title: Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates.
abstract: 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 repeats (TRs) 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 (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 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, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and own a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each other determined followed by cluster analysis based on neighbor joining. The resulting phenogram showed that B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore various parameters of B. bovis populations such as genetic diversity, infection dynamics and their structure under different epidemiological situations, which are of crucial importance for improved control strategies.
authors.affiliation: Institute of Pathobiology, CICVyA, INTA-Castelar, Buenos Aires, Argentina.
authors.forename: Agustina
authors.initials: A
authors.lastname: Perez-Llaneza
authors.forename: Marina
authors.initials: M
authors.lastname: Caballero
authors.forename: Eugenia
authors.initials: E
authors.lastname: Baravalle
authors.forename: Maria
authors.initials: M
authors.lastname: Mesplet
authors.forename: Juan
authors.initials: J
authors.lastname: Mosqueda
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
authors.forename: Ignacio
authors.initials: I
authors.lastname: Echaide
authors.forename: Frank
authors.initials: F
authors.lastname: Katzer
authors.forename: Gabriela M
authors.initials: GM
authors.lastname: Pacheco
authors.forename: Monica
authors.initials: M
authors.lastname: Florin-Christensen
authors.forename: Leonhard
authors.initials: L
authors.lastname: Schnittger
mesh_terms: D000818:Animals; D001118:Argentina / Q000453:epidemiology; D016793:Babesia bovis / Q000235:genetics*; D001404:Babesiosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology / Q000469:parasitology*; D008800:Mexico / Q000453:epidemiology; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013781:Texas / Q000453:epidemiology