bulk-milk samples, and the subsequent introduction of an eradication programme for BLV. The aim is to eliminate the infection from Swedish herds in 5 to 10 years.

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

The early stage of bovine leukaemia virus (BLV) infection was studied in experimentally infected calves in order to assess the diagnostic applicability of a double polymerase chain reaction (PCR). In addition, the kinetics of infection and virus distribution were evaluated. To simulate the natural route of virus transmission, the calves were infected by transferring two different infectious doses of whole blood from a BLV infected cow. The establishment of infection was determined by the double PCR and syncytia formation assay and by indirect serological methods including indirect ELISA, gp51/p24 ELISA, agar gel immunodiffusion (AGID) and Western blotting. BLV antibodies were first detected in ELISA on post infection (p.i.) day 26. Close agreement was found between the results of the various indirect methods. BLV infection was first detected in peripheral blood lymphocytes (PBL) by the PCR on p.i. day 7. No animal became seropositive to BLV prior to direct detection of BLV infection by the PCR. At slaughter, urine and saliva specimens as well as various organs were collected from the calves and tested by the double PCR. Several of the organs yielded positive results: e.g. spleen, uterus, liver, kidney, abomasum, and lymph nodes. Nine out of eleven spleen suspensions were positive by the PCR, including the spleen from one calf, which otherwise remained negative in all tests throughout the experiment. This phenomenon indicates that an animal may be infected without detectable levels of BLV proviral DNA in PBLs and without circulating antibodies, further emphasizing the diagnostic importance of the PCR. The findings indicate that the PCR is the most rapid method for the early detection of BLV infection in cattle and a valuable tool for studying the tropism of the virus.

Bovine leukemia virus: early reflections in blood after an experimental infection of calves.

In order to study early alterations in the blood following infection with bovine leukemia virus (BLV) in the natural host, 15 calves were inoculated with blood from a BLV-positive donor cow. The humoral immunological response was followed by ELISA for 2 months. Seroconversion to BLV was demonstrated at 4-5 weeks post-infection. Total and differential leukocyte counts were performed. Acute lymphocytosis was observed at the time of seroconversion in the majority of the experimental calves. By the aid of monoclonal antibodies (mAbs), the proportion as well as the total number of lymphoid cells were studied in four of the calves, applying analytical flow cytometry. At the time of seroconversion the percentage of B-cells increased from 19.1 +/- 7.5% to 37.9 +/- 15.8%, and the T-cells (CD2+) decreased from 36.7 +/- 7.3% to 22.7 +/- 6.0%, the latter being attributable to decreases in the percentage of CD4+ as well as CD8+ T-cells for the infected calves together. Subsequently, altered B/T ratios were observed. In one of the calves an increase in the absolute number of CD5+ cells coincided with an increase in total B-cells. The early phenotypic alterations in lymphocyte subsets, before and after seroconversion to BLV, were comparable to those of non-lymphocytotic and persistent lymphocytotic cattle, respectively. Sera from 15 calves were tested for the presence of interferon (IFN), as measured by antiviral activity. BLV does not appear to induce the production of IFN.

Direct detection of bovine leukemia virus infection: practical applicability of a double polymerase chain reaction.

A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.

Bovine herpesvirus 1: rapid diagnosis of infection by direct filter hybridization.

Direct filter hybridization (DFH) was applied as a simple method of nucleic acid hybridization to diagnose bovine herpesvirus 1 (BHV-1) infection without previous purification of nucleic acids from the specimens. The DNA of BHV-1 was cleaved with the restriction endonuclease Pst I and randomly cloned into pKH47 plasmids. The clones were labelled with 32P or biotin and selected on uninfected and infected cells for the highest specific activity to detect BHV-1 infection. Two clones, which detected about 10 infected cells, were selected for the diagnosis of BHV-1 in cattle. On specimens collected during experimental and natural disease, the DFH showed to be in concordance with the standard method of virus isolation. This simple hybridization technique proved to be a sensitive and rapid alternative to virus isolation. Specific diagnosis of BHV-1 infection can be made even in simply equipped laboratories within 10 h.