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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.

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 IC 50 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 IC 100 , suggesting Draxxin ® is a highly effective anti-Babesia/Theileria drug. Importantly, the IC 50 calculated for Draxxin ® for the Babesia/Theileria parasites tested is lower that the IC 50 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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Babesia bigemina and Babesia bovis, are the two major causes of bovine babesiosis, a global neglected disease in need of improved methods of control. Here, we describe a shared method for the stable transfection of these two parasites using electroporation and blasticidin/blasticidin deaminase as a selectable marker. Stably transfected B. bigemina and B. bovis were obtained using a common transfection plasmid targeting the enhanced green fluorescent protein-BSD (egfp-bsd) fusion gene into the elongation factor-1α (ef-1α) locus of B. bigemina and B. bovis under the control of the B. bigemina ef-1α promoter. Sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of parasite-infected red blood cells, demonstrated that the egfp-bsd gene was expressed and stably integrated solely into the ef-1α locus of both, B. bigemina and B. bovis. Interestingly, heterologous B. bigemina ef-1α sequences were able to drive integration into the B. bovis genome by homologous recombination, and the stably integrated B. bigemina ef-1α-A promoter is fully functional in B. bovis. Collectively, the data provides a new tool for genetic analysis of these parasites, and suggests that the development of vaccine platform delivery systems based on transfected B. bovis and B. bigemina parasites using homologous and heterologous promoters is feasible.

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 IC 50 between 5.1 and 10.1μM at 72h. 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 15μM, and TFLA 2, 11 and 13 had no detectable toxic effects on bovine and equine erythrocytes at 15μM, 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., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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.

Equine piroplasmosis, caused by the parasites Theileria equi and Babesia caballi, is a globally important disease, affecting a large percentage of the world's horses. This article serves as a review of these divergent parasites. Discussed are the clinical presentation of disease, diagnosis, and treatment. Special attention is given to the current disease status specifically in North America., (Published by Elsevier Inc.)

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis.

Antimicrobial treatment of persistent infection to eliminate transmission risk represents a specific challenge requiring compelling evidence of complete pathogen clearance. The limited repertoire of antimicrobial agents targeted at protozoal parasites magnifies this challenge. Using Babesia caballi as both a model and a specific apicomplexan pathogen for which evidence of the elimination of transmission risk is required for international animal movement, we tested whether a high-dose regimen of imidocarb dipropionate cleared infection from persistently infected asymptomatic horses and/or eliminated transmission risk. Clearance with elimination of transmission risk was supported by the following four specific lines of evidence: (i) inability to detect parasites by quantitative PCR and nested PCR amplification, (ii) conversion from seropositive to seronegative status, (iii) inability to transmit infection by direct inoculation of blood into susceptible recipient horses, and (iv) inability to transmit infection by ticks acquisition fed on the treated horses and subsequently transmission fed on susceptible horses. In contrast, untreated horses remained infected and capable of transmitting B. caballi using the same criteria. These findings establish that imidocarb dipropionate treatment clears B. caballi infection with confirmation of lack of transmission risk either by direct blood transfer or a high tick burden. Importantly, the treated horses revert to seronegative status according to the international standard for serologic testing and would be permitted to move between countries where the pathogen is endemic and countries that are free of the pathogen.

The tropical horse tick, Dermacentor nitens, is a natural vector of Babesia caballi in the Americas. B. caballi, one of the etiologic agents of equine piroplasmosis, occurs widely throughout the world, but the United States and a few other countries are considered to be free of infection. B. caballi is transovarially transmitted by the one-host tick D. nitens; we tested the hypothesis that B. caballi can persist in multiple generations of D. nitens in the absence of opportunity to reacquire infection from a susceptible equine host. Partially engorged female D. nitens were collected from a B. caballi-infected horse in Puerto Rico and allowed to reattach and feed on an uninfected horse, successfully transmitting the infection. Three subsequent generations of ticks were reared on calves (nonsusceptible hosts for B. caballi), testing for B. caballi infection in each generation by feeding a sample of the larvae on naive horses. The first generation ofD. nitens reared on a nonsusceptible host transmitted B. caballi, whereas the second and third failed to transmit to naive horses, showing that D. nitens infection with B. caballi was restricted to one generation in the absence of alimentary reinfection. These results imply that, in the event of the introduction of this pathogen into areas of the continental United States where D. nitens occurs, the tick could become a short-term reservoir of B. caballi, making control of introduced infections more complex.

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of > or =10(4) organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.

In this study, the kinetics of specific immunoglobulin G (IgG) isotypes were characterized in Babesia equi (Theileria equi)-infected horses. IgGa and IgGb developed during acute infection, whereas IgG(T) was detected only after resolution of acute parasitemia. The same IgG isotype profile induced during acute infection was obtained by equi merozoite antigen 1/saponin immunization.

Babesia divergens-like parasites identified in human babesiosis cases in Missouri and Kentucky and in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, share identical small subunit ribosomal RNA gene sequences. This sequence is 99.8% identical to that of Babesia divergens, suggesting that the U.S. parasite may be B. divergens, a causative agent of human and bovine babesiosis in Europe. Holstein-Friesian calves were inoculated with cultured Nantucket Island Babesia sp. (NR831) and B. divergens parasites and monitored by clinical signs, Giemsa-stained blood films, PCR, and culture. The NR831 recipients did not exhibit clinical signs of infection and remained negative for all assays. The B. divergens recipients developed clinical infections and became positive by all assays. NR831 recipients were fully susceptible upon challenge inoculation with B. divergens. This study confirms that the Nantucket Island Babesia sp. is not conspecific with B. divergens based on host specificity for cattle.

Equi merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equi proteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileria spp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equi transitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equi stages in the midgut lumen and midgut epithelial cells of Boophilus microplus nymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equi in salivary glands of adult Boophilus microplus. In addition, quantification of B. equi in the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equi organisms in the midgut. In contrast, there was no conclusive evidence that B. equi EMA-1 was expressed in either the Boophilus microplus midgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equi EMA-2 in Boophilus microplus provides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesia proteins for testing transmission-blocking immunity.

Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.

The experimental vector competence of five laboratory-reared ixodid tick species representing three genera [Amblyomma americanum (L.), Boophilus microplus (Canestrini), D. andersoni Stiles, D. occidentalis Marx, and D. variabilis (Say)] for Babesia equi (Laveran 1901) was evaluated by delayed transfer of male ticks from infected to susceptible equids or by infesting the latter animals with adult ticks previously fed as nymphs on infected equids. After feeding for 5, 6, or 13 d on acquisition hosts, ticks were forcibly removed and held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 6, 12, or 27 d. Intrastadial transmission to susceptible ponies by D. variabilis males, and transstadial transmission to susceptible burros by B. microplus adults, was demonstrated by blood smear and indirect immunofluorescence serology. The data indicated that male D. variabilis and adult B. microplus, tick species that occur on equids in North America and, in the case of the latter tick, also extensively in tropical and subtropical regions of the world, may be competent natural vectors of B. equi

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis. [ABSTRACT FROM AUTHOR]

Abstract: Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4+ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4+ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity. [Copyright &y& Elsevier]

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis. [ABSTRACT FROM AUTHOR]

Abstract: Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species’ unique host and life-cycle. [ABSTRACT FROM AUTHOR]

Abstract: Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast origins using multiple previously reported apicoplast sequences as well as several sequences recently reported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary origin of the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The data strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage. [Copyright &y& Elsevier]