Determination of telomere length and telomerase activity in cattle infected with bovine leukaemia virus (BLV).

Telomeres are repetitive sequence structures at the ends of chromosomes. They consist of the double stranded DNA repeats followed by the short single stranded DNA. In humans and other verterbrates the telomeric sequence is composed of tandem of TTAGGG repeats. With each cells division telomeres shorten by up to 200 base pairs. Telomerase is an enzyme responsible for continuous cell growth and is repressed in most somatic cells, except proliferating progenitor cells, but in more than 85% of cancer cells telomerase expression is observed. Tumour cells with metastatic potential may demonstrate a high telomerase activity, allowing cells to escape from the inhibition of cell proliferation due to shortened telomeres. Determination of telomerase expression was performed with the use of PCR ELISA in samples isolated from bovine leukaemia virus (BLV) infected cows. Telomerase activity was found in almost all investigated samples. The relative telomerase activity (RTA) was higher in infected cows than in healthy animals and the differences were statistically significant (α=0.05). In blood lymphocytes of BLV-infected cows the mean values of telomerase expression determined in real-time PCR were 3534.12 copies, in the healthy group there were 1010.10 copies and these differences were also statistically significant. For telomere length evaluation the Telomere PNA/FITC FISH and Telomere PNA/FITC FISH for flow cytometry were used. The mean fluorescence intensity of telomere sequences calculated on the surface of interphase nuclei of leukaemic blood lymphocytes was lower than that in the control animals and the difference was statistically significant. The mean length of telomeres in BLV- infected and healthy cows was 31.63 ± 12.62 and 38.4 ± 4.03, (p=0.112), respectively.

Determination of cytokine profiles in populations of dendritic cells from cattle infected with bovine leukaemia virus.

Dendritic cells (DCs) due to their ability to present antigens are essential during the immune response to infections. The aim of the study was to evaluate the influence of bovine leukaemia virus (BLV) infection on DC properties. Cytokine profiles of myeloid, plasmacytoid and mono- cyte derived DCs from BLV infected cattle were analysed. Concentrations of IL-6, IL-10, IL-12, IFN-γ, and TNF-α in DC cultures were measured by flow cytometry. Obtained results indicated activation of pDCs population, where a significant increase in production of the IFN-γ was shown. Meanwhile, a decrease in production of IFN-γ and increase in production of IL-10 were shown in mDCs; the main population responsible for antigens presentation. This may indicate a contribu- tory role of the population during the process of persistent infection. In MoDCs population a significant elevation in secretion of proinflammatory cytokines - IL-6 and TNF-α was noted.

Cytokine profiles of dendritic cells (DCs) during infection with bovine leukaemia virus (BLV).

BLV is an agent of enzootic bovine leukaemia (EBL), an infectious disease affecting cattle worldwide. BLV infection has been associated with immune system disorders and discrepancies in the cytokine network. The significance of dendritic cells in the pathogenesis of BLV infection is largely unknown, but considering their fundamental role in immune response it may be crucial. DCs precursors were isolated with the use of immunomagnetic beads from BLV-infected and BLV-free cows. From these precursors cultures of monocyte derived dendritic cells (MoDCs) were generated with the use of a cytokine cocktail (IL-4 and GM-CSF). Additionally, parallel DCs from BLV-negative animals were infected in vitro. The level of cytokines: IL-6, IL-10, IL-12(p40), IL-12(p70) was determined in DC cultures: infected in vitro, originating from naturally infected cattle and BLV-free cattle. The investigation showed significant changes in almost all analyzed populations of BLV-infected DCs. Cytokine profiles of blood MoDCs indicated activation of these groups during infection. In the case of spleen MoDCs and lymph node MoDCs a decrease in production of IL-12(p40) and IL-12(p70) in favour of IL-6 and IL-10 was noted, suggesting promotion of BLV infection development.

Blood dendritic cells in cattle infected with bovine leukemia virus (BLV): isolation and phenotyping.

Dendritic cells (DCs) are most potent antigen presenting cells (APCs) with unique ability to prime effective immune responses. They express higher levels of MHC class II and accesory molecules on their surface, than other professional APCs. The investigations were performed on DCs generated from blood with the use of microbeads magnetically labeled with mouse anti human CD14. Flow cytometry was applied for determination of DCs immunophenotype in healthy and naturally infected with BLV cattle. For immunophenotyping mouse monoclonal antibodies anti bovine: CD11a, CD11b, CD11c, MHC-I and MHC-II were used. Our results demonstrated that dendritic cells infected with BLV expressed very high percentage of determinants: CD11a, CD11b, CD11c, MHC-I and MHC-II class. Leukaemic DCs exhibited DCs morphology and had a phenotype of mature DCs. The expression of gp51 glycoprotein of BLV on leukaemic DCs was detected in flow cytometry investigations.

Experimental infection of sheep with bovine leukemia virus (BLV).

Nine groups of sheep have been experimentally infected with different doses of BLV and by various routes. An immunological response of animals, as well as clinical symptoms of leukosis in the experimental sheep were investigated within several years or months. The animals inoculated with the virus showed appearance of BLV-antibodies in 94 per cent of cases, lymphocytosis in 47 per cent and clinical symptoms of the disease in 33 per cent. In conclusion, sheep have been demonstrated to be animals suitable for experimental study of leukosis.