Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates

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

VETERINARY PARASITOLOGY

ELSEVIER SCIENCE BV

Año: 2010 vol. 167 p. 196 - 204

0304-4017

Mini and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeat sequences that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct 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. Fourteen tandem repeat sequences could be reliably amplified in all isolates and were found to be polymorphic. All primers used were specific for B. bovis and did not amplify genomic DNA from B. bigemina, the principal co-infecting bovine parasite in the Americas, allowing their use in field surveys. The fourteen satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n=3), chromosome 2 (n=2), chromosome 3 (n=5), and chromosome 4 (n=4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, and one with four and five alleles, respectively. The frequency of microsatellite sequences within the B. bovis genome seems to be significantly reduced in comparison to other eukaryotes, as suggested by the identification of only two polymorphic microsatellites out of the fourteen polymorphic markers identified in this study. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each of them determined. A cluster analysis based on Neighbor Joining was carried out and a phenogram constructed. B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore, in different epidemiological situations, genetic parameters of B. bovis populations such as genetic diversity, infection dynamics and population structure which are of crucial importance for improved control strategies.