Development of an Immunosensor for the Diagnosis of Bovine Anaplasmosis

SILVA M; WILKOWSKY S; ECHAIDE S; FARBER M; OLIVA A

Hanoi, Vietnam

Congreso; 8th Biennial Conference of the Society for Tropical Veterinary Medicine; 2005

Society for Tropical Veterinary Medicine

Development of an Immunosensor for the Diagnosis of Bovine Anaplasmosis Marta G. Silva1, Silvina Wilkowsky2, Susana Echaide3, Marisa Farber2, Abel Oliva1 Marta G. Silva1, Silvina Wilkowsky2, Susana Echaide3, Marisa Farber2, Abel Oliva1 1Biosensors Laboratory, Instituto de Biologia Experimental e Tecnológica (IBET) / Instituto de Tecnologia Química e Biológica (ITQB), Apartado 12, P-2781-901 Oeiras, Portugal. 2Instuto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina 3Estacion Experimental Agropecuaria, Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina Bovine anaplasmosis, a vector-borne rickettsial disease of cattle, is caused by Anaplasma marginale and Anaplasma centrale. Ticks biologically transmit Anaplasma species, while biting flies and fomites can mechanically transfer infected blood to susceptible cattle. Infection occurs in tropical and subtropical areas of the world where ticks are enzootic. Clinical disease, caused by replication of the rickettsiae in erythrocytes, is characterized by anemia, weight loss, and death. Previous work demonstrated that major surface protein 5 (MSP5) is present in all recognized Anaplasma spp. and that the MSP5 protein is broadly conserved among Anaplasma spp. and isolates. The data presented here resulted from testing the hypothesis that an MSP5-based immunosensor would detect anti-Anaplasma sp. antibodies in acute infection as well as in vaccinated and experimentally infected cattle. This study was performed using recombinant MSP5 covalently immobilised in controlled pore glass (CPG) beads to detect anti- MSP5 antibodies in serum samples. The quantification is based on the measurement of the Cy5 fluorescence of the detection antibody, anti bovine IgG, after reaction with serum. Sera were collected in enzootic and tick-free regions of Argentina. The samples were the following: A) sera from an outbreak of Anaplasma from a non-endemic area, B) sera from vaccinated bovines from non-endemic area; C) negative sera from non-endemic area; D) sera from vaccinated bovines from endemic area; E) sera from A. centrale-vaccinated animals before and after being challenged with A. marginale. All sera were also analized with an indirect ELISA using the same MSP5.  The results showed that the MSP5 immunosensor detected antibodies in acute infections as well as in vaccinated animals, with higher sensitivity when compared with ELISA using the same antigen. Besides, the immunosensor can also quantitate the anti MSP5 levels in experimentally infected animals. A detection range of 1.2 μg/ml to 48 μg/ml of antibody in sera was obtained. The optical immunosensor developed is suitable for quantification of antibodies in sera of naturally or experimentally infected animals. Besides, it presents advantages, to the standard immunoassay techniques like simplicity and rapidity of measurement, sensitivity and reproducibility and offers an alternative to conventional serologic methods. Detailed description of the results will be presented in the paper. This work was financially supported by Secretaría de Ciencia y Tecnología (SECyT), Argentina and Gabinete de Relações Internacionais da Ciência e do Ensino Superior (GRICES), Portugal, as an international cooperation grant.