A NEW SET OF MOLECULAR MARKERS FOR THE GENOTYPING OF BABESIA BOVIS ISOLATES

MORETTA R; MOSQUEDA J; GIL G; ECHAIDE I; LÍA V; FALCON NERI A; FLORIN CHRISTENSEN, M; FARBER M; WILKOWSKY S. E

Buenos Aires, Argentina

Congreso; VI International Conference on Ticks and Tick-borne Pathogens (TTP 6); 2008

A NEW SET OF MOLECULAR MARKERS FOR THE GENOTYPING OF BABESIA BOVIS ISOLATES   Moretta R1., Mosqueda J3*., Gil G1., Echaide I. 4, Lía V1., Falcón Neri A.3, Florin Christensen M.2, Farber M1., Wilkowsky S.E1   1- Inst. de Biotecnología and 2-Patobiología CICVyA, INTA Castelar, Buenos Aires, Argentina 3-CENID-PAVET, Cuernavaca, Mexico 4- EEA INTA-Rafaela, Santa Fe, Argentina 5-ADRU-USA, Pullman, USA *Current address: Universidad Autónoma de Querétaro, Campus Juriquilla, México. Acknowledgments: The technical assistance of Daniel Aguirre, Roberto Neumann, Bibiana Cetrá and Graciela Draghi from INTA is gratefully acknowledged.   This work was financially supported by a grant from INTA-ANPCyT PICT 08-12920 and by a bilateral cooperation project between SECYT (Argentina) and CONACYT (México). M.F., V.L., M.F.C., and S.E.W are researchers of CONICET (National Research Council of Argentina)   ABSTRACT   Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constrain for the development of cattle industries worldwide. In this hemoparasite, the existence of different strains and subpopulations with different degree of pathogenicity and tick transmisibility has long been described. However, few molecular markers have been reported that could be applied for strain genotyping and characterization of reference strains or field isolates. Minisatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. In this work we report a set of 4/5?? molecular markers containing minisatellites that have shown a variable degree of polymorphism in several argentinean, mexican and USA strains. We have used a bioinformatic approach to search for sequences contained in open reading frames using the B. bovis ESTs Sequencing Project. Five genes were chosen between the multiple candidates found by the program and the corresponding primers in conserved regions flanking the repeat region were designed. Two of the genes were the previously described Bv80 and TRAP. The other two genes were named p200 and Antigen 3). Amplification by PCR, sequencing and comparative analysis of 11 strains from Argentina, Brazil, Uruguay, Mexico and USA determined that the tandem repeats varied in number and sequence among the isolates. Although some degree of similarity can be observed, these differences were not obviously related to their geographical origin. Genome analysis of the four markers described in this work revealed that the molecular markers were single copy and localized in chromosomes 2 and 3. When Bv80, Antigen 3 and TRAP were analyzed in an experimental infection with the argentinean S2P strain, both markers were identical in sequence at the beginning and at the end of the experiment (206 days post infection), indicating the stability of these markers during the course of infection. Although a large number of natural isolates must be tested to asess the feasibility of these methods, the markers proposed in this work could provide new molecular tools for the genotyping of B. bovis isolates.   Key words: Babesia bovis, minisatellites, repeats