Effect of pasteurization and heat treatment on bovine leukemia virus.

Four sheep inoculated with mixtures of bovine leukemia virus (BLV) and milk that was treated by a simulated high-temperature short-time pasteurization procedure did not become infected with BLV or develop tumor. Four sheep inoculated with unpasteurized BLV-mil mixtures became infected with BLV and 3 died with tumor at 15, 21, and 27 months, respectively, after inoculation. Fluid from a BLV-infected cell culture was heated to 56, 60, 65, and 73 C for 1/2 minute and 1 minute (in separate trials) and transferred to noninfected cells. Culture fluid heated to 60 C and higher did not infect the cells.     

Detection of bovine leukemia virus in blood and milk by nested and real-time polymerase chain reactions.

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle (n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle (n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle (n = 21), most likely because of early detection before seroconversion.     

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.     

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

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.     

Effects of bovine leukemia virus infection on production and reproduction in dairy cattle.

The purpose of this study was to determine the effects of bovine leukemia virus (BLV) infection on production, reproduction and longevity in dairy cattle. The study population was a commercial Holstein dairy herd of approximately 400 milking cows. Cattle were tested for antibodies to BLV at least annually for three years and when culled. Four groups of culled cows were compared: seronegative cows (n = 79), seropositive cows without lymphocytosis (n = 176), seropositive cows with lymphocytosis (> or = 9,000 lymphocytes/microliter) (n = 74), and seropositive cows with lymphosarcoma (n = 29). Seropositive groups of cows were bred more times and had longer calving intervals than seronegative cows. The seropositive groups had greater 305-day ME (mature equivalent) FCM (3.5% fat-corrected milk) per lactation and were older when culled than seronegative cows. However, the percent fat per lactation was greater in seronegative cows. In the last complete lactation, differences in 305-day ME FCM, days open and cull age between groups were reduced and none were significant (p > 0.05). In the cull lactation, only cows with lymphocytosis had reduced milk production relative to seronegative cows, although this difference was not significant. After adjustment for initial production and reproductive values, only seropositive nonlymphocytotic cows were culled at a significantly older age than seronegative cattle. Lymphocytotic cows were culled four months younger on average than nonlymphocytotic seropositive cows. Hence, BLV infected cows had greater milk production on average than uninfected cows. Adverse effects of BLV infection were primarily limited to lymphocytotic cows which were culled earlier and had reduced milk production in the cull lactation.     

Further phenotypic characterization of target cells for bovine leukemia virus experimental infection in sheep

To determine the phenotype of target cells for bovine leukemia virus (BLV) infection in sheep, we analyzed blood lymphocytes from BLV-infected clinically healthy and leukemic sheep by use of monoclonal antibodies. In clinically healthy and leukemic sheep that were BLV-infected, the blood concentration of T lymphocytes was within normal values, but the number of B lymphocytes was increased in several cases. In addition, the number of blood lymphocytes expressing the BLV antigen correlated well with that of B lymphocytes. Double immunofluorescence staining demonstrated that lymphocytes expressing BLV antigens bore B-cell but not T-cell surface markers. Moreover, neoplastic cells in the lymph nodes of leukemic sheep were stained immunohistochemically with an anti-B monoclonal antibody but not with any of anti-T monoclonal antibody tested, indicating that tumor cells are of B-lymphocyte origin. Collectively, these results show that BLV antigen-positive cells obtained from BLV-infected sheep that have no clinical signs and BLV-induced lymphosarcoma cells belong to the B-lymphocyte lineage.     

Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds.

Vertical transmission of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) was investigated in five dairy cattle herds in Hokkaido, where 36.1 and 17.0% of cattle were BLV and BIV seropositive, respectively, and 9.9% of dams were co-infected with both BIV and BLV. Twenty six cases of offspring born from dams infected with only BLV (17 cases) or with both BIV and BLV (9 cases) were examined for the presence of BLV and BIV before and after colostrum feeding by polymerase chain reaction (PCR) and syncytium assay. After birth, all calves were separated immediately from their dams. The offspring born from BLV-positive dams were BLV-negative before colostrum feeding, suggesting that no transplacental transmission had occurred. Thereafter, these offspring were fed colostrum or milk from their dams, but still remained BLV-negative. The other offspring born from BLV-positive dams were fed with BLV-negative colostrum, or with pasteurized BLV-positive colostrum. All these calves remained negative for BLV infection, suggesting that in utero transmission of BLV is negligible. In the case of offspring born from dams co-infected with BLV and BIV, calves were BIV-positive before colostrum feeding at 1 day after the birth, indicating in utero transmission of BIV. After colostrum feeding from their dams, newborn calves became BLV-positive. In addition, one calf was BLV-positive even before colostrum feeding. These results suggest that BIV can be transmitted to offspring in utero, and that BLV can be transmitted through colostrum or milk if dams are infected with both BIV and BLV.     

Use of milk samples and monoclonal antibodies directed against BLV-p24 to identify cattle infected with bovine leukemia virus (BLV)

An indirect double-antibody sandwich (IDAS) enzyme-linked immunosorbent assay (ELISA) using milk samples was developed to identify cows infected with bovine leukemia virus (BLV). Two monoclonal antibodies (McAbs) were used. One, which was directed against BLV core protein p24, was used to coat ELISA plates; the other was used to prepare a horseradish peroxidase (HRP) conjugate directed against bovine immunoglobulin. The IDAS-ELISA detected antibodies directed against BLV-p24 in 97% of the milk samples collected from known seropositive cows identified by the agar gel precipitation test (AGTP). Even when milk samples were diluted 1:50, 93% of the seropositive cows were identified. Only 0.43% of the 4000 milk samples collected in The Netherlands reacted nonspecifically. Nonspecific binding disappeared, however, when these samples were diluted 50 times in BLV-negative milk. In a comparative evaluation of BLV test-kits in various European laboratories, our IDAS-ELISA using McAb directed against p24 was one of the most sensitive.     

To compare the incubation period following intratracheal and subcutaneous inoculation of bovine leukosis virus infected lymphocytes and to study their clearance from the circulation

The migration of fluorescein isothiocyanate labelled lymphocytes through the tracheobronchial mucosa has been studied in cattle. Following intratracheal inoculation of labelled non-infected autologous lymphocytes and bovine leukosis virus (BLV) infected heterologous (presumed allogeneic) lymphocytes, the labelled lymphocytes appeared in the blood circulation between 4 and 7 days post inoculation. Following intravenous inoculation of labelled autologous lymphocytes, the cells could be detected in the circulation for 10 days post inoculation whereas BLV infected and non-infected heterologous lymphocytes could be detected for only 2 days. The migration of BLV-infected heterologous lymphocytes through the tracheobronchial mucosa caused a delay in the appearance of labelled lymphocytes in the circulation and a corresponding delay in the appearance of BLV antibodies. Comparison was made of the effect of two different routes of inoculation, subcutaneous and intratracheal on the incubation period as indicated by the detection of antibody. Subcutaneous inoculation of 1 X 10(4), 5 X 10(3), 1 X 10(3) of lymphocytes from a BLV infected cow caused seroconversion whereas 5 X 10(2) cells did not. Intratracheal inoculation of 5 X 10(3) cells caused sero-conversion. One animal did not develop BLV antibody until 30 weeks after inoculation although BLV could be isolated from the blood at 24 and 26 weeks post inoculation.     

Associations between farm management practices, productivity, and bovine leukemia virus infection in Ontario dairy herds

The objective of this study was to investigate the associations between herd-level bovine leukemia virus (BLV) status and herd-level management and production variables. The study population consisted of 1330 cows sampled from 102 Ontario dairy herds. The individual-cow prevalence of BLV infection in the population (based on AGID testing) was 23%, with 69.6% of herds having one or more positive animals. The herd-level explanatory variables were divided into two datasets containing winter housing variables and all non-seasonal variables, and summer housing variables and all non-seasonal variables. In both datasets, multivariable analyses found a negative association between herd-level milk production and BLV status, and positive associations between weaning age and purchasing animals from outside sources, and BLV status. Housing pre-weaned calves in hutches or separate calf buildings in either season was associated with an increased risk of BLV. The model containing winter housing variables also included positive associations between contact with older animals and BLV status, and between BLV status and the facilities used to house dry cows in the winter.     

Bovine leukemia virus-associated antigens in lymphocyte cultures.

Short-term lymphocyte cultures from bovine leukemia virus (BLV)-infected cattle were tested for BLV-associated antigens at various times after incubation. Several immunologic methods were used, including fluorescent antibody tests, immunodiffusion, and radial immunodiffusion. Antigens were not detected in uncultured lymphocytes. The BLV-associated antigens were detected as early as 3 hours, with maximum antigen production occurring at 18 to 24 hours after incubation. These results indicate that culturing of lymphocytes in vitro is necessary for the expression of the virus.     

A case of an embryo transfer calf infected with bovine leukemia virus from the recipient cow

A case was discovered where the embryo transfer (ET) calf had been infected with bovine leukemia virus (BLV) from the recipient cow. The embryo was transferred from the BLV-uninfected donor cow to the recipient cow. However, the BLV test had not been performed to the recipient cow before ET was performed. The ET calf was raised in a calf hatch from birth to 1-month old and was given the recipient cow's colostrum and milk artificially. The ET calf was raised with the two other calves from a 1-month old to a 6-month old. The BLV test was performed to the ET calf by agar gel precipitation (AGP) and passive haemagglutination (PHA) assay when the ET calf was 6 months old. Because the ET calf was positive, the BLV test was performed to the recipient cow, the two other calves raised with the ET calf and the two dams of the two other calves. Because the recipient cow only was positive at the time of the first test, we judged that the ET calf had been infected with BLV from the recipient cow. The importance of the BLV test being carried out on the recipient cow for the prevention of enzootic bovine leukemia in a case of ET was recognised.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

Enumeration of T and B lymphocytes in bovine leukemia virus-infected cattle, using monoclonal antibodies

Monoclonal antibodies and microfluorimetry were used to determine the absolute number of B and T lymphocytes in the blood of bovine leukemia virus (BLV)-infected cows. The blood lymphocyte populations from BLV-infected cows were significantly higher than those from BLV-negative cows. The increase in the lymphocyte population in 3 BLV-infected nonlymphocytotic cows was attributed to a significant increase in the number of T lymphocytes; in 3 BLV-infected persistently lymphocytotic cows, the increase was attributed to a significant increase in the number of B and T lymphocytes. One persistently lymphocytotic cow had a high lymphocyte count, and lymphocytes from this cow contained cells that appeared to stain with markers specific for bovine B and T lymphocytes. We concluded that infection of cattle with the B-cell lymphotropic retrovirus, BLV, not only affected B cells, but also T cells.     

An attempt to protect calves against experimental bovine leukosis using allografts

Six calves sensitised by implanting skin from a calf were later inoculated with lymphocytes from the same calf after the calf had been infected with bovine leukosis virus (BLV). Two out of 6 calves challenged did not develop BLV antibodies and BLV was not isolated from these animals, whereas all of the 5 control calves became infected with BLV.     

In situ hybridization for the demonstration of bovine leukemia virus transcripts in lymphosarcoma cells using biotinylated probes.

Bovine leukemia virus (BLV) RNA was demonstrated in peripheral blood lymphocytes treated with concanavalin A or phytohemagglutinin from a BLV-infected ox, and in lymphosarcoma cells cultured without mitogen from an enzootic bovine leukosis cow by in situ hybridization using biotinylated DNA probes.