Rap1-c gene as a molecular marker for Babesia bigemina

THOMPSON, C; BARAVALLE, E; SHKAP, V; TORIONI DE ECHAIDE, S; MANGOLD, A; ECHAIDE, I

Buenos Aires. Argentina

Conferencia; VI International Conference on Ticks and Ticks-Borne Pathogens; 2007

The hemoparasite Babesia bigemina (phylum: Apicomplexa) is one of the causative agents of bovine babesiosis. To establish the genetic diversity of B. bigemina in natural populations, molecular markers may be useful tools as they have been successfully applied for this purpose to a range of parasite species. Rhoptry associated proteins (RAP) are encoded by a family of 11 genes located in a single genomic region. The gene family is arranged in five copies of the tandemly arranged genes rap1-a and rap1-b, and the downstream rap1-c gene locus. The last two genes were found not to be expressed, and rap1-c showed polymorphism among different strains (Suarez et al., 2003). The genetic polymorphism of rap1-c, predominantly situated in the region encoding the carboxy-terminal part of RAP-1, was used by Hilpertshauser et al. (2007) for the molecular characterization of B. bigemina isolates from different geographic regions. In the present work, the rap1-c polymorphism was used to compare sixteen strains and isolates with different phenotypes from Argentina and other countries. Primers designed by Hilpertshauser were used and the generated rap1-c amplicon yielded a fragment of 284 bp from all strains and isolates as determined by sequencing. The rap1-c sequences were aligned by Clustal W® (software Bioedit) and a Neighbor-joining analysis carried out using Mega 4®. In the alignment 26 point mutations were identified accounting for 12 different alleles. The rap-1c sequences of all attenuated strains displayed a similarity of 100 % and grouped into a single cluster. Interestingly, with a similarity of only 93.8 %, the most divergent rap-1c-sequences were found to belong to the attenuated and the pathogenic Israeli strain. Seven point mutations were identified in Argentinean strains and isolates, with two of them being synonymous and three neutral. The sequence diversity between strains was found to be rather related with their phenotypic differences than with their geographic origin. Results obtained indicate that despite the low genetic diversity of less than 0.1 %, rap1-c may be used as a primary characterization marker. However, further studies need to validate its use in the characterization of variation of B. bigemina from naturally infected bovines.(Acknowledments: Work supported by ANPCyT, PICT-O12920 and TCP INTA EEA-Rafaela-Asoc. Coop. 426100. We acknowledge Babesia DNA to Dr. Lew, A. from Dept. Primary Industries & Fisheries, Qld. Australia)