Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.

Genome-wide association study for host response to bovine leukemia virus in Holstein cows.

The mechanisms of leukemogenesis induced by bovine leukemia virus (BLV) and the processes underlying the phenomenon of differential host response to BLV infection still remain poorly understood. The aim of the study was to screen the entire cattle genome to identify markers and candidate genes that might be involved in host response to bovine leukemia virus infection. A genome-wide association study was performed using Holstein cows naturally infected by BLV. A data set included 43 cows (BLV positive) and 30 cows (BLV negative) genotyped for 54,609 SNP markers (Illumina Bovine SNP50 BeadChip). The BLV status of cows was determined by serum ELISA, nested-PCR and hematological counts. Linear Regression Analysis with a False Discovery Rate and kinship matrix (computed on the autosomal SNPs) was calculated to find out which SNP markers significantly differentiate BLV-positive and BLV-negative cows. Nine markers reached genome-wide significance. The most significant SNPs were located on chromosomes 23 (rs41583098), 3 (rs109405425, rs110785500) and 8 (rs43564499) in close vicinity of a patatin-like phospholipase domain containing 1 (PNPLA1); adaptor-related protein complex 4, beta 1 subunit (AP4B1); tripartite motif-containing 45 (TRIM45) and cell division cycle associated 2 (CDCA2) genes, respectively. Furthermore, a list of 41 candidate genes was composed based on their proximity to significant markers (within a distance of ca. 1 Mb) and functional involvement in processes potentially underlying BLV-induced pathogenesis. In conclusion, it was demonstrated that host response to BLV infection involves nine sub-regions of the cattle genome (represented by 9 SNP markers), containing many genes which, based on the literature, could be involved to enzootic bovine leukemia progression. New group of promising candidate genes associated with the host response to BLV infection were identified and could therefore be a target for future studies. The functions of candidate genes surrounding significant SNP markers imply that there is no single regulatory process that is solely targeted by BLV infection, but rather the network of interrelated pathways is deregulated, leading to the disruption of the control of B-cell proliferation and programmed cell death.

[Biological cycle of radionuclides and dynamics of cattle leukemia in the Orenburg region].

Global technogenic radionuclides pollution of environment, migration of radionuclides from soil into animal feeds stimulate additional contamination doses. Concentration of natural and of technogenic radionuclides in the investigated objects was determined as result of radiochemical and radiometric studies. It was established that maximum concentrations of 40K, 232Th, 226Ra (Bk/kg) radionuclides in the soil of the eastern zone was 391.6 +/- 77.64; 20.8 +/- 0.71; 16.7 +/- 0.8 (respectively) without exceeding permissable levels. It is also found that the density of soil pollution with 137Cs in the western zone is 0.28 +/- 0.075 mBk/m2 (76.8 +/- 20.4 mKi/km2), such soils are to be considered as locally polluted soils. The number of cattle infected with cattle leucosis is obviously higher in ecologically unsafe areas: 19.3% on the western farms, 12.95% in the central areas and 9.70% on the farms of the eastern zone. Long-term exposure to ecologically unsafe conditions may lead to the reduction of populations immune status expressed in lower disease resistance and in incidence of carcinogenic pathology.

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.

The eradication of enzootic bovine leucosis in a large farm population.

In the years 1992 to 2002 the ID and ELISA systemic serological examinations of infected cows from a large farm brought advantageous results. A total of 468 cows from three farms from Opole province were examined. In the years 1997 to 2002 there were four times more positive serological results obtained than in the years 1992 to 1996. The elimination of BLV infected animals from farms and watering of serologically negative calves with milk substitute feed were very useful methods. In the year 2002 the control of all cattle gave negative ELISA results based on anti-gp51 monoclonal antibodies. The farm demonstrated BLV-free status after 10 years as a result of regular laboratory testing and successful elimination of all positive animals.

Bovine leukemia virus SU protein interacts with zinc, and mutations within two interacting regions differently affect viral fusion and infectivity in vivo.

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.

Monoclonal antibodies to a tumour-associated antigen isolated from circulating immune complexes of bovine leukemia virus-infected cattle.

From circulating immune complexes (ICs) of BLV-infected cattle, an antigen preparation was produced that contained some virus-specific proteins and a tumour-associated antigen. Eleven hybridoma clones were produced that secreted monoclonal antibodies (MoAbs) to this tumour-associated antigen, and two of them, MoAbs 1B4 and 1E9, were used in further studies. Most antibodies were of IgG1 subclass and showed no cytotoxic activity towards lymphocytes of BLV-positive cattle or to certain tumour cells. The MoAbs 1B4 and 1E9 recognized an antigen of about 75 kD on the cell surface of bovine lymphosarcoma cells and circulating lymphocytes from BLV-infected cattle with persistent lymphocytosis. The results presented indicate that the circulating immune complexes from BLV-positive cattle contain a tumour-associated antigen that is expressed on tumour cells and on lymphocytes from cattle with persistent lymphocytosis.

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus.

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.

Polymorphism in BoLA-DRB3 exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus.

The relationship between polymorphism of the bovine lymphocyte Ag (BoLA)-DRB3 gene and resistance and susceptibility to persistent lymphocytosis (PL) caused by bovine leukemia virus (BLV) was investigated. Exon 2 of the BoLA-DRB3 gene was cloned from animals with BoLA haplotypes previously found to be associated with resistance and susceptibility to PL. Sequence analysis revealed the presence of the amino acids Glu-Arg (ER) at putative Ag binding residues 70 and 71 only in BoLA haplotypes associated with resistance to PL. This correlation was confirmed in a case control study (n = 26) using an allele-specific polymerase chain reaction for the detection of ER at residues 70-71. These results provide a molecular basis for Ir gene control of resistance and susceptibility to PL and suggest that the cellular immune response is important in preventing the in vivo spread of BLV infection.

Decreased insulin-like growth factor-I (IGF-I) receptor sites on circulating mononuclear cells from cows with persistent lymphocytosis.

Plasma IGF-I concentrations and IGF-I receptor binding on mononuclear cells have been studied on bovine leukemia virus (BLV)-negative (CO), BLV-infected aleukemic (AL) cows or cows with persistent lymphocytosis (PL). No significant differences in plasma IGF-I concentrations were demonstrated among the three groups of animals. However, a linear negative correlation existed between the number of circulating mononuclear cells and the number of IGF-I binding sites on these cells from control cows. In addition, mononuclear cells from PL cows had fewer IGF-I binding sites per cell when compared with control cows. These results suggest involvement of IGF-I in etiology of BLV infection and progression and warrant further studies to establish whether IGF-I plays a major physiological role in these conditions.