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.     

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three calves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.     

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.     

Bovine leukosis virus transmission with mouthparts from Tabanus abactor after interrupted feeding

A successful attempt was made to mechanically transmit bovine leukosis virus (BLV) from a BLV-infected cow with a normal lymphocyte count to sheep by inoculation with horse fly (Tabanus abactor) mouthparts. After interrupted natural feeding, horse flies were anesthetized with CO2. Mouthparts were severed and pooled into a tissue grinder containing medium. Five inocula containing the mouthparts of 10 flies each, and 5 inocula containing the mouthparts of 20 flies each, were prepared and inoculated SC in the right axilla of 10 BLV antibody-negative sheep. Five additional sheep served as controls. Serum samples were collected at 2-week intervals and tested by agar gel immunodiffusion for BLV antibodies. One sheep injected with 20 mouthparts developed antibodies to BLV at 10 weeks after inoculation. Six months after inoculation with fly mouthparts, 1 BLV antibody-negative sheep was randomly selected from each treatment group and injected, in the left axilla, with 3 ml of blood from the donor cow to confirm susceptibility of the sheep. All 3 sheep developed antibodies to BLV within 4 weeks.     

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.     

Detection of enzootic bovine leukemia virus using the syncytium test

BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method.     

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).     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by mosquitoes

Bovine leukosis virus (BLV) was transmitted to sheep in a simulated mechanical transmission experiment, using the following species of mosquitoes; Anopheles freeborni, A stephensi, A quadrimaculatus, and A albimanus. Mosquitoes were fed on blood taken from a BLV-infected cow with persistent lymphocytosis. Mouthparts and heads of mosquitoes were removed immediately after feeding, placed in RPMI 1640 medium, and inoculated subcutaneously into sheep. Nine sheep were inoculated with mouthparts and heads from 37 to 122 mosquitoes. Infection was determined serologically. Three monthly serum samples were collected from the sheep and were tested for the presence of antibodies to BLV, using the agar-gel immunodiffusion (AGID) test. Sera that were negative by AGID at 3 months were tested by radioimmunoassay. Results from radioimmunoassay agreed with those obtained by AGID. Four of the 9 sheep developed antibody to BLV. Sheep that seroconverted were inoculated with mouthparts and heads from as few as 54 mosquitoes.     

Tumor-associated antigen on bovine leukemia virus-induced bovine lymphosarcoma

Specific tumor-associated antigen (TAA) was detected on enzootic bovine leukosis (EBL) cells by monoclonal antibodies against TAA. One of the monoclonal antibodies, c143, reacted with all EBL tumor cells tested but not with bovine leukemia virus (BLV) antigens. c143 reacted slightly with bovine fetal thymus and mitogen-stimulated lymphocytes from BLV-free cows but not with normal bovine lymphoid cells. TAA may be a good tumor marker of EBL tumor cells. We sacrificed eight TAA-positive but clinically normal animals and examined them in order to elucidate whether or not they had gross or histological tumors. At necropsy, four animals had tumors macroscopically. Three animals had no tumors histologically but had initial lesions showing follicular hyperplasia and the TAA on affected lymph nodes. The one remaining showed medullary hyperplasia in the spleen but there were no findings of tumors. Thus, c143 is a useful tool not only for diagnosing EBL, but also for screening of BLV-infected cattle with potential to develop tumors in the future.     

Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction.

The early diagnosis of bovine leukosis virus (BLV) infection, the aetiological agent in enzootic bovine leukosis, is important for the implementation of control measures. BLV infection is currently assessed by the detection of circulating antibodies against the viral envelope protein, gp51. However, this approach has shortcomings in the time taken to detect anti-BLV antibodies (three to four weeks after infection), and in the failure to detect antibodies in some animals. Clearly a technique such as the polymerase chain reaction (PCR), which directly detects the presence of viral DNA, has advantages over methods designed to measure host antibodies. The use of PCR for the detection of proviral DNA in an affected DNA sample with as little as 10(-5) micrograms of host DNA using agarose gel electrophoresis followed by ethidium bromide staining is described here. It was possible to improve the sensitivity of this assay by using hybridisation analysis with a BLV gene probe. PCR used in combination with hybridisation analysis will provide a sensitive diagnostic assay to detect BLV when antibody tests give weakly positive or equivocal results.     

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.     

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.     

Observations on blood leucocytes and lymphocyte subsets in sheep infected with bovine leukaemia virus: a progressive study

Haematological parameters and reactivity of lymphocyte antigens to monoclonal antibodies were studied over a 10-month period in sheep experimentally infected with bovine leukaemia virus (BLV). BLV-inoculated animals seroconverted within 1 month and showed a significant lymphocytosis 2-6 weeks after infection. Control animals inoculated with BLV-free lymphocytes showed a stronger and more immediate neutrophil response than those inoculated with BLV-positive lymphocytes. One month after infection, BLV-inoculated sheep showed a relative increase of cells bearing antigens T4, T6, T8 and T19, and 10 months into the trial, MHC II lymphocytes increased, T6 remained elevated, but T4 helper cells were significantly decreased in number. Lymphoma tissue showed the presence of T8 cells, and lymph nodes from seroconverted sheep had areas of concentrated T4 staining cells. These results demonstrate responses in cellular immune mechanisms to infection with BLV.     

Detection of bovine leukosis virus in bronchoalveolar lung washings and nasal secretions

Cattle and sheep persistently infected with bovine leukosis virus (BLV) were studied for the presence of the virus in bronchoalveolar lung washings and nasal secretions. The virus was demonstrated in the cellular fraction of the lung washings in six out of nine cattle and in one out of six sheep. In no instance was bovine leukosis isolated from the cell-free bronchoalveolar lung washings. The virus was isolated from the nasal secretion of only one of six naturally infected milking cows despite frequent sampling; the virus-infected nasal secretion was from a sick 10-year-old cow. Bovine leukosis virus was not isolated from cellular fractions of nasal secretions.     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by stable flies, horn flies, and tabanids

The ability of stable flies (Stomoxys calcitrans), horn flies (Haematobia irritans), and tabanids (Diptera: Tabanidae) to transmit bovine leukosis virus (BLV) was investigated. Stable flies and horn flies were fed on blood collected from an infected cow, and the flies' mouthparts were immediately removed, placed in RPMI-1640 medium, ground, and inoculated into sheep and calves. Infection of sheep occurred with mouthparts from as few as 25 stable flies or 25 horn flies. However, sheep were not infected when removal of stable fly mouthparts was delayed greater than or equal to 1 hour after blood feeding. Infection of calves occurred after inoculation of mouthparts removed immediately after feeding from as few as 50 stable flies or 100 horn flies. Infected blood, applied by capillary action to the mouthparts (labella) of 15 deer flies (Chrysops sp) and a single horse fly (Tabanus atratus) caused infection in each of 2 sheep. Infection did not occur in 2 calves inoculated daily for 5 days with mouthparts from 50 horn flies collected after feeding on a BLV-infected steer. Four calves receiving bites from 75 stable flies interrupted from feeding on a BLV-positive cow also were not infected. Seronegative cattle held for 1 to 4 months in a screened enclosure with positive cattle in the presence of biting flies were not infected with BLV. The feeding behavior of each insect is discussed to assess their potential as vectors of BLV.     

Studies on Bovine Leukosis

Summary

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT‐BLV gp) were further used to test 633 bovine sera for antibodies to BL V. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGIDT‐BLV gp. On the other hand 11 sera were scored negative in the AGIDT‐BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (I) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.     

Properties of nine continuous B-cell lines established from enzootic bovine leukosis tumors.

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.     

The zoonotic potential of bovine leukemia virus

Many workers have investigated the possibility that bovine leukemia virus (BLV) might be transmissible to man. The epidemiological studies were designed to examine for associations between human leukemia and a rural environment, cattle farming, veterinary activities, or bovine leukosis. The serological studies were used to test serum samples from human cancer patients and from persons with potential occupational exposure to BLV, among others, for evidence of BLV antibodies. All these studies are critically reviewed. It is concluded that there is no epidemiological or serological evidence from human studies to indicate that BLV can infect man.     

Protective effects of vaccination with bovine leukemia virus (BLV) Tax DNA against BLV infection in sheep

A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.