Search

Background The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Babesiosis is a tick-borne disease with global impact caused by parasites of the phylum Apicomplexa, genus Babesia. Typically, acute bovine babesiosis (BB) is characterized by fever, anemia, hemoglobinuria, and high mortality. Surviving animals remain persistently infected and become reservoirs for parasite transmission. Bovids in China can be infected by one or more Babesia species endemic to the country, including B. bovis, B. bigemina, B. orientalis, B. ovata, B. major, B. motasi, B. U sp. Kashi and B. venatorum. The latter may pose a zoonotic risk. Occurrence of this wide diversity of Babesia species in China may be due to a combination of favorable ecological factors, such as the presence of multiple tick vectors, including Rhipicephalus and Hyalomma, the coexistence of susceptible bovid species, such as domestic cattle, yaks, and water buffalo, and the lack of efficient measures of tick control. BB is currently widespread in several regions of the country and a limiting factor for cattle production. While some areas appear to have enzootic stability, others have considerable cattle mortality. Research is needed to devise solutions to the challenges posed by uncontrolled BB. Critical research gaps include risk assessment for cattle residing in endemic areas, understanding factors involved in endemic stability, evaluation of parasite diversity and pathogenicity of regional Babesia species, and estimation of whether and how BB should be controlled in China. Research should allow the design of comprehensive interventions to improve cattle production, diminish the risk of human infections, and increase the availability of affordable animal protein for human consumption in China and worldwide. In this review, we describe the current state of BB with reference to the diversity of hosts, vectors, and parasite species in China. We also discuss the unique risks and knowledge gaps that should be taken into consideration for future Babesia research and control strategies.

Background Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. Results In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. Conclusions Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis. Graphical abstract

The incidence and prevalence of babesiosis in animals and humans is increasing, yet prevention, control, or treatment measures remain limited and ineffective. Despite a growing body of new knowledge of the biology, pathogenicity, and virulence of Babesia parasites, there is still no well-defined, adequately effective and easily deployable vaccine. While numerous published studies suggest that the development of such anti-Babesia vaccines should be feasible, many others identify significant challenges that need to be overcome in order to succeed. Here, we review historic and recent attempts in babesiosis vaccine discovery to avoid past pitfalls, learn new lessons, and provide a roadmap to guide the development of next-generation babesiosis vaccines.

Background Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. Methods The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. Results Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. Conclusions These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission. Graphical abstract

Background The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. Methods The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. Results We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis. Graphical Abstract

Background: Thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis. Methods: The 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays. Results: Three members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro. Conclusions: We have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina. Fil: Montenegro, Valeria Noely. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Paoletta, Martina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Jaramillo Ortiz, José Manuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Suarez, Carlos Esteban. Washington State University; Estados Unidos Fil: Wilkowsky, Silvina Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina

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.

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. Electronic supplementary material The online version of this article (10.1186/s13071-018-3052-9) contains supplementary material, which is available to authorized users.

Babesia bovis, Babesia bigemina and Theileria equi are worldwide tick-borne hemoprotozoan that cause diseases characterized by fever, anemia, weight loss and abortion. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the host for life, and becoming a reservoir for tick transmission. The live-attenuated vaccines for B. bovis and B. bigemina are not available for worldwide use due to legal restrictions and other concerns such as potential erythrocyte antigen and pathogen contamination, and a vaccine for T. equi is not available. The use of chemotherapeutics is essential to treat and control these diseases, but several studies have shown the development of drug-resistance by these parasites, and safe and effective alternative drugs are needed. Tulathromycin, a macrolide antibiotic, has proven to be effective against a vast range of bacteria and Plasmodium yoelli, a Babesia and Theileria related intra-erythrocytic apicomplexan. Draxxin® (tulathromycin) is currently licensed to treat infections that cause respiratory diseases in cattle in several countries. In this study, the activity of Draxxin® was tested in vitro on cultured B. bovis, B. bigemina and T. equi. Addition of the drug to in vitro cultures resulted in cessation of parasite replication of the three species tested, B. bovis, B. bigemina and T. equi, with estimated IC50 of 16.7 ± 0.6 nM; 6.2 ± 0.2 nM and 2.4 ± 0.1 nM, respectively, at 72 h. Furthermore, neither parasites nor parasite DNA were detectable in cultures treated with IC100, suggesting Draxxin® is a highly effective anti-Babesia/Theileria drug. Importantly, the IC50 calculated for Draxxin® for the Babesia/Theileria parasites tested is lower that the IC50 calculated for some drugs currently in use to control these parasites. Collectively, the data strongly support in vivo testing of Draxxin® for the treatment of bovine babesiosis and equine piroplasmosis. Keywords: Draxxin®, Tulathromycin, Babesia bovis, Babesia bigemina, Theileria equi, In vitro inhibition growth

Background Bovine babesiosis caused by Babesia bovis is a tick-borne hemoparasitic disease of global impact, and improved control is needed. In B. bovis, spherical body protein 2 (SBP-2) truncated copies 7, 9 and 11 (sbp2t7, sbp2t9 and sbp2t11) gene transcripts were recently reported to be significantly upregulated in two geographically distinct attenuated B. bovis strains. In the present work, additional virulent and attenuated B. bovis strain pairs were compared in order to corroborate this finding. Results Sequences of the sbp2t7, sbp2t9 and sbp2t11 genes were not fully conserved among geographically distinct B. bovis strains, and varied between 70.6–93.3% sequence identity in all three genes. Comparisons among transcript levels of the three sbp2t genes of distinct virulent-attenuated B. bovis strain pairs confirmed that upregulation of the sbp2t11 gene was exclusively associated with an attenuated phenotype in the studied strain pairs. This rejects sbp2t7 and sbp2t9 as reliable attenuation markers. In addition, SBP2t11 protein was found to be significantly overexpressed in Texas attenuated B. bovis in comparison to the Texas virulent strain. Finally, sbp2t11 was differentially expressed in blood stages of the parasite but undetectable in Texas strain kinetes. Conclusions Sbp2t11 is a strong candidate as a reliable attenuation marker for B. bovis, based on its consistent pattern of upregulation in four distinct attenuated strains when compared to their virulent parental strains. Sbp2t11 may only have functional roles associated with erythrocyte infection. Identification of attenuation markers will lead to future research focused on the production of novel and safer subunit and genetically defined vaccines against B. bovis.

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. Electronic supplementary material The online version of this article (10.1186/s13071-019-3531-7) contains supplementary material, which is available to authorized users.

Background Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected cattle would affect parasite transmission to the next generation of ticks. Results Silencing expression of the vitellogenin receptor in the ovary by RNA interference, resulted in reduced tick fertility. We observed reduced egg production (i.e. reduced weight of eggs), a lower rate of embryonic development, and a reduction in hatching. Analysis of individual larvae by PCR confirmed that RNAi mediated downregulation of the R. microplus vitellogenin receptor and also interfered with transovarial transmission of B. bovis. None of the larvae (0/58) from the RmVgR dsRNA-injected group were PCR-positive, whereas 12% (7/58) and 17% (10/58) of larvae from the non-injected and buffer-injected control groups, respectively, were infected with B. bovis. Conclusions The combined effects of reduced fecundity and reduced infection in surviving larvae resulting from silencing indicate that vitellogenin receptor is essential for tick reproduction and may play a vital role in B. bovis transmission.

Background Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis. Results The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. Conclusions The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies. Electronic supplementary material The online version of this article (10.1186/s13071-018-3164-2) contains supplementary material, which is available to authorized users.

Babesia bovis, a tick-borne apicomplexan parasite responsible for bovine babesiosis has a complex life cycle including sexual development in its Rhipicephalus microplus vector. Understanding the molecular mechanisms involved in sexual development is essential for developing future-generation transmission blocking vaccines (TBVs) and/or non-transmissible attenuated live vaccines. The widely conserved members of the 6-Cys gene family likely play roles in the development of sexual stages of B. bovis, and are candidates for developing novel TBV. The recently defined sexual markers 6-CysA and 6-CysB of B. bovis are strain-conserved and exclusively surface-expressed in tick-stage parasites. However, the high level of sequence identity among the 6-Cys A and 6-Cys B proteins (52% identity), together with similar 6-Cys domain distribution and sub-cellular localization, are suggestive of redundant function. We hypothesized that disruption of both 6-CysA and 6-CysB in B. bovis would result in unaltered ability of the parasite to invade and grow in red blood cells (RBCs), with concomitant loss of the transmission phenotype. Taking advantage of their contiguous genome localization, we generated a double gene-knockout system to disrupt a 3287 bp region encompassing both 6-CysA and 6-CysB genes using a single transfection plasmid. The resulting red-fluorescent ΔAΔB 6-Cys B. bovis transgenic parasite line was able to grow continuously in bovine RBCs in vitro at a similar rate to wild-type parasites, demonstrating that the 6-CysA and 6-CysB genes are not required for the development of blood-stage parasites. This novel gene manipulation approach will allow future experiments aimed at determining the tick-transmission phenotype of parasites lacking tick-stage genes. Parasites deficient in genes required for sexual reproduction could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis. Developing a non-tick transmissible live vaccine based on attenuated parasites unable to express critical 6-Cys genes and including a molecular vaccine marker could help reduce the burden of bovine babesiosis globally.

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.

This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasite's two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques.

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.

Background Babesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line. Methods In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5′ and 3′ regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1α promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies. Results Full genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites. Conclusions Overall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2143-3) contains supplementary material, which is available to authorized users.

Background Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establishing better control protocols. Methods A modified indirect enzyme-linked immunosorbent assay (MI-ELISA) was developed using the spherical body protein-4 (SBP4) of B. bovis to detect antibody against diverse strains through all infection stages in cattle. This SBP4 MI-ELISA was evaluated for sensitivity and specificity against field sera from regions with endemic and non-endemic B. bovis. Sera were also evaluated from cattle infected experimentally with various doses and strains during acute and persistent infection with parasitemia defined by nested PCR. Results The format variables for SBP4 MI-ELISA were optimized and the cutoff for positive and negative interpretation was determined based on receiver operating characteristic curve analysis using B. bovis positive and negative sera tested in the reference immunofluorescence assay (IFA). The diagnostic specificity of the SBP4 MI-ELISA using IFA-negative sera collected from Texas was 100%, significantly higher than the cELISA (90.4%) based on an epitope in the rhoptry-associated protein-1 (RAP-1 cELISA). The diagnostic sensitivity of the SBP4 MI-ELISA was 98.7% using the IFA-positive sera collected from several areas of Mexico, in contrast to that of the RAP-1 cELISA at 60% using these same sera. In cattle infected with low and high doses of three B. bovis strains, the SBP4 MI-ELISA remained antibody positive for 11 months or more after initial detection at 10 to 13 days post-inoculation. However, the RAP-1 cELISA did not reliably detect antibody after eight months post-inoculation despite the fact that parasitemia was occasionally detectable by PCR. Furthermore, initial antibody detection by RAP-1 cELISA in low-dose infected animals was delayed approximately nine and a half days compared to the SBP4 MI-ELISA. Conclusions These results demonstrate excellent diagnostic sensitivity and specificity of the novel SBP4 MI-ELISA for cattle with acute and long-term carrier infections. It is posited that use of this assay in countries that have B. bovis-endemic herds may be pivotal in preventing the spread of this disease to non-endemic herds. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2016-9) contains supplementary material, which is available to authorized users.

Bovine and equine babesiosis caused by Babesia bovis, Babesia bigemina and Babesia caballi, along with equine theileriosis caused by Theileria equi are global tick-borne hemoprotozoan diseases characterized by fever, anemia, weight losses and abortions. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the hosts for life. Antiprotozoal drugs are important for managing infection and disease. Previous research demonstrated that trifluralin analogues, designated (TFLAs) 1–15, which specifically bind to regions of alpha-tubulin protein in plants and protozoan parasites, have the ability to inhibit the in vitro growth of B. bovis. The inhibitory activity of TFLAs 1–15 minus TFLA 5 was tested in vitro against cultured B. bigemina, B. caballi and T. equi. The four TFLAs with greatest inhibitory activity were then analyzed for hemolytic activity and toxicity against erythrocytes. All TFLAs tested in the study showed inhibitory effects against the three parasite species. TFLA 2, TFLA 11, TFLA 13 and TFLA 14 were the most effective inhibitors for the three species tested, with estimated IC50 between 5.1 and 10.1 μM at 72 h. The drug’s solvent (DMSO/ethanol) did not statistically affect the growth of the parasites nor cause hemolysis. Also, TFLA 2, 13 and 14 did not cause statistically significant hemolytic activity on bovine and equine erythrocytes at 15 μM, and TFLA 2, 11 and 13 had no detectable toxic effects on bovine and equine erythrocytes at 15 μM, suggesting that these drugs do not compromise erythrocyte viability. The demonstrated ability of the trifluralin analogues to inhibit in vitro growth of Babesia spp. and Theileria equi, and their lack of toxic effects on erythrocytes supports further in vivo testing and eventually their development as novel alternatives for the treatment of babesiosis and theileriosis.

Bovine babesiosis, caused by Babesia bovis, is a global tick borne hemoprotozoan parasite disease characterized by fever, anemia, weight losses and ultimately death. Several babesicidal drugs that have been in use in cattle for years have proven to be only partially effective and the development of alternative chemotherapeutics that are highly specific and have low toxicity against babesiosis is needed. Trifluralin derivatives specifically bind alpha-tubulin in plants and protozoa parasites causing growth inhibition. A set of 12 trifluralin analogues (TFLA) has previously been shown to be inhibitory for the growth of Leishmania species. The conservation of several key amino acids involved in the trifluralin binding site of alpha-tubulin among Leishmania sp. and B. bovis provides rationale for testing these compounds also as babesiacides. The previously tested Leishmania inhibitory, TFLA 1-12 minus TFLA 5, in addition to three novel TFLA (termed TFLA 13-15), were tested against in vitro cultured B. bovis parasites. While all of the TFLA tested in the study showed inhibition of B. bovis growth in vitro TFLA 7, TFLA 10 and TFLA 13, were the most effective inhibitors with estimated IC50 (μM) at 72h of 8.5±0.3; 9.2±0.2; 8.9±0.7, respectively for the biologically attenuated cloned B. bovis Mo7 strain, and 13.6±1.5; 18.7±1.6; 10.6±1.9, respectively for the virulent B. bovis T3Bo strain. The differences found between the two strains were not statistically significant. Importantly, these drugs displayed low levels of toxicity for the host erythrocytes and bovine renal arterial endothelial cells at the doses tested. The demonstrated ability of trifluralin analogues to inhibit in vitro growth of B. bovis parasites combined with their low toxicity for host cells suggests that these compounds may be further developed as novel alternatives for the treatment of bovine babesiosis. publishersversion published

Apicomplexa tick-borne hemoparasites, including Babesia bovis, Babesia microti, and Theileria equi are responsible for bovine and human babesiosis and equine theileriosis, respectively. These parasites of vast medical, epidemiological, and economic impact have complex life cycles in their vertebrate and tick hosts. Large gaps in knowledge concerning the mechanisms used by these parasites for gene regulation remain. Regulatory genes coding for DNA binding proteins such as members of the Api-AP2, HMG, and Myb families are known to play crucial roles as transcription factors. Although the repertoire of Api-AP2 has been defined and a HMG gene was previously identified in the B. bovis genome, these regulatory genes have not been described in detail in B. microti and T. equi. In this study, comparative bioinformatics was used to: (i) identify and map genes encoding for these transcription factors among three parasites’ genomes; (ii) identify a previously unreported HMG gene in B. microti; (iii) define a repertoire of eight conserved Myb genes; and (iv) identify AP2 correlates among B. bovis and the better-studied Plasmodium parasites. Searching the available transcriptome of B. bovis defined patterns of transcription of these three gene families in B. bovis erythrocyte stage parasites. Sequence comparisons show conservation of functional domains and general architecture in the AP2, Myb, and HMG proteins, which may be significant for the regulation of common critical parasite life cycle transitions in B. bovis, B. microti, and T. equi. A detailed understanding of the role of gene families encoding DNA binding proteins will provide new tools for unraveling regulatory mechanisms involved in B. bovis, B. microti, and T. equi life cycles and environmental adaptive responses and potentially contributes to the development of novel convergent strategies for improved control of babesiosis and equine piroplasmosis., Author Summary The tick-borne apicomplexan parasites Babesia and Theileria are responsible for costly and devastating diseases globally. Improved control is needed, but the biology of these parasites remains poorly understood. Significant gaps include better understanding of the mechanisms involved in control of gene expression and the events leading to parasite development among hosts, including the production of sexual stages in their definitive tick vector hosts. Similar to other better-studied eukaryotic cells, it is likely that regulatory genes coding for DNA binding proteins such as members of the Api-AP2, HMG, and Myb families play crucial roles as transcription factors in these processes, but these genes remain uncharacterized in these three related parasites. In this study, we describe the presence and genomic organization of these three types of genes in Babesia bovis, Babesia microti, and Theileria equi, highlighting the importance of the conservation of these genes and their possible contributions to parasite development through their different life stages. We also describe the occurrence of a previously unreported HMG gene in B. microti, an important emerging human pathogen; define the repertoire of eight conserved Myb genes; and describe the pattern of transcription of the regulatory AP2, HMG, and Myb genes in B. bovis intra-erythrocytic stages for the first time. It is expected that these findings will elicit additional research in this field and contribute to the development of converged intervention strategies for the improved control of these devastating and generally under-studied diseases.

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

Graphical abstract Investigation of type II fatty acid and isoprenoid biosyntheses in Babesia resulted in the identification of two major components within the apicoplastic lumen. Highlights ► This study illustrates a four membrane babesid apicoplast. ► Babesia bovis apicoplast resides adjacent to the nucleus. ► Acyl carrier protein and LytB are transcribed and translated in Babesia bovis. ► Isoprenoid biosynthesis likely exists in Babesia bovis. ► Type II fatty acid biosynthesis may not be present in Babesia bovis., The apicoplast is a highly specialized organelle that mediates required functions in the growth and replication of apicomplexan parasites. Despite structural conservation of the apicoplast among different parasite genera and species, there are also critical differences in the metabolic requirements of different parasites and at different stages of the life cycle. To specifically compare apicoplast pathways between parasites that have both common and unique stages, we characterized the apicoplast in Babesia bovis, which has only intraerythrocytic asexual stages in the mammalian host, and compared it to that of Plasmodium falciparum, which has both asexual intraerythrocytic and hepatic stages. Specifically focusing on the type II fatty acid (FASII) and isoprenoid (MEP) biosynthesis pathways, we searched for pathway components and retention of active sites within the genome, localized key components [acyl carrier protein (ACP) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB)] to the apicoplast, and demonstrated that the N-terminal bipartite signals of both proteins are required and sufficient for trafficking to the apicoplast lumen. Using specific pharmacologic inhibition, we demonstrated that MEP biosynthesis may be disrupted and its presence is required for intraerythrocytic growth of B. bovis asexual stages, consistent with the genomic pathway analysis and with its requirement in the asexual erythrocytic stages of P. falciparum. In contrast, FASII biosynthesis may or may not be present and specific drug targets did not have any inhibitory effect to B. bovis intraerythrocytic growth, which is consistent with the lack of requirement for P. falciparum intraerythrocytic growth. However, genomic analysis revealed the loss of FASII pathway components in B. bovis whereas the pathway is intact for P. falciparum but regulated to be expressed when needed (hepatic stages) and silent when not (intraerythrocytic stages). The results indicate specialized molding of apicoplast biosynthetic pathways to meet the requirements of individual apicomplexan parasites and their unique intracellular niches.

Background Ticks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms of osmoregulation. Members of the AQP family are critical for blood-feeding arthropods considering the extreme osmoregulatory changes that occur during their feeding. We investigated the pattern of expression of a newly identified AQP2 gene of R. microplus (RmAQP2) in different tick tissues and stages. We also examined in vivo the biological implications of silencing expression of RmAQP2 silencing during tick feeding on either uninfected or B. bovis-infected cattle. Methods In silico gene analyses were performed by multiple alignments of amino acid sequences and topology prediction. Levels of RmAQP2 transcripts in different tick tissues and stages were analyzed by reverse transcriptase quantitative PCR. Patterns of expression of RmAQP2 protein were investigated by immunoblots. Gene silencing was performed by RNA interference and in vivo functional analyses carried out by feeding ticks on either uninfected or B. bovis-infected cattle. Results RmAQP2 transcripts were found in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged females; however, of all tick tissues and stages examined, RmAQP2 protein was found only in salivary glands of partially engorged females. RmAQP2 silencing significantly reduced tick fitness and completely abrogated protein expression. The effect of RmAQP2 silencing on fitness was more pronounced in females fed on a B. bovis-infected calf than in ticks fed on an uninfected calf and none of their larval progeny survived. Conclusions Collectively, considering the gene expression and tick fitness data, we conclude that RmAQP2 is critical for tick blood feeding and may be a suitable candidate target for the development of novel strategies to control R. microplus and tick-borne parasites. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1226-2) contains supplementary material, which is available to authorized users.

SUMMARYObjective.TheBabesia bovisgenome encodes arap-1related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA.Methods.Phylogenetic analysis was performed using the program Phylip. Expression ofrrawas analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in anin vitroneutralization assay.Results.RRA is more closely related to RAP-1b ofBabesia bigeminathan toB. bovisRAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers ofrratranscripts compared torap-1.Immunoprecipitation of metabolically labelledB. bovisproteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present inB. bovishyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion inin vitroneutralization tests, suggesting functional relevance for parasite survival.Conclusion.B. bovisexpress a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the B. bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family contains six genes termed Bbo-6cys-A, B, C, D, E and F encoding for proteins containing an arrangement of 6 cysteine residues. The Bbo-6cys genes A, B, C, D, and E are tandemly arranged as a cluster of Chromosome 2 in the B. bovis genome, whereas gene F is located in a distal region in the same chromosome. The Bbo-6cys-E gene, with higher homology to PFS230, was selected for further examination. Immunoblot analysis using recombinant Bbo-6cys-E protein and B. bovis-positive bovine serum demonstrated expression by the parasite and immunogenicity during B. bovis infection. Immunofluorescence analysis using anti-Bbo-6cys-E antibodies confirmed expression of Bbo-6cys-E in in vitro blood stages of B. bovis. In addition, polyclonal antisera against both recombinant Bbo-6cys-E and specific synthetic peptides containing predicted B-cell epitopes of Bbo-6cys-E, significantly inhibited erythrocyte invasion by B. bovis in in vitro neutralization assays, suggesting an important functional role for this protein. Identification of this new gene family in B. bovis and further investigation on its biological significance may aid our understanding of the bovine, tick and parasite relationships and the development of improved control methods against B. bovis infection in cattle.

The complex life cycle of Babesia bovis includes erythrocytic stages in the bovine host and other stages occurring inside its common tick vector Rhipicephalus microplus. In related apicomplexa, changing environmental conditions affect the expression of ribosomal RNA, but it remained unknown whether the polymorphic A, B, and C rRNA coding units of B. bovis are differentially expressed. Northern blot analysis confirmed that polymorphic regions in the B. bovis 18s and ITS-2 rRNA coding units are transcribed. Then, rRNA transcript expression profiles were compared by analyzing cDNA libraries generated from total RNA extracted from in vitro cultured parasites, B. bovis infected cattle, R. microplus larvae and egg sources. The 18s and ITS-2 expression profiles indicate that rRNA unit B is almost exclusively expressed in cultured parasites while units A, B, and C are co-transcribed in the in vivo total RNA sources. Collectively, the data indicate that differential transcription of rRNA occurs in B. bovis, depending on the life stage of the parasite and on the environment.

Background Babesiosis threatens the development of the cattle and buffaloes industries in Egypt and improved control is needed. The main objectives of this study are surveying the presence of bovine babesiosis in distinct selected bovine and buffalo populations in Egypt using novel molecular and previously validated serological methods, while also comparing the occurrence of hematological alterations among Babesia infected cattle and buffalos. Methods A total of 253 and 81 blood samples from apparently healthy cattle and buffaloes, respectively, were randomly collected from diverse locations in Egypt. All samples were tested for Babesia bovis and B. bigemina infection using blood film examination, competitive ELISA (cELISA) and PCR. Novel semi-nested and nested PCR assays for the detection of B. bovis and B. bigemina respectively, were developed and used to analyze DNA extracted from bovine and buffalo samples. Hematological profiles were studied using a hematological analyzer. Results Blood films examination revealed 13.8 % and 7.4 % Babesia infection rates in cattle and buffaloes, respectively. However, in cattle, the cELISA detected 32.8 %, 21.3 % and 10.7 % infection rates with B. bigemina, B. bovis and mixed infection, respectively. In addition, cELISA identified 22.2 %, 22.2 % and 6.2 % infection rates with B. bigemina, B. bovis and mixed infection, respectively in buffaloes. The semi-nested PCR assay showed that 15 % of the tested samples were positive for B. bovis in cattle, but just 3 % in buffaloes. Infections with B. bigemina were also found in cattle (32.4 %), but not in buffaloes upon nested PCR analysis. Sequencing analysis confirmed the identity of the PCR amplicons and showed that Egyptian genotypes of B. bigemina and B. bovis highly resemble sequences previously deposited in GenBank. Hemograms performed on the sampled animals revealed macrocytic hypochromic anemia associated with reduced platelet counts in infected cattle with babesiosis. In addition, marked increases in total leukocyte and granulocytic counts and decreases in lymphocytic counts were found in infected cattle. In contrast, no such hematological anomalies were found in presumably Babesia-infected buffaloes. Conclusions Frequent occurrence of babesiosis among apparently healthy bovines in Egypt, suggests the need for appropriately designed prevalence studies in this country. Infected bovine, but not buffalo, populations often present hematological disorders compatible with intravascular hemolysis and thrombocytopenia.

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.

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

Polyclonal and monoclonal antibodies to native Neospora caninum tachyzoite surface protein NcSRS2 were generated and tested in vitro for their ability to neutralize tachyzoite attachment to and invasion of host cells. Host cells included Vero cells and a newly cloned, immortalized ovine trophoblast cell line obtained from primary cultures of ovine placenta. The ovine trophoblasts had morphology consistent with fetal trophoblasts and expressed mRNA for interferon-tau, a marker for trophoblasts. Native NcSRS2 was used to immunize mice to obtain monospecific anti-NcSRS2 polyclonal serum and anti-NcSRS2 monoclonal antibodies. Compared to irrelevant antibodies, monospecific anti-NcSRS2 serum and two anti-NcSRS2 monoclonal antibodies, 100.2.4.4 and 119.4.9.10, significantly blocked invasion of tachyzoites into both trophoblasts and Vero cells. Parasite attachment, assessed by IFA, was significantly reduced by anti-NcSRS2 mAb 100.2.4.4 and monospecific serum. The findings provide rationale to investigate a role for antibodies to NcSRS2 in prevention of N. caninum transplacental transmission in vivo.

Members of the variable merozoite surface antigen (vmsa) gene family ofBabesia bovisencode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containingmsa-2, a member of thevmsafamily, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies ofmsa-2-related genes were found in the locus. The four genes, designatedmsa-2a1(which corresponds to the originally describedmsa-2gene),msa-2a2,msa-2b, andmsa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a1) amino acids. MSA-2a1and -2a2are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a2and -2b. In contrast,msa-2cis most closely related to the previously describedbabr 0.8gene in Australia strains ofB. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain ofB. boviswith the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a1, -2a2, -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a1, -2b, and -2c. The results support the evolution of themsa-2locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

Bovine babesiosis caused by the tick-transmitted haemoprotozoans Babesia bovis, Babesia bigemina and Babesia divergens commonly results in substantial cattle morbidity and mortality in vast world areas. Although existing live vaccines confer protection, they have considerable disadvantages. Therefore, particularly in countries where large numbers of cattle are at risk, important research is directed towards improved vaccination strategies. Here a comprehensive overview of currently used live vaccines and of the status quo of experimental vaccine trials is presented. In addition, pertinent research fields potentially contributing to the development of novel non-live and/or live vaccines are discussed, including parasite antigens involved in host cell invasion and in pathogen-tick interactions, as well as the protective immunity against infection. The mining of available parasite genomes is continuously enlarging the array of potential vaccine candidates and, additionally, the recent development of a transfection tool for Babesia can significantly contribute to vaccine design. However, the complication and high cost of vaccination trials hinder Babesia vaccine research, and have so far seriously limited the systematic examination of antigen candidates and prevented an in-depth testing of formulations using different immunomodulators and antigen delivery systems. Fil: Jacobsen, Monica Ofelia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Suarez, Carlos E.. Washington State University; Estados Unidos Fil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Flores, Daniela Agustina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Cientifíca y Tecnológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Glycosylphosphatidylinositol-anchored proteins are abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work, the relevance of GPI-anchored proteins for erythrocyte invasion of the cattle hemoparasite Babesia bovis was studied. We show that cleavage of GPI-anchored antigens from the merozoite parasite stage by phosphatidylinositol-specific phospholipase C abolished invasion of erythrocytes demonstrating the importance of this class of molecules for parasite propagation. In addition, the repertoire of GPI-anchored proteins of B. bovis was predicted with high fidelity by searching its genome with available web-based bioinformatic tools. Altogether 17 GPI-anchored proteins were identified, 5 of which represent the already characterized variable merozoite surface antigens (VMSAs). Fifteen of the identified GPI-anchored proteins contain 2–26 amino acid repeats indicating that they are likely involved in functions of recognition, adhesion, or transport. Repeats were found to contain an increased frequency of proline, indicative of unstructured regions; and were estimated to be 3.21 times more hydrophilic than non-repeat regions. This suggests that they might represent eminent antibody epitopes. The majority of the putative GPI-anchored antigens reported in this work have so far remained unnoticed, though they may represent potential candidates for inclusion in a subunit vaccine. Fil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Flores, Daniela Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Cientifíca y Tecnológica; Argentina Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Suarez, Carlos Esteban. Washington State University; Estados Unidos Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine delivery platform.

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i-RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis.

Cystic echinococcosis (CE) is the most prevalent zoonosis in Tierra del Fuego. In 1997, ulrasonography (US) was selected as the method of choice for the development of population surveys for epidemiological surveillance and early diagnosis in rural population. The aim of this work was to present the results of the epidemiological surveillance of CE by means of US in rural population of Tierra del Fuego, Argentina between 1997 and 2006. The ultrasonographic diagnostic was realized once a year. The population was stratified in children (4 to 17 years) and adults. From each individual, name, age, sex, actual residence and origin were registered. The images compatible with cysts were graded according to location, number and characteristics. A total of 1400 rural inhabitants were examined for CE. From the total of studied individuals, 27 (1.9%) exhibited images compatible with cysts on the abdominal ultrasound scan. Thirteen of these persons were finally diagnosed as having CE. The overall prevalence of CE was 0.9%. This value is in accordance with the decrease in the prevalence observed in the definitive host and the intermediate hosts (sheep and cattle). The absence of cases in children during the studied period, evidence no transmission of the disease to humans in the recent past.

The complexity of multigene families encoding rhoptry proteins and the generation of new variants in these families are constraints to development of vaccines incorporating rhoptry proteins. For example, the Babesia bigemina rhoptry associated protein (rap)-1 locus is composed of tandemly arranged genes including four polymorphic rap-1a genes and two classes of divergent genes, rap-1b and rap-1c. B. bigemina rap-1 polymorphism reflects recombination and gene conversion and results in multiple RAP-1 proteins with unique B- and T-cell epitopes. Is this complex locus structure and recombination a required feature of the rap-1 gene family among Babesia species? We addressed this question by analysis of the rap-1 locus in B. bovis. Sequence analysis of an 11 kb genomic clone representing the B. burn rap-1 locus revealed only two identical and continuous rap-1a gene copies, rap 1a-1 and rap-1a-2, located in a similar head to tail orientation. Using the conserved ig gene as a marker for the 3' boundary of the rap-1 locus, we conclude that divergent rap-1b and rap-1c genes, present in B. bigemina, are not similarly cis-linked to the B. bovis rap-1 locus. Analysis of the rap-1a genes 1 and 2 from each of multiple B. bovis strains from North and South America demonstrated RAP-1 size conservation with very limited amino acid sequence variation. The results suggest that the simple two gene arrangement in the B. bovis rap-1 gene family was generated by gene duplication and, in contrast to the B. bigemina rap-1 locus, both genes evolved together using homogenization mechanisms with point mutation as the single mechanism for gene variation. Three discontinuous non-rap-1 genes are closely cis-linked to the B. bovis rap-1 locus and the presence of multiple introns in these genes may limit rap-1 gene variation due to unequal crossing over. The different mechanisms likely involved in the evolution of the rap-1 family in B. bigemina versus B. bovis are reflected in the marked structural and antigenic polymorphism in the B. bigemina RAP-1 molecules as compared with the essentially monomorphic RAP-1 in B. bovis.

Among important candidates for babesial vaccines are apical complex proteins, including rhoptry-associated protein 1 (RAP-1) from Babesia bovis and B. bigemina , which have been shown to induce partial immunity. Four variant B. bigemina rap-1 transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-α1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes in B. bigemina RAP-1α1 protein (I. Hötzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89–99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains of B. bigemina , raising the possibilities that the T-cell epitopes may vary antigenically and that CT-1 may be differentially expressed with respect to the other RAP-1 CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains of B. bigemina . We further show that the RAP-1α1-immunized cattle were protected against homologous B. bigemina challenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.

Despite convincing evidence that T cells are critical for both cellular and humoral immunity against haemoprotozoan parasites, the difficulty of performing meaningful experiments in cattle that would define the role of T cells in immunity to Babesia spp. has impeded research in this area. However, experiments performed ex vivo with immune T cells can reflect in-vivo events, and provide valuable insight into the nature of immunogenic proteins and the responding lymphocytes. The progress made towards identification of the immunogenic proteins and epitopes that stimulate anamnestic CD4+ type-1 (interferon-gamma-producing) T-cell responses in cattle immune to challenge with Babesia bovis or B. bigemina is the subject of the present review.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4+ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4+ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99% nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3′ ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

Babesia merozoite polypeptides bear surface exposed and neutralization-sensitive B cell epitopes and have been shown to induce partial protection against experimental challenge. Variation in these epitopes has been examined in a limited number of strains. In this study, utilizing strains of Babesia bovis and Babesia bigemina from Matto Grosso do Sul in Brazil, we examined the conservation of epitopes bound by monoclonal antibodies developed against Mexico strains of B. bovis and B. bigemina . Apical complex B-cell epitopes, previously shown to be species-specific but common among otherwise antigenically distinct strains, were also conserved between clones of the Mexico strains and the Matto Grosso do Sul strains of each Babesia species. Mexico strain polypeptides bearing these epitopes were recognized by sera from cattle infected with the Matto Grosso do Sul strains. Two distinct epitopes on the B. bovis neutralization-sensitive merozoite surface antigen-1 (MSA-1) were also conserved between the Mexico Mo7 clone and the Matto Grosso do Sul strain, in contrast to previous studies which demonstrated variability among strains. Sera from cattle with B. bovis infections naturally acquired in Matto Grosso do Sul bound Mexico Mo7 MSA-1, demonstrating that conserved MSA-1 epitopes were recognized by the bovine immune system. Similarly, merozoite surface epitopes on the B. bigemina 45 kDa and 55 kDa glycoproteins were conserved on the Matto Grosso do Sul strain of B. bigemina .

Theileriosis in horses and cattle is caused by tick-borne Apicomplexa parasites and results in death or life-long infection in their respective hosts. Transmission risk associated with persistent infection severely limits movement of horses and cattle resulting in economic losses. The recent reemergence of Theileria equi infection in U.S. horses demonstrates the continual threat Apicomplexa parasites represent to global animal health. A paucity of data concerning equine immune responses to T. equi, including antigens recognized by antibodies in clinically asymptomatic, persistently infected horses, precludes vaccine development. Therefore, this investigation was initiated to characterize antigens recognized by the equine antibody response to T. equi. This goal was accomplished by defining T. equi merozoite antigens that are recognized by antibodies in horses infected with distinct T. equi isolates. Previously it was shown that equine post-infection serum consistently recognized at least five T. equi merozoite antigens, but their precise identity remained unknown. To determine specificity of antibody target identification, T. equi merozoite antigens were first isolated using equine post-infection serum in affinity chromatography. Proteins recognized by the equine antibodies were then isolated from two-dimensional electrophoresis gels, and analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) using the recently available T. equi genome database. Five T. equi antigens were identified and include Equi Merozoite Antigen-2 (EMA-2), EMA-3 and EMA-6, a previously uncharacterized protein annotated as “signal peptide containing protein”, and 40S ribosomal protein S12.

A Mo7-derived Babesia bovis line stably transfected with the gfp-bsd gene was inoculated into two four to five months old calves, while two additional calves were inoculated with Mo7 parasites. Similar mild clinical signs were detected in all calves. B. bovis rap-1 was identified in the bloodstream by PCR four days post inoculation (dpi), and consistently over ten months thereafter. Transfusion of blood from experimentally infected calves into four naive splenectomized calves at 212 dpi resulted in acute disease in recipients, confirming persistent infection in the four donor animals. The proportion of GFP expressing parasites recovered from a splenectomized recipient calf is undistinguishable from transfected parasites that were maintained in long term culture under blasticidin selection. Furthermore, the sequences of transfected genes in recovered parasites remained unaltered. Together, the data demonstrates that exogenous B. bovis transgenes can be expressed and remain stable throughout acute and persistent infection in calves.

The Babesia bovis and B. bigemina apicomplexan protozoa in conjunction with the rickettsia Anaplasma marginale are intraerythrocytic pathogens that are responsible for the most prevalent and costly tick borne diseases (TBD's) of cattle worldwide. These organisms are historically associated as they can cause clinically related hemolytic diseases in cattle, are all transmitted by Rhiphicephallus (Boophilus) ticks, and share an uncanny ability to evade the immune systems of the vertebrate hosts, causing persistent disease. In addition, acute babesiosis and anaplasmosis can be prevented quite effectively by combining tick control and vaccination with living attenuated organisms. However these methods of control have numerous limitations and improved approaches are needed. Importantly, immunizations of cattle with inactivated experimental Babesia and Anaplasma vaccines can elicit variable degrees of protection, indicating the feasibility for the development of inactivated or subunit vaccines. A new research toolbox that includes full genome sequencing combined with the improved ability to genetically modify the organisms is enhancing our understanding of their biology. An emerging paradigm is the use of recently developed Babesia and Anaplasma transfection methods for functional gene characterizations and for vaccine development. Promising recently identified subunit vaccine candidates are also emerging, including babesial proteases, putative rhoptry, microneme, and sexual stage antigens, as well as subdominant, conserved, A. marginale outer membrane major surface proteins. However, significant knowledge gaps on the role of key parasite molecules involved in cell invasion, adhesion, asexual and sexual reproduction, tick transmission, and evasion of the immune system, remain. A better understanding of the biology of these organisms and the protective immune responses will positively contribute toward the goal of developing improved immunological and pharmacological interventions against these elusive pathogens that are responsible for the most devastating TBD's of cattle. Importantly, the currently available research toolbox provides basic research instruments for helping close current knowledge gaps which will aid the design and production of effective vaccines and alternative pharmacological interventions.