Universal primers suitable to assess population dynamics reveal apparent mutually exclusive transcription of the Babesia bovis ves1α gene

Abstract

Babesia bovis is an intraerythrocytic hemoparasite of widespread distribution, which adversely affects livestock production in many regions of the world. This parasite establishes persistent infections of long duration, at least in part through rapid antigenic variation of the VESA1 protein on the infected-erythrocyte surface. To understand the dynamics of in vivo antigenic variation among the parasite population it is necessary to have sensitive and broadly applicable tools enabling monitoring of variation events in parasite antigen genes. To address this need for B. bovis, "universal" primers for the polymerase chain reaction have been designed for the ves1α gene, spanning from exon 2 to near the 3′ end of cysteine-lysine-rich domain (CKRD) sequences in exon 3. These primers robustly amplified this segment, with minimal bias, from essentially the entire repertoire of fulllength ves1α sequences in the B. bovis Mexico isolate genome, and are equivalently present in other isolates. On purified genomic DNA, this primer set can achieve a sensitivity of 10 genome equivalents or less. When applied to the amplification of cDNA derived from the B. bovis C9.1 clonal line evidence consistent with mutually exclusive transcription of the ves1α gene was obtained, concomitant with detection of numerous mutational events among members of the parasite population. These characteristics of the primers will facilitate the application of polymerase chain reaction-based methodologies to the study of B. bovis population and antigenic switching dynamics.

Characterization of the Unusual Bidirectional ves Promoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

“…Transcription of ves genes in B. bovis occurs in a monoparalogous fashion at the locus of active ves transcription (LAT) (60). This site is organized as a pair of divergently oriented ves1␣ and ves1␤ genes (2), as is the case for the majority of this gene family (9).…”

“…Previously, reverse transcriptase-PCR analysis of steady-state RNA isolated from bulk cultured parasites strongly suggested that under normal in vivo conditions, a single ves locus is active at any one time (60). The standard conditions used in this study involved analysis of transfectants following a 24-h incubation period.…”

“…A majority of members are organized as headto-head, divergent gene pairs and most often include one ves1␣ and one ves1␤ gene, although pairs containing two ves1␣ genes have also been observed (2,9). Transcription of ves genes appears to be monoparalogous, with both ves1␣ and ves1␤ transcripts arising from a single locus (60). Although it is likely that many such loci are capable of driving transcription, to date only a single locus, termed the "locus of active ves transcription" (LAT) (2), has been observed to be transcriptionally active.…”

“…Despite their overall similarities, ves genes bear distinct branch-associated asymmetries in sequence and organization, including within the IGr. We hypothesized that this complexity was key to regulation of the locus for the previously observed monoparalogous transcription (60).…”

Assessing bovine babesiosis in Rhipicephalus (Boophilus) microplus ticks and 3 to 9-month-old cattle in the middle Magdalena region, Colombia

“…Some amplification products, after selected randomly, were purified and bidirectional sequenced. The DNA sequences were edited, assembled and aligned using Geneious Pro 5.4.4 (Zupanska et al 2009). A BLASTn (NCBI, GenBank) analysis was performed by following the same program and using the sequences obtained and reported by diverse studies all over the world, and provided by the GenBank for B. bovis and B. bigemina strains .…”