Detection of BLV in frozen semen samples by PCR assay.

Intermitent detection of BLV in frozen semen simples 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. Introduction: The 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 present[1-6]. 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 & methods: Samples: 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. Results: Two 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 & conclusions: The 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. References : 1. 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

Detección del BLV en muestras de semen de toros seropositivos

Detección del BLV en muestras de semen de toros seropositivos Rossich Luciano1; Gutierrez Geronimo1; Rodriguez Sabrina1; Lefebvre Eduardo2; Trono Karina1; Dus Santos Maria Jose1. (1) Instituto de Virología, CICVyA, INTA-Castelar. CC 77. Buenos Aires, Argentina. (2) Centro de Inseminación Artificial La Elisa. Argentina. La principal ruta de transmisión del Virus de la Leucosis Bovina (BLV) es horizontal y esta dada por la exposición directa de fluidos biológicos contaminados con linfocitos infectados; principalmente sangre. A pesar de que antígenos virales y ADN proviral han sido identificados en semen, leche y calostro; la transmisión natural a través de estas secreciones no ha sido demostrada. El impacto sanitario y económico de la infección por BLV esta asociada con la interferencia en el movimiento internacional del ganado vacuno y su germoplasma. Datos experimentales soportan la improbabilidad de que el semen congelado de toros seropositivos para BLV puedan infectar vacas receptoras, sin embargo, aun hoy existen restricciones para la comercialización de semen de animales infectados. Dado que no existe vacuna ni tratamiento aplicable, la erradicación y control de la enfermedad esta exclusivamente basada en el diagnóstico temprano y segregación de los animales infectados. En este contexto la especificidad y la sensibilidad del diagnóstico utilizado constituyen un punto crítico. Hemos desarrollado previamente una PCR con alta sensibilidad y especificidad para detectar el genoma de BLV en muestras de semen congelado. El objetivo de este trabajo es el estudio del patrón de excreción natural de BLV en semen. Muestras de semen congelado de 30 toros seropositvos fueron analizados. Fue posible detectar ADN proviral en 144 de 752 muestras. La detección del genoma del BLV ha ocurrido en varias colecciones pero en forma alternada con períodos de no detección. Adicionalmente, la presencia de ARN proviral en sangre y muestras de sangre fue evaluada

Understanding interactions of organic nitrates with the surface and bulk of organic films: implications for particle growth in the atmosphere

Understanding impacts of secondary organic aerosol (SOA) in air requires a molecular-level understanding of particle growth via interactions between gases and particle surfaces. The interactions of three gaseous organic nitrates with selected organic substrates were measured at 296 K using attenuated total reflection Fourier transform infrared spectroscopy. The organic substrates included a long chain alkane (triacontane, TC), a keto-acid (pinonic acid, PA), an amorphous ester oligomer (poly(ethylene adipate) di-hydroxy terminated, PEA), and laboratory-generated SOA from α-pinene ozonolysis. There was no uptake of the organic nitrates on the non-polar TC substrate, but significant uptake occurred on PEA, PA, and α-pinene SOA. Net uptake coefficients (γ) at the shortest reaction times accessible in these experiments ranged from 3 × 10-4 to 9 × 10-6 and partition coefficients (K) from 1 × 107 to 9 × 104. Trends in γ did not quantitatively follow trends in K, suggesting that the intermolecular forces involved in gas-surface interactions are not the same as those in the bulk, which is supported by theoretical calculations. Kinetic modeling showed that nitrates diffused throughout the organic films over several minutes, and that the bulk diffusion coefficients evolved as uptake/desorption occurred. A plasticizing effect occurred upon incorporation of the organic nitrates, whereas desorption caused decreases in diffusion coefficients in the upper layers, suggesting a crusting effect. Accurate predictions of particle growth in the atmosphere will require knowledge of uptake coefficients, which are likely to be several orders of magnitude less than one, and of the intermolecular interactions of gases with particle surfaces as well as with the particle bulk