Enzootic bovine leukosis: Preparation of an antigen for the agar gel immunodiffusion test and statistical comparison with other antigens

The preparation of an antigen for the agar gel immunodiffusion test prepared by ultrafiltration of cell culture supernatant (chronically infected fetal lamb kidney cells) is described. The antigen obtained was compared by the single radial immunodiffusion test with an antigen prepared by ammonium sulfate precipitation (30%) and the commercial antigens (Pitman Moore, Seromed). The positive serum was used either undiluted or diluted 1:16 to test for the internal (P 24) and the external antigens (Gp 60) respectively.The results obtained indicated that the antigen prepared by ultrafiltration appeared to be much superior to the others, and contained Gp 60 which detects the commonly found antibodies in bovine leukosis virus-infected cattle.     

Bovine leukemia virus infection in Taiwan: evaluation of the enzyme-linked immunosorbent assay and agar gel immunodiffusion test.

I evaluated an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test simultaneously for the detection of bovine leukemia virus (BLV) antibodies. Total 1,293 serum samples were tested for ELISA and AGID test and the results were compared. The results of ELISA and AGID agreed by 1,156 out of 1,293 (89.4%). All of AGID-positive 356 sera were positive by ELISA. However, of 451 ELISA-positive sera, 95 sera were either negative or equivocal by AGID test. Eleven animals which showed ELISA-positive but AGID-negative or equivocal became AGID-positive in a year. It may be inferred that ELISA detects infected cattle earlier and with greater sensitivity than AGID.     

Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and western blotting for the detection of BLV antibodies

An indirect enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine leukaemia virus (BLV) infection was developed and compared with the agar gel immunodiffusion test (AGIDT). Western blotting (WB) was used as confirmatory test. ELISA and AGIDT had specificities that were comparable with that of WB, however, ELISA showed a higher sensitivity than AGIDT. The ELISA was useful for screening a large number of samples, whereas WB was important for detecting the antibody response against the individual BLV-proteins. Different types of positive serological reactions were discerned in WB, that correlated with reactions of sera in AGIDT and ELISA. The most important antigen in WB and ELISA was the BLV protein p24, whereas the BLV glycoproteins gp51 and gp30 were of special importance in AGIDT. The relevance of repeatedly testing the antibody response in BLV-infected herds for control and eradication programmes using assays with higher sensitivity than AGIDT was demonstrated.     

Comparison of the agar gel immunodiffusion test and ELISA in the detection of bovine leukosis virus antibody in cattle persistently infected with bovine virus diarrhoea virus

When six cattle persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with lymphocytes infected with bovine leukosis virus (BLV), a depressed antibody response to BLV was observed by ELISA which was due to a decrease in IgG1 synthesis. The ELISA was more sensitive and more reliable than the agar gel immunodiffusion (AGID) test in detecting BLV infection in cattle persistently infected with BVDV. Decreased antibody responses were manifested in the AGID test by negative, inconclusive or weakly positive reactions: only two of the six cattle developed antibodies that generated positive AGID reactions.     

Enzyme-linked immunosorbent assay for the detection of bovine papillomavirus

An enzyme-linked immunosorbent assay has been developed to detect and quantitate bovine papillomavirus in partially purified and in purified viral preparations, using rabbit antiserum against group-specific papillomavirus structural antigens and alkaline phosphatase-labeled affinity purified goat antibody to rabbit immunoglobulin G. Viral detection correlated well with negative-stain electron microscopy of the various preparations and peroxidase-antiperoxidase staining of paraffin sections of the original fibropapillomas. The technique is rapidly done and will detect minute amounts of viral protein (1 ng/ml).     

Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.     

An enzyme-linked immunosorbent assay for detection of antibodies to maedi-visna virus in sheep. II. Comparison to conventional agar gel immunodiffusion test.

A study was conducted to compare the indirect enzyme-linked immunosorbent-assay (i-ELISA) test using antigen prepared by a simple technique using sodium dodecyl sulfate (SDS) treatment to the conventional agar gel immunodiffusion test (AGID). Ten specific-pathogen-free (SPF) sheep were inoculated with maedi-visna virus (MVV) and serum antibody titers compared over a period of 14 weeks. All the sheep seroconverted by the i-ELISA compared to 90% by the AGID. The i-ELISA detected antibody at a mean of 2.6 weeks prior to the AGID. In both tests, fluctuations were observed in the serum antibody response of two sheep. The i-ELISA had a specificity of at least 98.8% and an increased relative sensitivity of 15.5% compared to the AGID, based on the analysis of sera from experimental sheep with MVV free status and sera from sheep from various sources. Of the sera from a seronegative flock which had been monitored with the AGID after a "test and remove" eradication program, 10.2% were positive by the i-ELISA. It was concluded that the AGID test may not be adequate to monitor samples for an eradication scheme.     

Comparison of the complement-fixation and agar gel immunodiffusion tests for diagnosis of subclinical bovine paratuberculosis

The performance of the serum complement fixation (CF) test was compared with that of a serum agar gel immunodiffusion (AGID) test on 74 subclinically infected and 154 uninfected cattle in 6 commercial midwestern dairy herds with Mycobacterium paratuberculosis infection and on 30 cattle in a herd that was free of infection. Infection status of cattle within herds was established by performance of a series of 3 or more fecal cultures and of ileocecal lymph node cultures of culled cattle. In cattle with subclinical infection detected by culturing, the sensitivity estimates of the CF and AGID tests were 10.8% (3.6% SE) and 18.9% (4.5% SE), respectively. In the cattle classified as disease free, the specificity estimates of the CF and AGID tests were 97.4% (1.3% SE) and 99.4% (0.6% SE), respectively. Neither set of estimates was significantly different. Negative test results obtained with the use of either test in apparently normal cattle from suspect herds should be interpreted with caution because both tests suffer from low sensitivities in subclinically infected animals. However, the AGID test may be more useful in regulatory situations in which the CF test is currently used because the AGID test is easier to perform and to interpret.     

Eradication of bovine leukemia virus infection in commercial dairy herds using the agar gel immunodiffusion test.

Demands for bovine leukemia virus test negative breeding cattle and for semen from bovine leukemia virus test negative bulls by several countries have encouraged the eradication of bovine leukemia virus infection from selected herds in Canada. This project was undertaken to evaluate the suitability of the agar gel immunodiffusion test, standardized to detect anti-bovine leukemia virus glycoprotein antibodies, for eradication of bovine leukemia virus from commercial dairy herds. Of nine participating herds, the prevalence rate of bovine leukemia virus infection was low (less than 10%) in three, medium (11-30%) in four and high (greater than 30%) in two. The herds were tested by the agar gel immunodiffusion test, reactors were removed and the herds were then retested at regular intervals. The results indicate that it is possible to eliminate bovine leukemia virus infection from the herds after two to three cycles of agar gel immunodiffusion tests and prompt removal of the reactors.     

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.     

Control of bovine leukemia virus infection in dairy herds by agar gel immunodiffusion test and segregation of reactors.

Canadian cattle intended for export, in future, may have to originate from herds which are serologically negative for bovine leukemia virus, in addition to being negative individually by the agar gel immunodiffusion test as currently required. In this study, agar gel immunodiffusion testing of herds and segregation of reactors were examined. The results demonstrated that bovine leukemia virus infection could be controlled when three groups: 1) bovine leukemia virus-positive, 2) bovine leukemia virus-negative and 3) replacement cattle were maintained at separate locations.     

Enzyme-linked immunosorbent assay for diagnosis of equine infectious anemia

An enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of specific antibody to equine infectious anemia (EIA) antigen. Sera from horses experimentally infected with EIA virus were assayed by ELISA, complement fixation (CF) and immunodiffusion (ID) tests for antibody to EIA antigen. The ELISA technique was found to be much more sensitive than CF and ID tests. In addition, EIA specific antibody could be detected by ELISA at an earlier stage of infection than by CF or ID techniques. The applicability of the technique to diagnosis of EIA is discussed.     

Diagnosis of enzootic bovine leukosis: a comparison of haematological and immunodiffusion tests.

A total of 657 cattle from three different herds were tested for the presence of specific antibodies against EBL antigens by means of the immunodiffusion test and the results compared with the classification according to the lymphocyte counts. All animals with pathologically raised lymphocyte counts showed a positive immunodiffusion test reaction. In the majority of these both antigens, 'p24' and 'gp', gave positive results; in a smaller number antibodies against the gp antigen only were detected, but never against the p24 antigen only. It is concluded that for field testing programmes the use of the gp antigen is sufficient. In future experiments, however, both antigens should be employed, because the lack of one of them might perhaps prove to be correlated with the future course of the disease. The absence of specific antibodies, together with a normal number of lymphocytes, in young animals should, however, not lead to the conclusion that they are free of the disease.     

The reasons for the occurrence of non-specific reactions in the immunodiffusion test for enzootic bovine leukosis

A batch of enzootic bovine leucosis antigen earmarked for immunodiffusion testing exhibited unspecific responses to cattle sera, which prompted an investigation of the underlying causes. Evidence was produced, for the first time ever, that such reactions were attributable to an antigen-antibody reaction caused by Mycoplasma arginini.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.