CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus that induces a chronic disease in cattle, often causing persistent lymphocytosis (PL), with lymphosarcomas developing in up to 10% of infected animals. Although there is still no vaccine commercially available, our group has been performing animal trials using a live-attenuated BLV strain obtained by genetic modification of the wild-type strain. This attenuated strain proved to be infectious in cows and replicated at low levels, with the development of a competent immune response, conferring protection from wild-type challenge. The attenuated strain was not transmitted to calves but maternal colostrum provided passive immunity. Since biosafety of this kind of vaccine is extremely important, we have carried out additional experiments in order to add knowledge in this regard.Objective: To evaluate the distribution of BLV provirus in various organs and tissues after long term infections.Methods: Three cows were studied. Cow A was naturally infected with a field strain of BLV, cows B and C were experimentally infected with the wild type and the attenuated strains, respectively. After 10 years of infection, animals were euthanized by exsanguination following stunning. Samples from different organs and muscle tissues, including those for human consumption, were collected aseptically. Whole blood sample was also collected from animals before euthanasia. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) and tissues, and tested by nested PCR for BLV provirus detection and real time PCR (qPCR) for proviral load (PVL) quantification. Results: At the time of euthanasia, PVL in PBMCs was high in cows A and B (452767 and 76888 copies/µg DNA, respectively), and extremely low (less than 100 copies/ µg DNA) in cow C. In animals A and B, BLV provirus was detected in all analyzed tissues, with higher PVL in lymph nodes, spleen, liver, uterus, bone marrow and heart. The provirus was also found in muscle tissues for human consumption although with lower proviral load values (700-3000 copies/ µg DNA). These cows were pregnant, and although proviral DNA was present in the udder fluid, it was undetectable in both embryos. BLV proviral DNA could not be detected in any tissue obtained from the cow infected with the attenuated strain.Conclusion: The undetectable levels of the attenuated strain after long term infection in organs and muscle tissues indicates that it is safe and suggests that could be considered as a tool for BLV clearance under a food safety point of view. Even though BLV transmission by consumption is not a key point under discussion, the analysis of safety is a necessary step for regulatory compliance of a modified strain.