• Intermitent detection of BLV in frozen semen simples by PCR assay

ROSSICH LUCIANO; GUTIERREZ GERONIMO; RODRIGUEZ SABRINA; LEFEBVRE EDUARDO; TRONO KARINA; DUS SANTOS MARIA JOSE

Melbourne, Australia

Simposio; 13th International Symposium of the World Association of Veterinary Laboratory Diagnosticians; 2007

wavld

Detection of BLV in frozen semen samples by PCR assay L. Rossich1, J. Gutierrez1, S. Rodriguez1, E. Lefebvre2, K. Trono1 and M. J. Dus Santos1* (1) Instituto de Virología, CICVyA, INTA-Castelar. CC 77. Buenos Aires, Argentina. (2) Centro de Inseminación Artificial La Elisa. Argentina. * mdussantos@cnia.inta.gov.ar IntroductionThe major route of transmission of BLV is horizontal by direct exposure to biological fluids contaminated with infected lymphocytes, mainly blood. Although viral antigens and proviral DNA has been identified in semen, milk and colostrums, natural transmission through these secretions has not been demonstrated. The sanitary and economic impact of BLV infection is associated with the interference in the international movement of cattle and their germ plasm. Although experimental data support the improbability that semen from BLV-positive bulls could infect recipient cows, restriction for commercialization of semen from infected animals is still present1-6. Since there is no vaccine or treatment available, eradication and control of the disease is exclusively based on early diagnostic and segregation of infected animals. In this context the specificity and sensitivity of the diagnostic test used is a critical point. We have previously developed a PCR assay with high sensitivity and specificity to detect BLV genome in frozen semen samples. The objective of this work was to study the detection of BLV in semen. Material & methodsSamples: Serum, whole blood and semen samples were obtained from CIALE (Artificial Insemination Centre La Elisa, Capitan Sarmiento, Buenos Aires, Argentina) Serology: The agar gel immunodifussion (AGID) test kit used to detect antibodies to BLV was produced by the Faculty of Veterinary, La Plata University, Argentina. An indirect ELISA using recombinant p24 as antigen was used to detect antibodies to BLV. This assay has been completely developed and standardized in the Institute of Virology, INTA-Castelar, Buenos Aires, Argentina. Detection of proviral DNA: DNA was extracted from PMBCs (purified from whole blood) and semen samples (fresh and straw). PCR assays that amplified a region of genes pol and gag and a nPCR that amplified a region of gene env were developed. ResultsTwo PCR assays were standardized for detecting BLV genome in semen and PMBC. The limit of detection of viral particles was assessed by the addition of purified pBLV344 (a plasmid containing the complete BLV genome, kindly provided by Dr. Willems, Faculté Universitaire des Sciences Agronomiques, Gembloux, Belgium) to DNA from semen or PMBCs of a seronegative bull. By gag-PCR and pol-PCR, it was possible to detect 60 viral particles, using bromide staining after electrophoresis separation of DNA. In order to increase analytical sensitivity, a nested-PCR was developed which amplified a region of env gene. The n-PCR was able to detected 6 viral particles. Assessment of the limit of detection was highly repetitive under the standardized conditions. Frozen semen samples from 30 seropositive bulls were remitted to the laboratory and analyzed in the period 2005-2007. It was possible to detect proviral DNA in 172 out of 862 samples. BLV genome detection occurred in several collections but in an alternated way with non detection periods. Fresh semen samples, straws and whole blood were also obtained from 5 seronegative and 5 seropositive bulls and tested together for the presence of BLV provirus. Results indicated that while detection of provirus was positive in PMBC from all seropositive bulls, detection of gag, pol and env genes in semen did not occurred in all the samples. Discussions & conclusionsThe results obtained suggest that BLV could present an intermittent pattern of excretion. Further studies should be done to confirmed the results obtained and to establish why the presence of BLV provirus in semen is not constant. References1. Choi, K, et al. 2002. Vet Diagn Invest 14: 403-406. 2. Kaja, R. and Olson, C.  1982. Theriogenology 18: 107-112. 3. Miller, J. and Van Der Maaten, M. 1979. J Natl Cancer Inst 62; 425-428. 4. Monke, D. 1986. JAVMA 188 (8): 823-826. 5. Romero, C., et al. 1983.  Tropical Animal Health and production. 15: 215-218. 6. Straub O. 1982. In: Fourth International Symposium of Bovine Leukosis. The Hague: Martinus Nijhoff Publishers: 299-308.