Identification of proteins expressed by Babesia bigemina kinetes

Abstract

Background: Babesia bigemina is an apicomplexan parasite transovarially transmitted via Rhipicephalus ticks that infect red blood cells and causes bovine babesiosis, a poorly controlled severe acute disease in cattle. New methods of control are urgently needed, including the development of transmission blocking vaccines (TBV). Babesia bigemina reproduces sexually in the gut of adult female R. microplus upon acquisition following a blood meal. Sexual reproduction results in zygotes that infect gut epithelial cells to transform into kinete stage parasites, which invade tick ovaries and infects the egg mass. The subsequent tick generation transmits B. bigemina upon feeding on bovine hosts. An important limitation for developing novel TBV is that the pattern of protein expression in B. bigemina tick stages, such as the kinete stage, remain essentially uncharacterized.

Results: We determined the protein expression profile of three B. bigemina putative tick stage candidates BbiKSP (BBBOND_0206730), CCp2 and CCp3. We found that BbiKSP expression was restricted to B. bigemina kinetes. CCp2 and CCp3, previously shown to be expressed by induced sexual stages, were also expressed by kinetes. Importantly, none of these proteins were expressed by B. bigemina blood stages.

Conclusions: Babesia bigemina kinetes express BbiKSP, CCp2 and CCp3 proteins, therefore, these proteins may play important roles during B. bigemina development within tick hemolymph and may serve as potential candidate targets for the development of TBV.

Keywords: Babesia bigemina; Kinete; Kinete stage-specific protein; Rhipicephalus microplus; in vitro induced sexual stages.

Fig. 1

Detection of BbiKSP transcripts during B. bigemina development in the mammalian and tick hosts. (a) Blood, midguts from day 0 to 6 post-incubation of engorged female ticks and infected hemolymph. To demonstrate that Bbiactin was specific for B. bigemina (b) B. bigemina actin was detected in infected blood but not in midgut from uninfected female tick. R. microplus α-Tubulin was used to demonstrate tick RNA quality. RT− and RT+ indicate the absence or presence of reverse transcriptase

Fig. 2

Detection of BbiKSP transcripts in non-induced blood and in vitro induced cultures at multiples time points. Bbiactin was used as a reference gene. RT− and RT+ indicate the absence or presence of reverse transcriptase

Fig. 3

Immunofluorescence assays demonstrating expression of BbiKSP (a), CCp 2 (b), CCp3 (c) by B. bigemina kinetes but not blood stages and bovine anti-KLH as control (d). Scale-bars: 3 and 5 µm