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Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Highlights • Defining vulnerabilities in the life-cycle of Babesia parasites may lead to designing new control measures for babesiosis. • Poorly understood interactions between Babesia parasites and their hosts can be effectively addressed with new technologies. • Effective control of babesiosis will require additional extensive integrative research strategies., The global impact of bovine babesiosis caused by the tick-borne apicomplexan parasites Babesia bovis, Babesia bigemina and Babesia divergens is vastly underappreciated. These parasites invade and multiply asexually in bovine red blood cells (RBCs), undergo sexual reproduction in their tick vectors (Rhipicephalus spp. for B. bovis and B. bigemina, and Ixodes ricinus for B. divergens) and have a trans-ovarial mode of transmission. Babesia parasites can cause acute and persistent infections to adult naïve cattle that can occur without evident clinical signs, but infections caused by B. bovis are associated with more severe disease and increased mortality, and are considered to be the most virulent agent of bovine babesiosis. In addition, babesiosis caused by B. divergens has an important zoonotic potential. The disease caused by B. bovis and B. bigemina can be controlled, at least in part, using therapeutic agents or vaccines comprising live-attenuated parasites, but these methods are limited in terms of their safety, ease of deployability and long-term efficacy, and improved control measures are urgently needed. In addition, expansion of tick habitats due to climate change and other rapidly changing environmental factors complicate efficient control of these parasites. While the ability to cause persistent infections facilitates transmission and persistence of the parasite in endemic regions, it also highlights their capacity to evade the host immune responses. Currently, the mechanisms of immune responses used by infected bovines to survive acute and chronic infections remain poorly understood, warranting further research. Similarly, molecular details on the processes leading to sexual reproduction and the development of tick-stage parasites are lacking, and such tick-specific molecules can be targets for control using alternative transmission blocking vaccines. In this review, we identify and examine key phases in the life-cycle of Babesia parasites, including dependence on a tick vector for transmission, sexual reproduction of the parasite in the midgut of the tick, parasite-dependent invasion and egression of bovine RBCs, the role of the spleen in the clearance of infected RBCs (IRBCs), and age-related disease resistance in cattle, as opportunities for developing improved control measures. The availability of integrated novel research approaches including “omics” (such as genomics, transcriptomics, and proteomics), gene modification, cytoadhesion assays, RBC invasion assays and methods for in vitro induction of sexual-stage parasites will accelerate our understanding of parasite vulnerabilities. Further, producing new knowledge on these vulnerabilities, as well as taking full advantage of existing knowledge, by filling important research gaps should result in the development of next-generation vaccines to control acute disease and parasite transmission. Creative and effective use of current and future technical and computational resources are needed, in the face of the numerous challenges imposed by these highly evolved parasites, for improving the control of this disease. Overall, bovine babesiosis is recognised as a global disease that imposes a serious burden on livestock production and human livelihood, but it largely remains a poorly controlled disease in many areas of the world. Recently, important progress has been made in our understanding of the basic biology and host-parasite interactions of Babesia parasites, yet a good deal of basic and translational research is still needed to achieve effective control of this important disease and to improve animal and human health.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.

Elongation factor 1 alpha (EF-1alpha) is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1alpha promoter has been utilised to drive exogenous gene expression in transfected cells. In this study, we identified and characterised the ef-1alpha locus of Babesia bovis, a causative agent of bovine babesiosis, and examined the transcriptional activity of the EF-1alpha promoter. The ef-1alpha locus in the T2Bo strain of B. bovis contains two identical ef-1alpha genes ('A' and 'B') arranged in a head to head orientation and separated by a 1.4 kb intergenic (IG) region containing a 260 bp terminal inverted repeat. Both ef-1alpha genes encode identical proteins with 448 amino acids and a calculated molecular mass of 49 kDa. While the B. bovis ef-1alpha-IG sequence is conserved among multiple strains of B. bovis, it is not significantly related to any regulatory sequence in the DNA databases. The IG region promotes expression of both ef-1alpha genes. Both fragment Ig-A containing 730 bp upstream of ef-1alpha open reading frame A and fragment Ig-B containing 720 bp upstream of ef-1alpha open reading frame B were able to promote luciferase in transient transfection. In the 5' to 3' orientation, the Ig-B fragment resulted in the highest level of luciferase activity, 10 times higher than positive control plasmid p40-15-luc containing the rap-1 IG region, suggesting that this fragment contains a very strong promoter. Analysis of ef-1alpha transcripts confirms that both ef-1alpha genes are transcribed in merozoites. Interestingly, in contrast to other related intra-erythrocytic apicomplexans, the ef-1alpha locus of B. bovis contains a 160 bp intron in the 5' untranslated region.

Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp. has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, a plasmid was designed to target integration of a gfp-bsd gene into the Babesia bovis ef-1alpha locus. Babesia bovis-infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids and selected in cultures with varying amounts of blasticidin 24h after electroporation. Several blasticidin-resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by reverse transcriptase-PCR, Western blot analysis and fluorescence microscopy for longer than 9 months after electroporation. No plasmid or episomal DNA could be detected in this line, and plasmid recovery in Escherichia coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1alpha locus in the transfected parasite line. Overall the results demonstrate, we believe for the first time, chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease and immunity of B. bovis, one of the three major tick-borne parasites that limit global livestock production.

Members of the Babesiarap-1 gene family are expressed during multiple parasite stages, and are regulated by both transcriptional and post-transcriptional mechanisms. In all Babesia species, tandemly arranged rap-1 gene copies are separated by an intergenic (IG) region that is hypothesized to regulate gene expression. In this study, we tested that hypothesis by determining whether the Babesia bovisrap-1 IG region could promote extra-chromosomal expression of exogenous genes introduced into merozoites by transfection, and whether a tandem arrangement of IG regions similar to the rap-1 locus enhances exogenous gene expression. Initially, electroporation conditions of B. bovis parasites were determined using expression of the reporter luciferase gene. Both B. bovis transfected by electroporation and Escherichia coli transformed with plasmid p40-15-luc containing the luciferase gene under the control of the B. bovisrap-1 IG and 3' flanking regions were able to express luciferase, indicating that the rap-1 IG region contains a functional promoter. The chromosomal organization of the B. bovisrap-1 locus includes two identical rap-1 open reading frames and IG regions in a head to tail orientation. To determine whether this orientation enhanced expression of exogenous genes, plasmid constructs containing two rap-1-IG regions controlling expression of the luc and human dihydrofolate reductase (hdhfr) genes, and oriented either in head to head (pLuc-H-13) or head to tail (pLuc-H-18) arrangement, were compared. The head to tail orientation of the gene cassettes resulted in a significant increase in the level of luciferase as compared to either head to head orientation or a single IG region construct (p40-15-luc). Thus, an organization that mimics the native structure of the rap-1 locus results in enhanced luciferase expression. These results are the first to demonstrate exogenous gene expression in B. bovis after transfection, and to confirm that the B. bovisrap-1 IG region can promote extra-chromosomal gene expression in vivo.