Molecular Tools for Babesia bigemina Detection

ROMINA PETRIGH; PAULA RUYBAL; CAROLINA THOMPSON; ROBERTO NEUMANN; ROSALIA MORETTA; SILVINA WILCOWSKY; GRACIELA DRAGHI; IGNACIO ECHAIDE; SUSANA TORIONI DE ECHAIDE; MARISA FARBER

Merida, Yucatan , Mexico

Conferencia; 9th Biennial Conference of the Society for Tropical Veterinary Medicine; 2007

Bovine babesiosis caused by Apicomplexa protozoa Babesia bigemina and Babesia bovis is considered one the most economically important tick-transmitted disease of cattle in tropical ands sub-tropical areas of the world.  In Argentina, acute infections are diagnosed by microscopic examination of blood smears. On the other hand, the currently serological tests used to determine the babesial infection statuses of herds are immunofluorescence (IFAT) and indirect ELISA based on crude parasite antigens recognition. The application of PCR-based assays to study the epidemiology of babesiosis is still incipient. Current molecular detection of B. bigemina involves a nested-PCR protocol (Figueroa et al., 1992), and Reverse Line Blot hybridization (RLB) assay based on the 18S gene (Gubbels et al., 1999). Moreover, RLB is even better as it enables the detection of other  pathogens  that coexist within the endemic area of babesiosis. In this study, we report two new molecular approaches to improve B. bigemina detection in bovine blood samples:- a one step PCR assay based in the amplification of rap-1a paralogous;- a RLB membrane including 4 new 18S probes. The commercially available RLB membrane from Isogen failed to detect B. bigemina isolates from Argentina. Analysis of PCR-product sequences showed nucleotide variations within the probe region. Furthermore, each isolate showed more than one sequence variation reflecting a mixed population composition. Based on these observations we designed a new membrane including this polymorphic probes and a new B. bigemina specie-specific probe. The B. bigemina rap-1 gene locus contains four  tandemly arranged copies of rap-1a genes. The highly conserved region was selected for designing specific primers so as to amplify a 400bp fragment. Specificity for both techniques was tested against different B. bigemina (BgM1A, BgM1P, BgM2P, BgS1A, BgS2P and BgBr1P), B. bovis (BvS2P, BvM2P, BvR1A and Corrientes), A.marginale (Virasoro) and A.centrale isolates. On the other hand, the sensitivity is being calculated by performing PCR and RLB assays on a blood sample with a known parasitemia that had been 10-fold serially diluted in parasite free bovine blood. Comparison among two laboratories is being performed in order to establish PCR robustness.   Finally, both assays are being used for testing field samples from Argentina enzootic region to evaluate the usefulness of each of them.