Multilocus Sequence Typing for Characterization of Babesia bovis and Babesia bigemina strains

GUILLEMI E.; RUYBAL P; GONZALEZ S; FARBER M; WILKOWSKY S

Zaragoza

Conferencia; TTP7: Ticks and Tick-borne pathogens International Conference; 2011

Seventh Ticks and Tick-borne Pathogens International Conference

The multilocus sequence typing (MLST) is a highly reproducible and discriminatory method of characterizing isolates on the basis of the nucleotide sequence of PCR fragments of six to seven genes coding for conserved metabolic functions. For each gene fragment, the different sequences are assigned as distinct alleles; therefore, each isolate is defined by the alleles at each of the six or seven loci. This is called allelic profile or sequence type (ST).. The aim of this study wasto develop an MLSTscheme for Babesia bovis and B. bigemina. Both protozoans are enzootic in north Argentina and cause major economic losses to livestock industry. We first started by pre-selecting 23 single-copy genes encoding hypothetical proteins from the annotated genome of Babesia bovis. These genes are well distributed in the 4 chromosomes of the parasite. For B. bigemina, 20 genes were identified by TBLASTN using the orthologousgenes of B. bovis as query. In both parasites, primers were designed to obtain a PCR fragment of 500 to 700bp. Seven genes for B. bovis (check, dnaJ, gpad, pkid, rcc, rip9 and rho4) and 6 for B. bigemina (cyp, dnaJ, rcc, sbp3, sbp4 and zfc) were finally chosen. The selection was based on: number of single nucleotide polymorphic sites (SNPs), chromosome distribution, absence of selective pressure (dN/dS<1) and specificity against heterologous DNA. We then analized 9 B. bovis isolates. These included the reference strain from the genome project (T2Bo), 5 argentine reference strains and 3 isolates from clinical acute cases. For B. bigemina, 13 isolates were studied: 7 reference strains from Argentina and 2 from Mexico and Brazil. We also included the australian virulent strain from the genome project. Besides, we analyzed a clinical acute case, a first passage through R. microplus  ticks and a second syringe passage in a splenectomized calf. Nine STs were found for the 9 isolates of B. bovis and the same number of STs were found for the 13 isolates studied in B. bigemina. Phylogenetic analyses were performed using Maximum Likelihood (ML) method for each locus separately and for the concatenated sequences using PhyML (version 2.4.4) with 500 bootstrap replicates. To further investigate if the topology observed for the concatenated sequences was supported by the distribution of the various haplotypes, a similar phylogenetic analysis was conducted on each locus separately. Tree topologies were different than those observed for the concatenated sequences, with different topologies for each locus. In B.bovis, the locus yielding the topology resembling the most that of the concatenated genes was rip9 while in B.bigemina was rcc In this parasite, we identified two distinct clusters for the argentinean strains, one for the pathogenic strains and the other  composed exclusively of attenuated strains. The 2 foreign strains grouped accordingly to this pattern. DNA polymorphism analysis was performed using the DnaSP 5.00.02 package and reflected very low number of haplotypes or alleles, with the exception of rip9 in B.bovis and cyp, rcc and zfc in B.bigemina which were significantly more diverse. Recombination events were detected for 3 genes in both parasites accompanied by low linkage disequilibrium figures and higher rates of synonymous substitutions compared with non-synonymous substitutions which may be indicative of negative selective pressure. In conclusion, an MLST scheme was developed for the first time for the genus Babesia and it allowed a precise genotyping of these protozoans. In the case of B. bigemina, this scheme also grouped strains according to phenotypic differences. In both organisms, a larger set of samples will allow us address issues such as genome plasticity and recombination present in co-circulating strains of Argentina. Financial support was obtained from ANPCYT- PICT 1634/CONICET