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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

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 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.

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.

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.

Background Equine piroplasmosis (EP) caused by Theileria equi, Babesia caballi, or both, contributes to significant economic loss in the equine industry and remains uncontrolled in Egypt. This study focuses on surveying T. equi and B. caballi infections and hematological disorders in equine populations in Egypt. Methods Theileria equi and B. caballi infections were assessed in blood from 88 horses and 51 donkeys in Egypt using light microscopy, indirect immunofluorescent antibody test (IFAT), nested PCR (nPCR), and competitive-ELISA (cELISA) assays. PCR products were examined for specificity by DNA sequencing. Hematological alterations were evaluated using a standard cell counter. Results Microscopic analysis revealed EP infection in 11.4 % and 17.8 % of horses and donkeys respectively. IFAT detected 23.9 % and 17.0 % infection of T. equi and B. caballi, respectively, in horses, and 31.4 % of T. equi and B. caballi in donkeys. T. equi cELISA detected 14.8 % and 23.5 % positive horses and donkeys, respectively, but the B. caballi RAP-1-based cELISA failed to detect any positives, a result hypothesized to be caused by sequence polymorphism found in the rap-1 genes. Nested-PCR analysis identified 36.4 % and 43.1 % positive horses and donkeys, respectively for T. equi and it also identified 19.3 % and 15.7 % positive horses and donkeys, respectively for B. caballi. The overall EP incidence found in the population under study was relatively high and comparable regardless of the diagnostic method used (56.8 % using nPCR and 48.9 % using IFAT). Hematologic analysis revealed macrocytic hypochromic anemia and thrombocytopenia in all piroplasma-infected horses. Conclusions The data confirm relatively high levels of EP, likely causing hematological abnormalities in equines in Egypt, and also suggest the need for an improved serological test to diagnose B. caballi infection in this region. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1539-9) contains supplementary material, which is available to authorized users.

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.

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.

This paper is the results of a workshop convened by the United States Department of Agriculture - Agricultural Research Service and the Center for Ecoepidemiology, Yale University School of Medicine on 22nd and 23rd of April 2009 in McAllen, Texas.-- Group for Emerging Babesioses and One Health Research and Development in the U.S.: et al., [Background]: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. [Results]: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. [Conclusions]: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

Background Tick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum. Methods The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5’ untranslated region was determined by 5’ Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes. Results The ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α genes. Plasmid constructs containing the 5’ and 3’ halves of the IG regions controlling the expression of the luciferase gene containing a 3’ region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina. Conclusions Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1859-9) contains supplementary material, which is available to authorized users.

Background Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of Babesia bovis, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival. Results Four papain-like cysteine proteases were found to be encoded by the B. bovis genome using the MEROPS database. One of them, the ortholog of Plasmodium falciparum falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in B. bovis chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several B. bovis isolates from North and South America. Synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids, Theileria parva and T. annulata, into families of 6 and 7 clustered genes respectively. The bovipain-2 g ene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated B. bovis strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of in vitro cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with B. bigemina-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six B. bovis-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages. Conclusions Overall, we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.