Invited review: Bovine leukemia virus-Transmission, control, and eradication.

Bovine leukemia virus (BLV) infection, endemic in North American dairy herds, has production-limiting effects. A literature review of available papers published since 1995 concerning BLV transmission and its control was conducted. Although confirmed transmission routes were reviewed (blood, natural breeding, in utero, colostrum, and milk), there is still a lack of detailed information on other specific risks for transmission (e.g., contact transmission and hoof-trimming knives). Eradication of BLV has been achieved by combined management, segregation, and culling approaches. In contrast, although sole implementation of best management practices aimed at prevention of BLV transmission has decreased within-herd BLV prevalence, it has not eradicated BLV from a herd. Therefore, control and eradication of BLV by best management practices only should be further investigated. Additionally, the role of proviral load in infected cattle was investigated. Cattle with a high proviral load seem to be more likely to infect others, whereas those with a very low proviral load seem to have low risks of transmitting BLV. Information on proviral load could be taken into account when controlling BLV in high-prevalence herds. In conclusion, there is a need for detailed, large-scale studies investigating roles of specific transmission routes, knowing proviral load of infected individuals.

Pilot implementation of a newly developed bovine leukemia virus control program on 11 Alberta dairy farms.

We developed a custom bovine leukemia virus (BLV) control program for the Alberta dairy industry, consisting of a risk assessment and a comprehensive list of best management practices (BMP) aimed at prevention of BLV transmission between cattle. This control program was implemented on 11 farms for approximately 1 yr. Blood samples were collected from all cattle ≥12 mo old, and serum was tested with a commercial ELISA. Risk assessments were performed on each farm, risk-connected on-farm management was identified, and management changes expected to prevent transmission of BLV between cattle were suggested by the first author and agreed upon with each farmer. Throughout the following year, all participating farmers were visited multiple times to identify and overcome barriers to implementation and to monitor progress. After approximately 1 yr of implementing BLV control, all cattle ≥12 mo old on farm with a negative or no previous test result were sampled, and the within-herd prevalence was determined. The median number of cattle on farm that were ≥12 mo was 195 (range 110-524). The initial prevalence averaged 39% (13-66%). On average, 5 BMP (3-7) were suggested to each farmer. On average, 4 BMP (1-7) were implemented. At the second sampling, the average within-herd prevalence of all animals that tested positive (including the previous sampling) was 36% (12-62%). Eight farms reduced their within-herd BLV prevalence, within-herd prevalence stayed constant on 1 farm, and it increased on 1 farm. The remaining farm terminated their participation before the second sampling. The number of seroconversions per farm ranged from 3 to 109, highlighting the success of some producers to minimize new infections. The risk assessment was proven to be a valuable tool to identify flaws in on-farm management, although risk assessment score was unrelated to the within-herd BLV prevalence. Finally, it appeared that implementation of BMP aimed at prevention of BLV transmission between cattle could reduce within-herd BLV prevalence when farmers committed to their implementation.

Efficacy of the spray-drying treatment to inactivate the bovine leukemia virus in bovine colostrum.

Previous studies have shown the presence of bovine leukemia virus (BLV) in colostrum and milk of naturally infected cows. The relationship between virus or provirus and specific antibodies in these secretions is particular to each infected cow and will probably determine whether the consumption of colostrum or milk from these naturally infected dams provides an infective or a protective effect in recipient calves. Our recent findings suggest that this issue is a key point in BLV transmission in very young calves. Based on this, the aim of the present study was to determine the effect of the spray-drying treatment of colostrum on BLV infectivity. The treatment was done on scale-down conditions, using fresh colostrum from BLV-negative cows spiked with infective BLV. Residual infectivity was tested in susceptible lambs. Lambs inoculated with colostrum spiked with BLV-infected cells or cell-free BLV showed evidence of infection 60 d after inoculation, whereas none of the lambs inoculated with spray-dried colostrum showed evidence of infection 60 d after inoculation. These results provide direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious BLV in colostrum. These findings suggest that the risk for BLV transmission could be reduced if milk and colostrum were treated by spray-drying prior to consumption in dairy facilities. The effect of spray-drying on the functional properties and stability of the antibodies present in colostrum under long-term storage should be further investigated.

Development and implementation of a risk assessment and management program for enzootic bovine leukosis in Atlantic Canada.

Over the past 30 yr, the prevalence of bovine leukemia virus (BLV) infection has increased in North America, including Atlantic Canada, at both the herd and individual cow levels. This has occurred despite increased awareness of the disease and its deleterious effects and despite implementation of management practices aimed at reducing disease transmission. Our objectives were to identify risk factors associated with the within-herd prevalence of BLV-infected cows by using a risk assessment and management program workbook, as well as to determine the current level of BLV prevalence in the Atlantic Canada region. We hypothesized that previously established risk factors, including management practices associated with calf rearing and fly control, would affect within-herd BLV prevalence. Bulk tank milk (BTM) samples were collected in January and April of 2016 and again during the same months in 2017 and 2018 from all dairy farms shipping milk in the region. Samples were tested with ELISA for levels of anti-BLV antibodies to estimate within-herd prevalence. Regional BLV prevalence at the herd level was 88.39% of dairy herds infected in 2016 and 89.30% in 2018. All dairy farms shipping milk and who had BTM samples collected in 2017 (n = 605) were eligible to participate in the risk assessment and management program questionnaire (RAMP), which was developed and distributed to all bovine veterinarians in Atlantic Canada. One hundred and six RAMP were returned, with representation from all 4 provinces. The RAMP results were combined with the mean BTM ELISA results, and univariable logistic regression followed by multivariable logistic regression was performed to investigate the association between RAMP risk factors and the estimated within-herd BLV prevalence. Factors in the multivariable model significantly associated with the odds of a herd being classified as >25% estimated within-herd prevalence included history of diagnosis of clinical BLV and calves receiving colostrum from cows with unknown BLV status. Differences in within-herd prevalence were not associated with hypodermic needle and injection practices, rectal sleeve practices, or using bulls for natural breeding, based on these 106 dairy farms.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Economic evaluation of 4 bovine leukemia virus control strategies for Alberta dairy farms.

Bovine leukemia virus (BLV) is a production-limiting disease common in North American dairy herds. To make evidence-based recommendations to Canadian dairy producers and their consultants regarding cost and financial benefits of BLV on-farm control, an economic model that takes the supply-managed milk quota system into account is necessary. Alberta-specific input variables were used for the presented analysis. A decision tree model program was used to evaluate economic aspects of decreasing a 40% BLV within-herd prevalence on dairy farms by implementing various control strategies over 10 yr. Investigated strategies were (1) all management strategies, including 3 options for colostrum management; (2) some management strategies; (3) test and cull; and (4) test and segregate. Each of these strategies was compared with a no control on-farm approach. The prevalence for this no-control approach was assumed to stay constant over time. Each control strategy incurred specific yearly cost and yielded yearly decreases in prevalence, thereby affecting yearly partial net revenue. Infection with BLV was assumed to decrease milk production, decrease cow longevity, and increase condemnation of carcasses at slaughter from cattle with enzootic bovine leukosis, thereby decreasing net revenue. Cows infected with BLV generated a yearly mean partial net revenue of Can$7,641, whereas noninfected cows generated Can$8,276. Mean cost for the control strategies ranged from Can$193 to Can$847 per animal over 10 yr in a 146-animal herd. Net benefits of controlling BLV on farm, as compared with not controlling BLV, per cow in a 146-animal herd over a 10-yr period for each strategy was: Can$1,315 for all management strategies (freezer); Can$1,243 for all management strategies (pasteurizer); Can$785 for all management strategies (powdered colostrum); Can$1,028 for some management strategies; Can$1,592 for test and cull; and Can$1,594 for test and segregate. Consequently, on-farm BLV control was financially beneficial. Even though negative net benefits were possible and expected for some iterations, our sensitivity analysis highlighted the overall robustness of our model. In summary, this model provided evidence that Canadian dairy farmers should be encouraged to control BLV on their farm.

Controlling bovine leukemia virus in dairy herds by identifying and removing cows with the highest proviral load and lymphocyte counts.

The objective of this field trial was to reduce bovine leukemia virus (BLV) transmission and prevalence in commercial dairy herds using proviral load (PVL) and lymphocyte count (LC) measurements as indicators of the most infectious animals for culling or segregation. Bovine leukemia virus causes lymphoma in <5% of infected cattle, and increased lymphocyte counts (lymphocytosis) in about one-third. Recent research has shown that dairy cows infected with BLV have altered immune function associated with decreases in milk production and lifespan. Recent findings show that a minority of infected cattle have PVL concentrations in blood and other body fluids of over 1,000 times that of other infected cattle. In combination with a high LC, these animals are thought to be responsible for most transmission of BLV in a herd. Milk or blood samples from adult cows in our 3 Midwestern dairy farm field trials were tested semiannually with ELISA for BLV antibodies, and ELISA-positive cattle were then retested using a blood LC and a quantitative PCR test for PVL to identify the animals presumed to be most infectious. Herd managers were encouraged to consider PVL and LC status when making cull decisions, and to segregate cows with the highest PVL and LC from their BLV ELISA-negative herd mates where possible. After 2 to 2.5 yr of this intervention, the incidence risk of new infections decreased in all 3 herds combined, from 13.8 to 2.2, and the overall herd prevalence decreased in all 3 herds combined from 62.0 to 20.7%, suggesting that this approach can efficiently reduce BLV transmission as well as prevalence. This is encouraging, because a very low prevalence of BLV infection would make it economically feasible to cull the remaining ELISA-positive cattle, as was achieved in national eradication programs in other countries decades ago.

Risk factors associated with within-herd transmission of bovine leukemia virus on dairy farms in Japan.

BACKGROUND: Although several attempts have been made to control enzootic bovine leukosis (EBL) at the local level, a nationwide control program has not been implemented in Japan, except for passive surveillance. Effective control of EBL requires that the transmission routes of bovine leukemia virus (BLV) infection should be identified and intercepted based on scientific evidence. In this cross-sectional study, we examined the risk factors associated with within-herd transmission of BLV on infected dairy farms in Japan. Blood samples taken from 30 randomly selected adult cows at each of 139 dairy farms were tested by enzyme-linked immunosorbent assay (ELISA). Information on herd management was collected using a structured questionnaire. RESULTS: Infected farms were defined as those with more than one ELISA-positive animal and accounted for 110 (79.1%) of the 139 farms in the study. Completed questionnaires obtained from 90 of these 110 farms were used for statistical analysis. Seroprevalence, which was defined as the proportions of animals that tested positive out of all animals tested on the farm, was 17.1%, 48.1%, and 68.5% for the 25th, 50th, and 75th percentiles, respectively. A mixed logistic regression analysis implicated a loose housing system, dehorning, and a large number of horseflies in summer as risk factors (coefficient = 0.71, 1.11, and 0.82; p = 0.03, < 0.01, and 0.01, respectively) and feeding of colostrum to newborn calves from their dams as a protective factor (coefficient = -1.11, p = 0.03) against within-farm transmission of BLV on infected farms. CONCLUSION: Control of EBL in infected dairy farms in Japan will be improved by focusing particularly on these risk and protective factors.

Effectiveness of on-farm continuous flow high-temperature short-time pasteurization for inactivation of bovine leukemia virus in milk.

The effectiveness of on-farm continuous flow high-temperature short-time (HTST) pasteurization (i.e., 72°C for 15 s) for the inactivation of bovine leukemia virus (BLV) in milk was investigated with a sheep bioassay. Four sheep that had been inoculated with completely pasteurized milk containing approximately 3.4 × 107 BLV-infected peripheral blood mononuclear cells (PBMC) and treated by either HTST pasteurization or laboratory-scale low-temperature long-time (LTLT) pasteurization (i.e., 60°C for 30 min), remained negative for BLV for at least 17 weeks after inoculation. In contrast, all sheep inoculated with unpasteurized or inadequately pasteurized milk containing the same number of BLV-infected PBMC were tested positive for BLV and anti-BLV antibodies within 3 weeks after inoculation. These results suggest that on-farm continuous flow HTST pasteurization was equivalent value with inactivated BLV on the LTLT procedure and can effectively inactivate BLV in the milk. Therefore, on-farm HTST pasteurization of the pooled colostrum or milk used in automated feeding systems is likely to protect group-housed preweaned calves from BLV infection, thereby improving animal health on dairy farms.

Vector control efficacy of fly nets on preventing bovine leukemia virus transmission.

Bovine leukemia virus (BLV) is horizontally transmitted among cattle through infected blood. This 3-year field study (2013-2016) aimed to confirm the potential of the blood-sucking stable fly as a risk factor of BLV transmission and to determine the efficacy of vector control on preventing the transmission of BLV. The BLV-positive conversion rate during summer was higher than that during winter in a model dairy farm, where many stable flies were observed during the summer. After fly nets were fixed onto the barn to prevent fly invasion, the BLV-positive conversion rate during the summer was significantly decreased compared with that in the absence of fly nets (P<0.01). These findings suggest that vector control using a fly net may inhibit BLV transmission.

The eradication experience of enzootic bovine leukosis from Lithuania.

Before 1985 the situation regarding enzootic bovine leukosis (EBL) in Lithuanian cattle was described only haphazardly. In 1986 serological investigations were initiated together with an eradication programme. The EBL bovine leukosis virus (BLV) situation was monitored by the Institute of Immunology Vilnius University, national and regional veterinary laboratories. Starting in 1986 all EBL-positive cattle were separated from negative cattle into BLV-infected and BLV-free herds. To create the latter, calves were fed pasteurized milk. The seroprevalence in 1990 was 7.29%, but it steadily declined to 0.32% in 2006.

Tuberculosis, Brucellosis and Leucosis in Cattle: A Cost Description of Eradication Programmes in the Region of Lazio, Italy.

The eradication of tuberculosis, brucellosis and leucosis in cattle has not yet been achieved in the entire Italian territory. The region of Lazio, Central Italy, represents an interesting case study to evaluate the evolution of costs for these eradication programmes, as in some provinces the eradication has been officially achieved, in some others the prevalence has been close to zero for years, and in still others disease outbreaks have been continuously reported. The objectives of this study were i) to describe the costs for the eradication programmes for tuberculosis, brucellosis and leucosis in cattle carried out in Lazio between 2007 and 2011, ii) to calculate the ratio between the financial contribution of the European Union (EU) for the eradication programmes and the estimated total costs and iii) to estimate the potential savings that can be made when a province gains the certification of freedom from disease. For the i) and ii) objectives, data were collected from official sources and a costing procedure was applied from the perspective of the Regional Health Service. For the iii) objective, a Bayesian AR(1) regression was used to evaluate the average percentage reduction in costs for a province that gained the certification. The total cost for the eradication programmes adjusted for inflation to 1 January 2016 was estimated at 18 919 797 euro (5th and 95th percentiles of the distribution: 18 325 050-19 552 080 euro). When a province gained the certification of freedom from disease, costs decreased on average by (median of the posterior distribution) 47.5%, 54.5% and 54.9% for the eradication programmes of tuberculosis, brucellosis and leucosis, respectively. Information on possible savings from the reduction of control costs can help policy makers operating under budget constraints to justify the use of additional resources for the final phase of eradication.

In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection.

Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

Reduced humoral immunity and atypical cell-mediated immunity in response to vaccination in cows naturally infected with bovine leukemia virus.

Bovine leukemia virus (BLV) is a retrovirus that is widely distributed across US dairy herds: over 83% of herds are BLV-infected and within-herd infection rates can approach 50%. BLV infection reduces both animal longevity and milk production and can interfere with normal immune health. With such a high prevalence of BLV infection in dairy herds, it is essential to understand the circumstances by which BLV negatively affects the immune system of infected cattle. To address this question, BLV- and BLV+ adult, lactating Holstein dairy cows were vaccinated with Bovi-Shield GOLD® FP® 5 L5 HB and their immune response to vaccination was measured over the course of 28days. On day 0 prior to vaccination and days 7, 14 and 28 post-vaccination, fresh PBMCs were characterized for T and B cell ratios in the periphery. Plasma was collected to measure titers of IgM, IgG1 and IgG2 produced against bovine herpesvirus 1 (BHV1), Leptospira hardjo and L. pomona, as well as to characterize neutralizing antibody titers produced against BHV1 and bovine viral diarrhea virus types 1 and 2. On day 18 post-vaccination, PBMCs were cultured in the presence of BHV1 and flow cytometry was used to determine IFNγ production by CD4+, CD8+ and γδ T cells and to investigate CD25 and MHCII expression on B cells. BLV+ cows produced significantly lower titers of IgM against BHV1, L. hardjo and L. pomona and produced lower titers of IgG2 against BHV1. γδ T cells from BLV+ cows displayed a hyper reactive response to stimulation in vitro, although no differences were observed in CD4+ or CD8+ T cell activation. Finally, B cells from BLV+ cows exhibited higher CD25 expression and reduced MHCII expression in response to stimulation in vitro. All together, data from this study support the hypothesis that BLV+ cows fail to respond to vaccination as strongly as BLV- cows and, consequently, may have reduced protective immunity when compared to healthy BLV- cows.

Peptide-based bovine leukemia virus (BLV) vaccine that induces BLV-Env specific Th-1 type immunity.

In controlling retrovirus infection and replication, cell-mediated immunity (CMI) is considered to be effective. To develop a synthetic peptide vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed and inoculated to BALB/c mice. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses, and a weak cytotoxic lymphocyte response. The spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. Mannan-coated liposome containing two kinds of peptides (the 121-140 and 142-161 regions of BLV Env gp51) also induced peptide-specific lymphocyte proliferative response and IFN-g production in C57BL/6 mice. Thus, the synthetic T-cell epitope peptide-liposome system augmented a strong Th-1 type immunity in mice.

Establishment of a bovine leukemia virus-free dairy herd in Korea.

In view of the high prevalence rate of bovine leukemia virus (BLV) infections in cattle over the entire country, a large dairy farm in Chungnam province was chosen and 'test and segregate' program was instituted. On July 1999, ELISA test was performed on 491 animals on the farm and only 163 cattle (139 adult cows, 18 female and 6 male calves)were BLV-seronegative. From February 2000 through April 2004, the seronegative group was placed in barns 1,500 to 2,000 m from seropositive group and thereafter tested at 3-to 5-month intervals by ELISA. Animals seroconverted in consecutive tests were removed from the seronegative group immediately after the detection of anti-BLV antibodies. The changes in management were aimed at preventing iatrogenic transfer of blood between cattle. Replacement heifers imported from other countries and calves born at the farm were repeatedly tested by ELISA, and only seronegative animals were introduced into the group. As of April 2004, there were 311 cattle in the BLV seronegative group of the farm. Twenty four cows of the initial 139 adult cows were seroconverted in 2000, and no seropositive animals were found since February 2001. Follow up of the group, from which all seropositive cattle were moved to a separate location, revealed no recurrence of BLV infection for three years. The approach in the present study might be valuable for Korean producers who would like to move toward a BLV-negative status.

Control and eradication programme of enzootic bovine leucosis (EBL) from selected dairy herds in Turkey.

Serum samples of 15,909 cattle from 31 dairy herds located in various regions of Turkey were tested for the presence of antibodies against bovine leucosis virus (BLV) using Agar Gel Immuno-diffusion technique (AGID). 48.3% (15/31) of the herds had seropositive animals and positivity rates were detected from 0.5-34.4% in these herds. In an EBL control/eradication programme all seropositive animals were culled in the infected herds. Thereafter, a total of 74,347 sera were tested for the presence of BLV specific antibodies. The serological results and detail of EBL control/eradication programme were shown in this paper.

Enzootic bovine leukosis: report of eradication and surveillance measures in Italy over an 8-year period (2005-2012).

Bovine leukaemia virus (BLV) is associated with enzootic bovine leukosis (EBL). BLV causes malignant lymphoma and lymphosarcoma; however, most BLV infections remain clinically silent in an aleukaemic state. EBL is a notifiable disease, and official control measures include screening or monitoring, precautions at borders, control of movement inside the country, and stamping out. The objective of this study was to evaluate EBL eradication and surveillance measures in Italy from 2005 to 2012. One-hundred twenty-three outbreaks were recorded (1 January 2006 to 31 December 2012) in the National Veterinary Information System (SIMAN) on 7 November 2013. Of these, 101 had occurred in southern Italy. An outbreak usually lasted for a few days, but sometimes lasted for weeks. Some areas were subjected to normal eradication measures, whereas others were subjected to additional eradication measures as a consequence of persisting EBL outbreaks. During the study period, we noted an overall annual decrease from 0.21% in 2005 to 0.08% in 2012 in the herd prevalence rate, from 0.06% in 2005 to 0.04% in 2012 in the herd incidence rate, and from 0.027% in 2005 to 0.015% in 2012 in the animal prevalence rate. Regions officially recognised as EBL-free areas were found to have their own surveillance plans. Differences in their surveillance plans include the type of sample (serum, milk, or both), age at which the animals must be tested (12 or 24 months), and test frequency of herds (annually or every 2, 3, 4, 5, or 6 years). The eradication programme for EBL is difficult to implement in some Italian areas because of several factors such as incomplete herd registry, geographical location and socio-economic conditions of the region.

Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants.

BACKGROUND: The early stages consecutive to infection of sheep (e.g. primo-infection) by Bovine leukemia virus mutants are largely unknown. In order to better understand the mechanisms associated with this period, we aimed at analyzing simultaneously three parameters: B-lymphocytosis, cell proliferation and viral replication. RESULTS: Sheep were experimentally infected either with a wild type BLV provirus or with selected mutants among which: a virus harboring an optimalized LTR promoter with consensus cyclic AMP-responsive elements, two deletants of the R3 or the G4 accessory genes and a fusion-deficient transmembrane recombinant. Seroconversion, as revealed by the onset of an anti-viral antibody response, was detected at 3 to 11 weeks after inoculation. At seroconversion, all sheep exhibited a marked increase in the numbers of circulating B lymphocytes expressing the CD5 and CD11b cluster of differentiation markers and, interestingly, this phenomenon occurred independently of the type of virus. The net increase of the absolute number of B cells was at least partially due to accelerated proliferation as revealed, after intravenous injection of bromodeoxyuridine, by the higher proportion of circulating BrdU+ B lymphocytes. BLV proviral DNA was detected by polymerase chain reaction in the leucocytes of all sheep, as expected. However, at seroconversion, the proviral loads were lower in sheep infected by the attenuated proviruses despite similar levels of B cell lymphocytosis. CONCLUSIONS: We conclude that the proviral loads are not directly linked to the extent of B cell proliferation observed during primo-infection of BLV-infected sheep. We propose a model of opportunistic replication of the virus supported by a general activation process of B lymphocytes.

Genetic heterogeneity among bovine leukemia virus genotypes and its relation to humoral responses in hosts.

The existence of bovine leukemia virus (BLV) genotypes was investigated by restriction fragment length polymorphism (RFLP) analysis using bovine peripheral blood leukocytes collected from different geographical areas of Japan. For this purpose a nested polymerase chain reaction (PCR) for a 444 bp fragment of the envelope (env) gene was used because it was previously reported that this region might be responsible for the serological status in the host. The PCR products from 60 samples of BLV-infected cells were digested with endonucleases BamH I, Bgl I, Bcl I, Hae III and Pvu II. RFLP analysis demonstrated that there were six different genotypes of BLV present among cattle in Japan. In some herds PCR-positive animals were infected with only one genotype, but in other herds a few genotypes were found. One genotype was dominant throughout infected cattle and it was also detected in neoplastic cells from three of four animals with lymphosarcoma and three cell lines persistently infected with BLV. Production of antibodies to BLV in each cattle was surveyed by agar gel immunodiffusion and indirect hemagglutination tests, and the results were compared with those obtained from PCR. No genotype related to decreased immunoreactivity was detected. The difference in anti-viral immune responses of each animal appears to be related to the infection stage and other host factors, not to genetic heterogeneity of the envelope gene.