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.     

Survey for antibodies to leukemia (C-type) virus in cattle.

Serums from 4,394 dairy cattle in 100 herds and from 2,794 beef cattle in 50 herds were tested for antibody to the bovine (C-type) leukemia virus (BLV), using the agar gel immunodiffusion test. Reactors were found in 66% of the dairy herds (10.2% of the cattle) and in 14% of the beef herds (1.2% of the cattle). The prevalence of reactors was examined with respect to age, herd size, and sex. Few of the reactors were less than 2 years old. There was a high percentage of reactors in small dairy herds (less than 50 cattle). In 22 dairy herds (1,354 cows and 96 bulls), the rate of infection in cows was compared with that in bulls. In those herds, 13.5% of the cows and 10.4% of the bulls were reactors.     

Prevalence of bovine leukemia virus antibody in seven herds of Holstein-Friesian cattle

Sera from 959 Holstein-Friesian cattle in 7 herds were tested for antibody to bovine leukemia virus, using the agar gel immunodiffusion test with glycoprotein antigen. Seropositive cattle were found in 6 herds; 120 (12.5%) were reactors. In 1 of the herds, the prevalence of seropositive cattle was significantly greater in purebred than in grade cows. Purebred cows raised on the premises and an overall antibody prevalence similar to purchased purebred cows. Purebred cattle more than 2 years old had a significantly greater prevalence of reactors than younger cattle. There was no difference in prevalence by sex.     

Establishment of a bovine leukosis virus-free dairy herd

A large dairy herd was established free of bovine leukosis virus (BLV) infection at the US Dairy Forage Research Center, Madison, Wis. Cattle introduced into the herd originated from BLV-infected herds, but only those negative for BLV antibodies by an agar gel immunodiffusion test were accepted there. Cattle that were found to be seropositive after their arrival at the new facility were promptly removed. Embryo transfer and artificial insemination were used to introduce new genetic stock into the herd. All recipients receiving embryos from BLV-positive donors and the 30 calves born from the successful transfers were seronegative at 21 months for BLV antibodies. Thus, under these conditions, embryo transfer and artificial insemination did not spread BLV. The agar gel immunodiffusion test was effective in screening cattle for BLV antibodies.     

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.     

Serological detection of multiple retroviral infections in cattle: bovine leukemia virus, bovine syncytial virus and bovine visna virus

Individual experimental animals used in our studies on bovine leukemia virus (BLV) are routinely screened for the presence of antibodies to the three bovine lymphotropic retroviruses. We utilized these screening methods to examine frozen sera from eight herds for antibodies to BLV, bovine visna virus (BVV) and bovine syncytial virus (BSV). Serum samples from 235 animals in four dairy and four beef herds were analyzed. Detection methods used included indirect fluorescent antibody tests of virus-infected cell cultures (BLV, BSV, BVV) and agar gel immunodiffusion (BLV). Sera from the BLV-infected animals in the dairy herds showed the highest single (50%, 49/97) and multiple (30%, 29/97) infections compared with 5% (7/138) and less than 1% (1/138), respectively in the beef herds. Single BVV infections were not detected in the dairy herds, but 11% (11/97) of the sera contained antibodies to BVV plus BLV or BSV. Five sera from beef cattle had antibodies only to BVV and four were obtained from one herd. Only one beef serum of the 138 tested demonstrated multiple antibodies (BLV, BVV).     

Sporadic Bovine Leukosis: A Description of Eight Calves Received at Animal Diseases Research Institute from 1974-1980

Eight calves with sporadic bovine leukosis are described. The common features were generalized lymphadenopathy, visceral involvement and raised total leukocyte and lymphocyte counts. Agar gel immunodiffusion tests for bovine leukemia virus antibodies were negative in eight animals and in all animals from three herds of origin. Lymphocytic nuclear pockets were found in the tissues of one calf but attempts to isolate bovine leukemia virus from two animals were unsuccessful.     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.     

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.     

Bovine leukemia virus infection in Taiwan: epidemiological study.

We conducted a seroepidemiological survey for antibodies to bovine leukemia virus (BLV), by using agar gel immunodiffusion technique, in dairy cows, water buffaloes, and yellow cattle throughout Taiwan. The positive reactors were 8.4% (376/4,459) in 1985 and 5.8% (1,277/22,190) in 1986, in 15 prefectures and 7 cities. Relatively high infection rate appeared in the northern and southern areas of Taiwan. Positive reactors increased gradually with age. The incidence of positive antibodies was 2 to 3 times higher in pasture-style farms than in housed-style farms. Among the 6,313 imported cattle, 302 (4.8%) showed positive reaction. Between 1985 and 1987, 5 cattle showed enzootic bovine leukosis among 351 sero-positive reactors in four highly positive prefectures. Survey of 134 water buffaloes and yellow cattle showed no positive reactors. This survey demonstrated that BLV-infection has increased over the years and spread throughout Taiwan.     

Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis

Contamination of a batch of tick fever (babesiosis and anaplasmosis) vaccine with bovine leucosis virus (BLV) was detected when a herd, in the final stages of an enzootic bovine leucosis (EBL) accreditation program, developed a large number of seropositive cattle following use of tick fever vaccine. Investigations incriminated a single calf used to produce Anaplasma centrale vaccine from which 13,959 doses were distributed. The failure of this calf to give a positive agar gel immunodiffusion (AGID) test before use was not fully explained. A total of 22,627 cattle from 111 herds receiving contaminated vaccine was tested to validate claims for compensation. Results showed infection rates of 62% and 51.8% in vaccinated dairy and beef cattle, respectively, compared with 6.1% and 1.5% in non-vaccinated cattle in the same herds. The results also indicated that infection did not spread from vaccinated to non-vaccinated in-contact cattle. Heavy reliance is now placed on purchase of calves for vaccine production from EBL accredited-free herds and on transmission tests from the calves to sheep to prevent a recurrence of contamination. The need for a BLV antigen detection test, with the sensitivity of the sheep transmission test but simpler and faster to perform, is evident.     

The role of virus dose in experimental bovine leukemia virus infection in sheep.

Twenty-four, six month old lambs were assembled into four groups of five animals each and one group of four animals. All groups were inoculated with lymphocytes from a single donor lamb infected with bovine leukemia virus. The inoculum varied from 250 to 250,000 lymphocytes, in tenfold increments. Animals were exposed by intradermal injection in the neck region immediately anterior to the left shoulder joint. All groups were monitored at 0, 3, 7 and 12 weeks after inoculation using the following procedures: a. Syncytia induction assay for detection of bovine leukemia virus in peripheral blood lymphocytes. b. Agar gel immunodiffusion against the gp51 antigen of bovine leukemia virus for the detection of antibovine leukemia virus gp51 antibody. c. Lymphocyte stimulation test for the assessment of cell-mediated immunity using mitogen, nonfractionated bovine leukemia virus antigen, and partially purified bovine lymphoma tumor-associated antigen for the in vitro activation of lymphocytes from bovine leukemia virus-inoculated and sham-inoculated, control animals. d. Routine hematological techniques for the assessment of total leukocyte and lymphocyte counts. The median infectious dose for lymphocytes from the single bovine leukemia virus-infected donor used in this study was determined to be 2000 cells. The syncytia induction assay detected more infected individuals (13/23) at an earlier time than did the agar gel immunodiffusion assay (10/23). Using either serological or virus isolation techniques, infected animals were first detected at three weeks postinoculation in the group receiving the high-dose inoculum and at seven weeks postinoculation in groups receiving low- or medium-dose inocula.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of the sensitivity and specificity of the agar gel immunodiffusion test for bovine leukemia virus: 1,296 cases (1982-1989).

A retrospective study of the results of 12,549 agar gel immunodiffusion tests for bovine leukemia virus, conducted on 1,296 dairy bulls over an 8-year period, was performed to estimate the sensitivity and specificity of the test. The number of tests performed on each bull ranged from 5 to 35, with a mean of 9.7 tests per bull. Bulls were categorized by their agar gel immunodiffusion test responses; 1,069 (82.5%) were noninfected and 227 (17.5%) were infected. Eighteen false-positive results were reported from the noninfected bulls. Test specificity was estimated to be 99.8%. Thirty-one false-negative results were reported from the infected bulls. Test sensitivity was estimated to be 98.5%. Fifty-six bulls had 1 or more positive responses when less than 6 months old. In 26 (46%), these results were thought to be attributable to colostral immunity.     

Evidence of bovine immunodeficiency virus in cattle in Turkey

A seroepidemiological study of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infections was conducted in four different cattle herds in Turkey. A total of 300 blood samples were analyzed and 12.3% were found to be positive for anti-BIV p26 antibodies by Western blot analysis and 1.6% positive for anti-BLV gp51 antibodies by an immunodiffusion test. BIV infection was confirmed with the detection of BIV-provirus DNA using the nested polymerase chain reaction. This is the first evidence for the presence of BIV in cattle in Turkey.     

Prevalence of bovine leukemia virus infection in Florida

A serologic study was undertaken to determine the prevalence of bovine leukemia virus (BLV) infection in dairy and beef cattle in Florida. Using the agar gel immunodiffusion test with a glycoprotein antigen 47.8% of 7,768 dairy cattle and 6.7% of 4,911 beef cattle were found to have antibodies to BLV. The prevalence of BLV antibodies increased significantly (P less than 0.0001) with increasing age. After data were adjusted for age, prevalence of BLV antibodies was significantly associated with dairy breed (P less than 0.05) but not with species (Bos taurus and B indicus) or sex.     

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.     

Seroprevalence of caprine arthritis-encephalitis virus in goats in the Cariri region, Paraiba state, Brazil

The seroprevalence of caprine arthritis–encephalitis virus (CAEV) in the Cariri Region of Paraíba State, Brazil, was determined in 60 goat herds using the agar gel immunodiffusion test. The overall seroprevalence was 8.2%, with seropositivity in 21/60 (35%) herds and 13/15 (86.6%) municipalities. Bucks had a significantly higher frequency of infection (28.3%) than does (5.9%), and bucks that originated in other states had a significantly higher frequency of infection (76.5%) than those from Paraíba State (9.3%).     

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.     

Evidence of bovine immunodeficiency virus (BIV) infection: serological survey in Argentina

This is the first report of serological evidence for bovine immunodeficiency virus (BIV) infection in Argentina. The analysis was performed in 589 dairy bovine sera samples, applying indirect enzyme-linked immunosorbent assay (I-ELISA) using a synthetic antigen (transmembrane peptide, TM) and Immunofluorescent assay (IFA). In this study, 9 dairy herds from 4 Argentinian provinces were evaluated and 12% of the animals tested positive for BIV. Seven of the 9 herds tested were BIV seropositive and the percentage of BIV seropositive animals in the herds ranged from 2% to 42%. Direct detection of BIV provirus applying nested PCR was not conclusive. Antibody detection against bovine leukemia virus (BLV) in all sera was also performed applying immunodiffusion (ID) assay and 59% resulted seropositive. Statistical analysis of the results was carried out and possible evidence of association between BIV and BLV infection was considered. Future studies should be performed including local field isolates strains of BIV.     

Eradication of bovine leukemia virus infection from a high-prevalence herd, using radioimmunoassay for identification of infected animals

Radioimmunoassay (RIA), using the virion glycoprotein antigen, was applied in an attempt to eradicate bovine leukemia virus (BLV) infection from a herd in which virtually all the adult cattle are infected. Considering that most calves born to BLV-infected cows are negative for BLV at birth and remain negative for the first several months of life, the eradication program was based on the identification and isolation of the BLV-free calves born to infected cows. Twenty-five calves raised on colostrum and milk from their infected dams were classified as BLV-free on the basis of negative results in the RIA at 6 to 8 and 9 to 11 months of age. These animals were maintained in either complete (10 calves) or partial (15 calves) isolation from infected cattle and were examined at regular intervals for BLV and BLV antibodies. With the exception of 1 calf in the group raised in partial isolation, the animals have remained free of BLV up to the time of the last evaluation, when they were 32 to 35 months old. At these ages, more than 90% of the nonisolated cattle in the herd are BLV-positive. The data also show that this eradication trial would have failed if, in the initial procedure used to classify the calves as BLV-free, the agar gel immunodiffusion test instead of the RIA had been used. Inasmuch as the 25 calves in this study were fed colostrum and milk from their dams, the fact that only 1 of the calves became infected during the 26 to 29 months of observation provides further evidence that milk-borne transmission of BLV is infrequent and perhaps inconsequential.