Identification of proteins expressed by Babesia bigemina kinetes.

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.

Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages.

BACKGROUND: Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia such as B. bigemina and B. bovis. These tick-borne pathogens have a complex life-cycle involving asexual multiplication in vertebrate hosts and sexual reproduction in invertebrate vectors. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. Understanding the mechanisms that underlie formation of extracellular Babesia tick stages is an important step towards developing control strategies for preventing tick infection and subsequent parasite transmission. RESULTS: We induced B. bigemina sexual stages in vitro by exposing parasites to Tris 2-carboxyethyl phosphine (TCEP). Subsequently, we identified a novel putative methyltransferase gene (BBBOND_0204030) that is expressed uniquely in all B. bigemina tick stages but not in blood stages. In vitro TCEP-exposed B. bigemina presented diverse morphology including parasites with long projections, round forms and clusters of round forms indicative of sexual stage induction. We confirmed the development of sexual stages by detecting upregulation of previously defined B. bigemina sexual stage marker genes, ccp2 and 3, and their respective protein expression in TCEP-induced B. bigemina cultures. Next, transcription analysis of in vitro TCEP-induced B. bigemina culture based on an in silico derived list of homologs of Plasmodium falciparum gamete-specific genes demonstrated differential expression of the gene BBBOND_0204030 in induced cells. Further examination of ex vivo infected ticks demonstrated that BBBOND_0204030 is transcribed by multiple stages of B. bigemina during parasite development in tick midgut, ovary and hemolymph. Interestingly, ex vivo results confirmed our in vitro observation that blood stages of B. bigemina do not express BBBOND_0204030 and validated the in vitro system of inducing sexual stages. CONCLUSIONS: Herein we describe the identification of a B. bigemina gene transcribed exclusively by parasites infecting ticks using a novel method of inducing B. bigemina sexual stages in vitro. We propose that this gene can be used as a marker for parasite development within the tick vector. Together, these tools will facilitate our understanding of parasite-tick interactions, the identification of novel vaccine targets and, consequently, the development of additional strategies to control bovine babesiosis.

Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.