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

GUILLEMI ELIANA; RUYBAL PAULA; GONZALEZ SERGIO; FARBER MARISA; WILKOWSKY SILVINA

Conferencia; TTP7 Ticks and Tick-borne pathogens international conference; 2011

The multilocus sequence typing (MLST) is a highly 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, and each isolate is defined by the alleles at each of the six or seven loci. This is called the allelic profile or sequence type (ST). The MLSTs system is highly reproducible and discriminative and allows a thorough analysis of the population structure. The aim of this study was to develop an MLST scheme 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 were well distributed in the 4 chromosomes of the parasite. For B. bigemina 20 genes were identified by TBLASTN using the orthologous genes of B. bovis as query genome. For both parasites, primers were designed to obtain a PCR fragment of 500 to 700-bp. Seven genes for B. bovis (check, dnaJ, gpad, pkid, rcc, rip9 and rho4) and six 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 (Ka/Ks 1) and specificity against heterologous DNA. The number of isolates analized was 14 for B. bovis. these include the reference strain from the genome project (T2Bo), 6 argentine reference strains and 7 isolates from clinical acute cases. For B. bigemina, 13 isolates were studied: 7 reference strains from Argentina and 2 from foreign countries. We also included the australian virulent strain from the genome project. Besides, we analyzed a clinical acute case, a first passage through ticks and a second syringe passage in a splenectomized calf.. Nine 14 STs were found for the 14 isolates of B. bovis analyzed and 10 STs were found for the 12 isolates studied in B. bigemina. Phylogenetic analyses were performed using Maximum Likelihood (ML) method for each locus separately and for the concatenated sequences using Mega 5.05 software with 1.000 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 .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 rcc and zfc in B.bigemina which were significantly more diverse. Recombination events, were detected for in 3 genes in both parasite accompanied by low linkage disequilibrium figures. The most striking features, however, were the almost complete absence of singletons and non-synonymous substitutions, indicative of negative selection and absence of population expansion; this seems to be the result of a very strong adaptation to a very specific cell type and cell environment.MLST scheme seems to be a useful tool for isolate discrimination. In the case of B. bigemina this scheme allowed us to discriminate strains with different phenotype as the dendogram split pathogenic strains from attenuated ones.  For the case of B. bovis, taking into account that the sample size is small, we can not rule out that associations will be detected after analyzing a larger number of samples.