Coinfection with antigenically and genetically distinct virulent strains of Babesia bovis is maintained through all phases of the parasite life cycle

Abstract

Antigenic polymorphism is a defining characteristic of the Babesia bovis variable merozoite surface antigen (VMSA) family. Sequence analysis strongly suggests that recombination between virulent strains contributes to VMSA diversity. While meiosis during the aneuploid stage of the parasite's life cycle in the tick vector Rhipicephalus (Boophilus) microplus is the most probable source of interstrain recombination, there is no definitive evidence that coinfection of the mammalian host or R. microplus ticks with more than one virulent strain occurs. Using allele-specific real-time quantitative PCR, we tested the hypotheses that cattle could support coinfection of two antigenically variant virulent tick-transmissible strains of B. bovis and that R. microplus ticks could acquire and transmit these two divergent strains. The results indicate that both calves and ticks can support virulent B. bovis coinfection through all phases of the hemoparasite's life cycle. Neither strain dominated in either the mammalian or invertebrate host, and larval tick progeny, which could be coinfected individually, were also able to transmit both strains, resulting in virulent babesiosis in recipients. While coinfection of the tick vector provides the context in which allelic antigenic diversity can be generated, recombination of VMSA genes could not be confirmed, suggesting that VMSA allelic changes are slow to accumulate.

FIG. 1.

Evaluation of the sensitivity and specificity of RTQ-PCR allele-specific primer/probe sets. (A) Standard curve generated by T2Bo-specific RTQ-PCR amplification of octuplet samples of T2Bo sbp-1-containing plasmids serially diluted from 1 × 10⁷ to 1 copy. r is the correlation coefficient. This curve is representative of those generated by the amplification of L sbp-1-containing plasmids with the L-specific primer/probe set. (B) RTQ-PCR amplification using T2Bo and L sbp-1 allele-specific primer/probe sets was performed on individual strains or mixtures of the T2Bo (T) and L (L) strains. E2 and E4 are 1 × 10² and 1 × 10⁴ genome equivalents, respectively. Each data bar represents the mean threshold cycle value per triplicate set of amplification reactions. Target DNA quantities are labeled above each bar. The left half of the graph shows threshold cycle values for amplification reactions carried out using the T2Bo-specific primer/probe set, and the right half of the graph shows threshold cycle values for amplification reactions carried out using the L-specific primer/probe set, as labeled.

FIG. 2.

Allele-specific RTQ-PCR analysis of calves with intact spleens coinfected with two virulent strains of B. bovis. T2Bo and L strain parasitemia levels on a log₁₀ scale per microliter of blood from calves c4537 (A) and c4574 (B) were charted against days post-intravenous inoculation with a stabilate containing approximately equal numbers of T2Bo and L strain-infected erythrocytes. ND, nondetectable target. Both calves were treated on day 13 postinoculation for clinical signs of babesiosis. Significant differences in infection level between the two strains occurred at 10 to 13 dpi in both calves (P < 0.05).

FIG. 3.

Allele-specific RTQ-PCR analysis of an acquisition feed splenectomized calf during the time of female adult tick engorgement. T2Bo and L strain parasitemia levels on a log₁₀ scale per microliter of blood from splenectomized calf 1084 are shown for multiple days post-intravenous inoculation with a stabilate containing approximately equal numbers of T2Bo and L strain-infected erythrocytes. ND, nondetectable target. Significant differences in infection level between the two strains occurred at 11, 12, and 14 dpi (P < 0.05).

FIG. 4.

Allele-specific RTQ-PCR analysis of a transmission feed splenectomized calf. T2Bo and L strain parasitemia levels on a log₁₀ scale per microliter of blood from splenectomized calf 1101 were charted against days postplacement of larvae derived from adult female ticks that had acquisition fed on coinfected calf 1084. ND, nondetectable target. The calf was euthanized on day 12 after the placement of larvae due to clinical signs of babesiosis. Significant differences in infection level between the two strains occurred on days 10 and 12 post-larval placement (P < 0.05).