Isolation of Parainfluenza virus 3 genotype B from water buffalo. Incorporation of this genotype in an ELISA to detect Parainfluenza virus type 3

CECILIA FERRUFINO; SILVINA SOLEDAD MAIDANA; VIVIANA PARREÑO; GUSTAVO CRUDELI; SONIA ALEJANDRA ROMERA

Puket

Congreso; 10th Congreso Mundial de Búfalos y 7th Congreso de Búfalo Asiático; 2013

Asociación internacional de búfalos

Parainfluenza virus type 3 (PIV3) is an enveloped, single-stranded negative sense RNA virus within the Respirovirus genus of the Paramyxoviridae family.During the period 2009-2011, one bubaline and six bovine PIV3 field isolates were obtained and phylogenetically analyzed. These preliminary results showed that PI3 isolates from bovines have a high identity to the genotypes A and C, and isolate from buffalo to the genotype B.Actually the hemagglutination inhibition is the gold standard technique to evaluate the immune status on a herd against PI3; however, this technique has low sensitivity. To overcome this point an indirect ELISA was developed to evaluate the presence of antibodies from both bovine and Guinea pig against PI3. With this purpose the 3 genotypes were amplified on Madin Darby bovine kidney (MDBK) cell culture, afterwards the supernatant was clarified and pelleted. Different blocking solutions and incubation times of sera were tested.ELISA results showed that there are some differences in the recognition of the field sera to each genotypes therefore was necessary to coating plates with all genotypes in concentration equal.While the optimal conditions of the ELISA for bovine antibodies were incubation with PBS-T OVA 1% as a blocking solution and 1 hour of incubation time for the serum, an additional step of pre-incubation with MDBK cells was necessary for guinea pig antibodies.We evaluated different sera samples from infected, vaccinated and negative animals and calculated the cut off. Positive sample was considered when the sera optical density is greater than 0.3. Regarding guinea pig ELISA cut off is necessary to increase the number of the samples for to establish the final cut off. This study reports the development of an alternative tool for the detection of bovine and guinea pig antibodies against PI3. This assay will be useful to evaluate the seroprevalence of PI3 in cattle and also to analyze the immunogenicity generated by vaccines in a guinea pig model before its use in natural host.