Introduction: Numerous mutations in the bovine tumour necrosis factor receptor type two (TNF-RII) gene have been identified, but their biological consequences remain poorly understood. The aim of this study was to determine whether polymorphism in the analysed loci of the bovine TNF-RII gene is linked with the size of cell subpopulations naturally infected with bovine leukaemia virus (BLV) which serve important immune functions in the host. Material and Methods: Samples originated from 78 cows. Polymorphisms in the studied gene were determined by PCR-RFLP and DNA sequencing by capillary electrophoresis. BLV infection was diagnosed by the immunofluorescence (IMF) technique and nested PCR. Cell subpopulations were immunophenotyped with IMF. Results: Similar and non-significant differences in the average percentages of TNFα+, IgM+TNFα+, and CD11b+TNFα+ cells infected with BLV were noted in individuals with various genotypes in the polymorphic sites g.-1646T > G and g.16534T > C of the TNF-RII gene, and significant differences in the percentages of these subpopulations were observed between selected microsatellite genotypes (g.16512CA(n)). Conclusion: STR polymorphism and the number of CA dinucleotide repeats in intron 1 of the TNF-RII gene influence the frequency of TNF+, CD11b+TNF+, and IgM+TNF+ subpopulations naturally infected with BLV. Polymorphism in the gene’s other two sites do not affect the size of these cell subpopulations.

Objective: To determine the reference interval for WBC counts in Holstein dairy cows from herds with high seroprevalence for anti–bovine leukemia virus (BLV) antibodies, analyze the correlation of total WBC counts and blood proviral load (bPVL) in BLV-infected animals, and determine whether total WBC count can be used a hematologic marker for in vivo infection. Animals: 307 lactating cows from 16 dairy herds with high BLV seroprevalence. Procedures: Blood samples were collected for assessment of plasma anti–BLV p24 antibody concentration (all cows), manual determination of WBC count (161 BLV-seronegative cows from 15 herds), and evaluation of bPVL (146 cows from another herd). Results: The WBC count reference interval (ie, mean ± 2 SD) for BLV-seronegative dairy cows was 2,153 to 11,493 cells/μL. Of the 146 cows used to analyze the correlation between WBC count and bPVL, 107 (73%) had WBC counts within the reference interval; of those cows, only 21 (19.6%) had high bPVL. Most cows with high WBC counts (35/39) had high bPVL. Mean WBC count for cows with high bPVL was significantly higher than values for cows with low or undetectable bPVL. White blood cell counts and bPVL were significantly (ρ = 0.71) correlated. Conclusions and Clinical Relevance: These data have provided an updated reference interval for WBC counts in Holstein cows from herds with high BLV seroprevalence. In dairy cattle under natural conditions, WBC count was correlated with bPVL; thus, WBC count determination could be a potential tool for monitoring BLV infection levels in attempts to control transmission.

Two agar gel immunodiffusion (AGID) kits for the serodiagnosis of bovine leukemia virus (BLV) were imported from Europe and were compared with North American kits. The BLV AGID kits from North America and from Europe differed significantly. The punches were different, as were the pattern distribution in the agar of the reference and the test sera, resulting in differences in the reading of the immunoprecipitation lines. Based on the testing of 1200 serum samples from cattle, the European kits gave a good correlation with the American kits, as indicated by their respective kappa values. However, the European kits were found to be less sensitive when evaluated against weakly positive samples from field specimens or following a dilution trial. Only 65% and 50% of the weakly positive samples detected by the American kit #1 were detected by the European kits #2 and #3, respectively. The American kit was also capable of detecting BLV antibodies in 45% of strongly positive samples diluted 1/50 in negative sera, while antibodies were detected in only 15% of the samples with the European kit #2 and in none of the samples with the European kit #3. False negatives were also detected with the European kits. Among the false negatives, the degree of expected reactions was weak (European kit #2) or of varying degrees of positivity (European kit #3). Besides the differences in format and performance, the BLV-AGID kits in Europe are evaluated with the National Standard Serum E4 while a proficiency panel composed of a quadruplicate set of 10 reference sera is used in Canada to monitor the kits. Based on the overall observations, we noted a lack of standardization between the BLV-AGID kits used in North America and in Europe.

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by, infection with bovine leukemia virus (BLV). The superficial cervical lymph nodes from 3 BLV-infected but apparently healthy sheep and 5 sheep with full-blown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (sIgM) and major histocompatibility complex (MHC) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5- B-cell lineage and to be classified as 2 types, CD5-CD4-CD8-B2+MHC class-II+sIgM+ and CD5-CD4-CD8-B2+MHC class-II+sIgM-. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

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 used 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.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), which is the most prevalent neoplastic disease of cattle worldwide. Few studies have been conducted on BLV detection in Egypt, and it is unknown whether BLV is prevalent in some areas. BLV seroprevalence has never been identified in Beheira province. Therefore, the main objective of this research was to determine the seroprevalence of BLV among cattle in Beheira. A total of 368 cattle plasma samples (219 dairy and 149 beef) from 6 dairy farms, 1 beef farm, and 9 slaughterhouses in eight districts covering most geographical areas of Beheira were investigated using a commercial ELISA for the detection of anti-gp51 antibodies. Data were analyzed, and the risk factors associated with BLV infection were evaluated. Out of the whole samples, 44 (11.9%) tested positive for BLV, and the seroprevalence rates in dairy and beef cattle were 31/219 (14.2%) and 13/149 (8.7%), respectively. Cattle breed had a significant risk factor on BLV seroprevalence, as in Holstein cattle, it was 21.65% (OR= 3.1, p <0.004) higher than mixed local breed (8.20%) in dairy cattle. However, Colombian cattle showed the highest seroprevalence (19.15%) among tested beef cattle breeds.  Additionally, neither age nor farming system had a potential risk on BLV seroprevalence in the tested dairy or beef cattle (p > 0.1). It is concluded that BLV infection is widespread among cattle in Beheira province's various localities, with a potential risk for cattle of foreign breeds to contract the BLV infection.

Hematologic investigations were made on the blood samples taken from bovine leukemia virus (BLV)-seropositive Holstein-Friesian cattle in Korea, and their absolute lymphocyte count was compared with that of BLV-seronegative cattle. The incidence of persistent lymphocytosis (PL) was also determined. The normal bovine lymphocyte count was established on the basis of studies of 656 blood samples taken three times from 297 seronegative animals aged from 0~6 months to over 5 years at 5~6-month intervals. The data were examined according to 7 age groups of samples placed into their respective age groups.

A specific polymerase chain reaction (PCR) assay was devised, allowing detection of 1 bovine leukemia virus (BLV)-infected cell in 10(4) bovine lymphocytes. The efficacy of field application of the developed method was verified by evaluating the rate of viral transmission to calves from infected cows, whether they have persistent lymphocytosis. With this objective, 43 calves were simultaneously tested at birth and at 6 months of age for viral antibodies in serum and for proviral DNA in lymphocytes. At birth, 36 calves were BLV-negative and 3 were BLV-positive by results of serologic and DNA-based assays. Conversely, results for 4 calves had lack of correlation between the diagnostic methods. In particular, 2 calves were DNA-positive and antibody-negative for BLV and 2 other calves had the opposite test results. At 6 months of age, when the immunologic pattern more closely reflects the status of calves' immune response, independent of maternal antibodies, all calves DNA-negative for BLV at birth (n = 38), were consistently PCR- and antibody-negative for BLV. On the contrary, the cattle DNA-positive for BLV at birth (n = 5), whether seropositive or not, were PCR- and antibody-positive for BLV, at the time of the second screening. Thus, these results indicate reliability of the PCR to diagnose perinatal BLV infection. Furthermore, the observation that all calves found to be infected at birth were born to BLV-positive cows with persistent lymphocytosis, indicates that the persistent lymphocytosis status of the cow may represent a factor associated with BLV infection in utero.

One hundred and fifty lactating Holstein cows from 2 genetic lines selected for high and average milk production were used in the study. Sera from 6 annual herd tests were analyzed by agar-gel immunodiffusion test for antibodies to bovine leukemia virus. Odds of being seropositive were analyzed by use of stepwise and backward logistic regression procedures. Analysis within birth year revealed that estimated ln odds increased by 0.19/year of age among cows of the high genetic line and by 0.43 among cows of the average genetic line. This was accompanied by a more important cohort effect among high producers than among average producers.

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.