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.     

Histopathology and distribution of cells harboring bovine leukemia virus (BLV) proviral sequences in ovine lymphosarcoma induced by BLV inoculation.

Six sheep with lymphosarcoma induced by hypodermic inoculation of bovine leukemia virus (BLV) materials were examined to elucidate the relation between pathologic lesions and integration of BLV provirus in cellular DNAs. Antibodies to BLV gp-antigens had been detected since the 3rd week after the inoculation, and BLV was positive when checked 3 months later. Lymphosarcomas followed the leukemic phase in 4 sheep. The other 2 sheep showed initial lesions of lymphosarcoma and were aleukemic clinically. Five animals were killed by enthanasia and autopsied at 2.5 to 3.5 years postinoculation (pi) because of their diseased condition. One animal died 10 years pi following the 4th leukemic episode. Sarcomatous lesions were confirmed grossly and histologically, and the proliferating neoplastic cells were classified into lymphocytic, prolymphocytic, lymphoblastic and histiocytic types. Integration of BLV provirus in cellular DNAs of the peripheral blood lymphocytes (PBL) and neoplastic cells of sarcomatous lesions was examined by Southern blotting technique. BLV provirus was demonstrated in the PBL of all infected animals and in most of the sarcomatous lesions of the spleen, kidney and lymph nodes except 4 lymph nodes showing slight neoplastic infiltration. The results indicated that ovine lymphosarcoma could be caused by BLV and the cells carrying proviral information seemed to be disseminated and proliferate in the lesions.     

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle.

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5-10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.     

The recent prevalence of bovine leukemia virus (BLV) infection among Japanese cattle

A seroepidemiological survey of bovine leukemia virus (BLV) infection was conducted in Japan in 2007 using an enzyme-linked immunosorbent assay (ELISA) and an agar gel immunodiffusion (AGID) test. A total of 5420 cattle (dairy, 3966; breeding beef, 797; fattening beef, 657) from 209 farms in seven prefectures in Japan were tested. The overall prevalence of BLV infection was 28.6%. The prevalence of BLV infection in dairy cattle (34.7%) was higher than for both fattening beef cattle (7.9%) and breeding beef cattle (16.3%). Age-specific prevalence showed that BLV prevalence increased with age in all types of cattle and was notably different between dairy and beef cattle under 1 year of age. Among 207 farms, 141 herds (68.1%) had one or more positive animals. The proportion of these positive farms was significantly higher among dairy farms (79.1%) than among beef breeding farms (39.5%) and beef fattening farms (51.9%) (P < 0.001). Dairy farms (40.5%) also showed a significantly higher within-herd prevalence than beef breeding (27.4%) and fattening (14.9%) farms (P = 0.001). This study indicated that BLV is more widely spread in dairy cattle than in beef breeding cattle in Japan. Given the prevalence of BLV infection in dairy and beef cattle was 8- and 1.7-fold higher, respectively, than rates previously found in 1980–1982, BLV appears to be spreading particularly among the dairy cattle population during the last two decades. Further investigation is required to determine the risk factors necessary to control BLV infection that take into account the different farming practices that exist between dairy and beef sectors.     

Humoral and cellular responses in calves experimentally infected with bovine leukemia virus (BLV)

In order to elucidate whether infection by Bovine Leukemia Virus (BLV) might induce an immunodeficient state, we inoculated sixteen calves with BLV. The calves were followed up for two years and were tested for humoral and cellular responses using various parameters, namely the appearance of antibodies to the BLV antigens, the changes in the numbers of lymphocytes involved, and the ratio between the two main populations of lymphocytes. Antibodies to the BLV antigens were of both the IgG and the IgM classes of immunoglobulins. The levels of antibodies of the IgM class were higher than those of IgG. There was a temporary decrease of reactive antibodies to the BLV antigens, to below detectable levels, during the 14-24 weeks post infection. A significant decrease in the level of plasma IgM was found in all BLV infected calves exhibiting lymphocytosis, while the level of IgG in the plasma of all experimental calves did not diverge significantly from the initial values, throughout the experiment. BLV infection was followed by lymphocytosis of B-cells in most infected calves, which persisted for the whole course of the experiment, while a decrease in the population of T-cells in peripheral blood was observed for a period of several months in all infected calves.     

Detection of bovine leukaemia virus (BLV) in tissue samples of naturally and experimentally infected cattle

Enzootic bovine leukaemia (EBL) which is caused by the bovine leukaemia virus (BLV) still plays a remarkable role despite a significant success in sanitation programmes. In the Federal Republic of Germany it was not possible to eradicate the disease until today. Sporadically during slaughter or necropsy of cattle neoplastic lesions of the lymphatic tissues are observed that need to be clarified with regard to BLV as etiological agent. Due to the fact that in most instances no serological data are available from the respective animals and blood drawings from the original holdings are not easy to obtain the polymerase chain reaction (PCR) opens new avenues as supplementary diagnostic tool to test unfixed lymphatic tissues for the presence of BLV proviral DNA. Lymph node tissues from 10 naturally or experimentally BLV-infected cattle, which have been monitored virologically and serologically, and tissues from 4 negative animals were processed, DNA was extracted and subjected to PCR to amplify BLV env gene specific sequences. The results show that in cattle with BLV-induced leukosis as well as in cattle, which were clinically healthy and unsuspicious at slaughter or at post-mortem, either with persistent lymphocytosis (PL) or without, BLV proviral DNA could be detected easily in samples of lymphatic tissues and in high concordance with serological data. In this article data from the National and OIE reference laboratory for EBL at the Friedrich-Loeffler-Institut (FLI, Germany) are presented. Elaborated laboratory protocols for processing of tissue samples and performing of BLV-PCR are recommended.     

Electron microscopy of bovine leukosis viruses (BLV) in a fetal lamb spleen cell line

The ultra-thin section method, the method of negative staining, and immunoelectron microscopy were used for detecting BLV and for determining its morphological characteristics in the FLS continual cell line used as a virus antigen producer for the ELISA test. Particles of C type, about 110 nm in size, having a structure corresponding to BLV, were detected in the FLS cells on the ultra-thin sections. The viruses were located extracellularly, in cytoplasmic vacuoles, and in different stages of maturation by budding from cell plasma membrane. BLV presence was also demonstrated by immunoelectron microscopy.     

Natural mode of horizontal transmission of bovine leukemia virus (BLV): the potential role of tabanids (Tabanus spp.)

In order to evaluate the potential role of hematophagous insects in the natural spreading of bovine leukemia virus (BLV) infection in cattle, a 2-year survey was carried out involving sequential serological tests on 3328 cattle in three different areas. A parallel entomological study was run over the same period, using continuous trapping, in order to determine both the density and variations of horsefly (Tabanus spp.) populations in the close vicinity of the herds. After statistical analysis, this space-time study showed that: (1) There is a significant positive geographical correlation between the rate of incidence of BLV infection and the density of the horsefly population. (2) Seasonal variations in the incidence rate exist; the highest rates are generally observed during summer (from July of September), and the lowest during winter, spring and early summer (from November to mid-July). (3) There is a time link between the rate of sero-conversion and the variations in activity of the horsefly population. All these data combined would appear to indicate that tabanids play a considerable role in the spread of BLV under natural conditions.     

S-phase evaluation with bromodeoxyuridine in lymphocytes from cattle infected with bovine leukemia virus (BLV)

Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase. In this paper a determination of the S-phase in BLV+ cattle with lymphocytosis has been performed by incorporating bromodeoxyuridine in the DNA. This evaluation was compared to the DNA content, demonstrating that i) bromodeoxyuridine incorporation is a reliable marker of S-phase in BLV+ cattle with lymphocytosis and ii) cytofluorimetry is the method of choice, together with immunocytochemistry, to demonstrate bromodeoxyuridine incorporation.     

Seroprevalence of bovine leukemia virus (BLV) infection in dairy cattle in Isfahan Province, Iran

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis (EBL) is an exogenous C-type oncovirus in the Retroviridae family. It causes significant economic losses associated with the costs of control and eradication programs due to carcass condemnation at slaughter and restrictions of export of cattle and semen to importing countries. The main objective of this research was to determine the seroprevalence of BLV infection in cattle herds in central region of Iran (Isfahan province) using a commercial enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies against BLV. Samples of blood serum were collected from 403 female dairy cattle (Holstein-Friesian) from 21 livestock farms and 303 animals (81.9%) were BLV seropositive. A significant association was found between age as a potential risk factor and BVL seroprevalence with animals ≥ 4 years (86.6%) having a significantly (χ(2) = 35.6, p < 0.001) higher seroprevalence compared to those < 4 years (54.2%). We found no significant statistical association between seroprevalence and pregnancy, lactation status and farming systems as potential risk factors in this study (p > 0.1). It is concluded that BLV infection is a very common problem in the study area. Hence, control measures should be instituted to combat the disease and further studies are required to investigate the impact of this disease on dairy production in the country.     

Immunohistological demonstration of virus and tumor associated antigens in tissues experimental and spontaneous bovine leukemia virus (BLV) infection

Expression of bovine leukemia virus (BLV) antigens in vivo has not been shown. After BLV infection, however, production of antibodies directed towards BLV proteins (e.g. gp51) can be easily demonstrated. Thus, production of BLV proteins has to take place somewhere in infected cattle. Tissues and organs of experimentally infected cattle were fixed in acetone and embedded in paraffin. Monoclonal antibodies directed to gp51 were used to demonstrate BLV expression immunohistologically by the peroxidase-antiperoxidase (PAP) method. The same samples were also used to demonstrate a tumor associated antigen (TAA) employing a monoclonal antibody. Our results indicate that very few cells, found in the intestinal mucosa, produce gp51 in vivo. The expression of TAA, however, increases significantly shortly after infection with BLV and remains high throughout life.     

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.     

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.