Molecular comparisons of Babesia odocoilei using the internal transcribed spacers of ribosomal RNA

Babesia odocoilei is an intraerythrocytic apicomplexan parasite which infects cervidae, sometimes causing babesiosis. It is vectored by the tick Ixodes scapularis and is distributed throughout the southeastern United States. The geographic and host range continue to extend as new incidence of infection is detected. A genomic DNA region spanning the internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and ITS2 of ribosomal RNA (rRNA) from 18 B. odocoilei isolates (speciation confirmed by small subunit rRNA analysis) was amplified using the polymerase chain reaction, cloned and sequenced. The isolates originated from 6 different cervidae or bovidae hosts in various U.S. geographic areas. Included in the analysis was a previously described reindeer B. odocoilei-like isolate, RD61, which showed only 99.0% identity in SSU rRNA analysis to B. odocoilei. Percent identity pairwise comparisons among the samples were calculated for both the full ITS1-5.8S-ITS2 and individual genomic regions. Identity values for all comparisons ranged from 90% to 100%, with the exception of RD61, which showed no higher than 88% identity for all gene regions. An analysis of fixed differences identified in the ITS1 and ITS2 gene regions of all clones revealed 21 fixed differences in ITS1, and only 11 in ITS2. Most isolates were found to have 2 overall patterns of fixed differences, although some had 1 or 3. Phylogenetic analysis of all sequences for the entire ITS1-5.8S-ITS2 gene region placed most isolates into 2 distinct groups corresponding to those observed in the analysis of fixed differences. This suggested the presence of at least 2 rRNA transcription units in B. odocoilei. ITS analysis failed to demonstrate host or geographic differences that might serve to pinpoint the source of outbreaks of B. odocoilei in farmed and managed host animals. This failure might result from genetic recombination of ITS genomic regions during the tick vector stage. Lack of conspecificity between the RD61 isolate and B. odocoilei was supported by this study; however, more data are needed to clarify the taxonomic status of this B. odocoilei-like isolate