Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.     

Decay of colostral antibodies to bovine leukemia virus with application to detection of calfhood infection

An estimated weighted-regression method was used to describe the decay of colostral bovine leukemia virus (BLV) antibodies in the calf, as measured by agar-gel immunodiffusion with glycoprotein antigen. The prediction equation, based on 473 observations from 130 animals, was log10 inverse titer = 1.29 -0.012 age (days). The half-life of BLV antibodies was estimated to be 25.8 days. Ages at which colostral antibodies were last detected were between 51 and 187 days. Normal limits of antibody decay were estimated and used to identify virus-induced active antibodies in calves during the colostral antibody period. Calves known to be infected were identified between 2 and 180 days of age, using 95% limits. Application of this procedure for the early serologic detection of BLV-infected calves in eradication or control programs is discussed.     

Bovine leukemia virus: Duration of BLV colostral antibodies in calves from commercial dairy herds

A study was conducted to determine the duration of colostral antibodies to bovine leukemia virus (BLV) in diary calves from commercial dairy farms. Sera of pregnant dams from four different dairy farms and sera of the calves of these dams were analyzed for BLV antibodies using the agar gel immunodiffusion (AGID) test. Precolostral serum samples were collected from the female calves of known BLV serologically positive dams. Postcolostral serum samples of the same calves were collected on Day 2 and biweekly until 2 consecutively negative AGID tests performed 4 weeks apart were obtained. Subsequently, the biweekly serum samples from each calf were analyzed quantitatively for BLV antibodies with the AGID test. End-point titers were determined using phosphate buffered saline to make two-fold dilutions. A logarithmic transformation of the inverse of the end-point titer was used to determine the regression line of antibody decay for each calf. Estimated weighted regression analysis was used to determine the least-squares regression line for 27 of the 38 calves sampled. The duration of colostral antibodies was calculated as 71 days using the prediction equation. The minium and maximum durations of colostral antibodies were 14 days and 147 days, respectively. The half-life of the antibodies was 36.05 days. Factors affecting the duration of BLV colostral antibodies and the practical applicability of this study were discussed.     

Protection of colostral antibodies against bovine leukemia virus infection in calves on a California dairy.

A three-year prospective study involving 244 calves was undertaken on a California dairy to evaluate the protective role of colostral antibodies against bovine leukemia virus (BLV) infection in calves. Calves were followed from birth to the time they left their individual hutch (TLIH), at about 90 days of age. The probability of being infected at TLIH and the daily risk of infection between birth and TLIH were modelled using the logistic and the Cox models, respectively. Calves with no detectable antibodies during the first week of life were up to 2.00 and 2.75 times more likely to be infected at TLIH compared to calves with low and high concentrations of antibodies during the first week of life, respectively (p = 0.01). When the daily risk was modelled, calves without antibodies at the estimated day of infection were up to 3.4 and 11.6 times more likely to become infected than calves with low and high concentrations of antibodies on that day, respectively (p less than 0.001). Results indicated that calfhood infection may be reduced by about 45% through the feeding of colostrum with BLV antibodies. Further reduction in infection may be possible by feeding calves milk powder, milk replacer, and/or milk from noninfected cows. Results also indicated that quantification of the effect of a time-dependent risk factor, such as colostral antibody concentration, might be affected if treated as a fixed factor.     

Radioimmunologic detection of antibodies to bovine leukemia virus

The radioimmunologic assay (RIA) was elaborated for a demonstration of serum antibodies to bovine leukosis virus. The procedure makes use of the viral antigen bond to the fixed phase of a polystyrene carrier. The method was compared with the ELISA method and pseudoneutralizing and immunodiffusion tests. High congruence of the results of the RIA and ELISA methods was achieved, making 95%. The RIA method is more sensitive than the immunodiffusion test.     

Effect of colostral antibody on bovine leukemia virus infection of neonatal calves

Pairs of newborn calves were exposed to bovine leukemia virus (BLV) when they were given their 1st colostrum feeding. Calves that were given 10(6) BLV-infected lymphocytes in colostrum free of BLV-specific antibody became infected. Calves that were fed 10(7), 10(8), or 10(9) infected lymphocytes in colostrum that contained BLV-specific antibody did not become infected. One of 2 calves inoculated intradermally with 250,000 infected lymphocytes was protected by colostral antibody, but the other was not. Colostral antibody titers in the unprotected calf decreased normally until the calf was 4 months old and then increased markedly; this pattern indicates that the presence of colostral antibody may have prolonged the latent period of the BLV infection.     

Detection of bovine leukemia virus antibodies using the cytotoxicity test in comparison with other serologic methods

In ninety-five serum samples taken in a herd of five-year to seven-year cattle that was heavily infected by bovine leukosis virus, the four serological assays were used for demonstration of the antibodies to bovine leukosis virus; cytotoxic test, immunodiffusion test in agar-agar, immunoenzymatic test and serum neutralizing test. The serum neutralizing test was found to be the most sensitive: further seven positive reagents were diagnosed in comparison with immunoenzymatic test; cytotoxic and immunodiffusion tests in agar-agar have the lowest sensitivity and the results of these tests are almost identical. It was found out in forty titrated samples that serum neutralizing test was by as much as 20 times more sensitive than immunoenzymatic test, the latter being about 50 times more sensitive than cytotoxic and immunodiffusion tests.     

An improved immunodiffusion test for antibodies to bovine leukemia virus antigens

A new immunodiffusion (ID) test for antibodies to bovine leukemia virus (BLV) antigens was established. This test, refered to as the tannic acid enhanced, indirect, double immunodiffusion (IID-T) test, includes the following steps: (a) double micro-immunodiffusion using diluted references reagents, (b) treatment of the gel plate with antiglobulin serum, (c) treatment of the gel plate with 1% tannic acid. The IID-T test has proved to be eight times more sensitive than the double immunodiffusion test (ID) commonly used for anti-BLV. Diagnostic efficiency at individual levels of the IID-T test for anti-gpBLV is higher than this parameter of the ID test for anti-gpBLV and radioimmunoassay (RIA) for anti-p24BLV. The IID-T test is simple, reproducible, and more economic than the ID test in the amount of the reference reagents required. The IID-T test is more reliable than the ID test especially when weak, low titer sera are tested. Thus, the IID-T test seems to be suitable for large scale serodiagnosis of BLV infection in cattle.     

Duration of active and colostrum-derived passive antibodies to bovine viral diarrhea virus in calves.

Duration of active and colostrum-derived passive antibodies to bovine viral diarrhea virus was studied in 14 calves. Five calves born with actively induced antibodies to bovine viral diarrhea virus retained high titers during the year of observation. Colostrum-derived antibodies to bovine viral diarrhea virus in nine calves declined at an expected rate for the first four to six months of age. However, titers of six of these calves increased at five to eight months of age and either remained constant or increased through one year of age. Bovine viral diarrhea virus antibody titers of the other three calves declined at a constant rate to less than 1:4 by nine to 12 months of age.     

A simple, rapid syncytial-inhibition test for antibodies to bovine leukemia virus.

Cocultivation of equal numbers of cells from a fetal lamb kidney line infected with bovine leukemia virus and African green monkey (Vero) cells results in the rapid production of syncytia. The effect was blocked or inhibited by serum containing antibodies to bovine leukemia virus. A serological test based on syncytial inhibition was compared to the agar gel immunodiffusion test and the modified direct complement fixation test for the detection of bovine leukemia virus antibodies in sera from leukosis-free cattle, cases of adult enzootic bovine lymphosarcoma and cattle from herds in contact with enzootic lymphosarcoma. The results showed the syncytial inhibition test to react positively with sera from all cases of adult enzootic lymphosarcoma, but to be much less sensitive than the other tests in detecting bovine leukemia virus antibodies in sera of exposed animals.     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

Transmission of bovine leukemia virus by Tabanus fuscicostatus

Bovine leukemia virus (BLV) was transmitted by horse flies, Tabanus fuscicostatus, from a cow with a lymphocyte count of 31,500/mm3 to goats and dairy calves. As few as 10 and 20 flies transmitted BLV to goats and calves respectively, but the minimal number of flies required to transmit the infection was not established. Groups of 150 and 100 T fuscicostatus transmitted BLV to beef calves from a cow with a lymphocyte count of 14,600/mm3. These results support a role for horse flies in the horizontal transmission of BLV.     

Use of a continuous feline cell line for virologic and serologic investigations of bovine leukemia virus infections

Simple, practical, reproductible methods for the detection of bovine leukemia virus (BLV) in peripheral blood leukocytes and BLV-neutralizing antibodies in bovine sera are described. Virus detection is based on syncytium formation in transformed feline (F-81) cells. The use of multiwell culture plates provides practical benefits in testing large numbers of samples. Results obtained in 2 farm herds show that the agreement of serologic and virologic test results may vary considerably from herd to herd.     

A serologic survey of Oklahoma cats for antibodies to feline immunodeficiency virus, coronavirus, and Toxoplasma gondii and for antigen to feline leukemia virus

A serologic survey was done on 618 cat sera submitted to the Oklahoma Animal Disease Diagnostic Laboratory between July 1, 1987 and June 30, 1988. The samples were collected from clinically normal and sick cats. The sera were tested for the presence of antibodies to feline immunodeficiency virus by a commercial immunoassay, to a coronavirus by an indirect fluorescent antibody test, and to Toxoplasma gondii by a commercial latex agglutination test and for the presence of feline leukemia virus antigen with one of 3 different commercial assay kits. Ten percent of the sera had antibodies to feline immunodeficiency virus, 35% had antibodies to a coronavirus, and 22% had antibodies to Toxoplasma gondii. Feline leukemia virus antigen was detected in 15% of the sera. Thirty-two percent of the sera had evidence of exposure to 2 or more of the agents.