Development of a satellite marker system to distinguish Babesia bovis geographic isolates.

PEREZ LLANEZA, A; CABALLERO, M; BARAVALLE, E; MESPLET, M; MOSQUEDA, J; ECHAIDE, I; SUAREZ, C; KATZER, F; FLORIN CHRISTENSEN, M; SCHNITTGER, L

Buenos Aires. Argentina

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

The tick-borne hemopathogen Babesia bovis is a major cause of bovine babesiosis in tropical and subtropical areas worldwide. Live vaccines are often used to prevent outbreaks in regions of enzootic instability. Although these are usually effective, vaccine breakthroughs are observed resulting in significant losses of cattle heads. It would thus be important to have a molecular marker system able to differentiate whether vaccine failures are due to mishandling or infection with other field strains escaping protection. Furthermore, it would be desirable to know the distribution of strains throughout geographical regions. Mini and microsatellite sequences have proven to be distinguished tools for the differentiation of strain populations in other protozoan parasites due to their high variability. In the present work we have identified 19 polymorphic satellite markers in B. bovis isolates originating from five different geographic locations of North, and South America (Texas, USA, (T2B); Santa Fe, Argentina (R1A); Salta, Argentina (S2P); Mexico (RAD); and Mexico (M07)). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. All primers used did not amplify genomic DNA from B. bigemina, the principal co-infecting bovine parasite in this geographical region, which allows their use in field surveys. The 20 satellite markers identified are distributed throughout the 4 chromosomes of B. bovis as follows: chromosome 1 (n=4), chromosome 2 (n=2), chromosome 3 n=8), and chromosome 4, (n=6). Within the five B. bovis isolates we identified 2 alleles in 6, 3 alleles in 11, and 4 alleles in 3 tested marker loci, respectively. The number of microsatellite sequences within the B. bovis genome seems to be significantly reduced in comparison to other eukaryotes, as suggested by the identification of 17 polymorphic minisatellites (period size: 6-21 nt) but only 2 polymorphic microsatellites (period size: 2-5 nt) in this study. The multilocus genotype of each of the 5 B. bovis isolates was assessed and the genetic similarity determined. In all cases a larger distance between their geographic origin corresponded with a lower genetic similarity. Acknowledgments: This work was supported by the EC (CT-2005-INCO 003691, MEDLABAB)