MOLECULAR METHODS FOR THE DISCRIMINATION OF BABESIA BOVIS STRAINS THROUGH THE USE OF MINISATELLITES

MORETTA R; ECHAIDE I; MOSQUEDA J; JACOBSEN M; SUAREZ C; FARBER M; WILKOWSKY S

Castelar, Buenos Aires, Argentina.

Congreso; Babesia World Summit; 2005

Centro de Investigaciones Veterinarias y Agronómicas, CICVyA

MOLECULAR METHODS FOR THE DISCRIMINATION OF BABESIA BOVIS  STRAINS THROUGH THE USE OF MINISATELLITES Moretta R1., Echaide I2., Mosqueda J5., Jacobsen M3., Suarez C4., Farber M1., Wilkowsky S1.1Inst. de Biotecnología,2 EEA INTA Rafaela, 3Inst. de Patobiología, CICV, INTA Castelar, Argentina. 4USDA, Pullman USA. E-mail: swilkowsky@cicv.inta.gov.ar Prevention against the tropical tick-borne parasite of cattle, Babesia bovis, is currently obtained by vaccination with attenuated strains. When vaccine failures sporadically happen, it would be convenient to differentiate whether they are due to vaccine mishandling or to infection with Babesia field strains against which the vaccinial strains are not protective. Strain-specific molecular markers could be useful tools for this purpose and, in addition, they could serve to correlate transmission or virulence with specific B. bovis subpopulations. Besides, these markers could be used to map strain distribution throughout geographical regions for epidemiological studies. This work presents preliminary data on the development of a simple method that could be applied to B. bovis strain identification. Our aim was to identify DNA sequences that contain tandem repeats (minisatellites) useful for the molecular typification of B. bovis strains. We have used the information provided by the B. bovis ESTs Sequencing Project of the Sanger Institute to select sequences contained in ORFs and the information of the Genome Sequencing Project at Washington State University to eliminate intron-containing sequences. The search was done with the Tandem Repeat Finder program. Of all the sequences detected, 2 were chosen for further analysis: Bv80 and Bv200. We have analized by PCR 11 isolates from Argentina, Mexico, Brazil, Uruguay and USA using primers within conserved regions flanking the minisatellites. In all cases, amplicons were obtained for both probes. No product was obtained when B. bigemina DNA was used. A clear polymorphism was observed in many strains, suggesting the association of these polymorphisms to the presence of a variable number of repeats. Direct sequencing of the Bv80 PCR products and further analysis confirmed that the different sizes of the Bv80 amplicons were due to a variable type and number of repeats Future studies will determine if the PCR tests developed in this work could be implemented as efficient tools for the identification and characterization of B. bovis field strains. Comparative genomic analysis will ascertain the functional role of these minisatellites.