Carlos E. Suarez

Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The ∼150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide

ABSTRACT The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasiteBabesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423–5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA fromEscherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-α, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli ≥ T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations,E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

Emerging perspectives in the research of bovine babesiosis and anaplasmosis.

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 (TBDs) 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 TBDs 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.

Characterization of Allelic Variation in the Babesia bovis Merozoite Surface Antigen 1 (MSA-1) Locus and Identification of a Cross-Reactive Inhibition-Sensitive MSA-1 Epitope

ABSTRACT The Babesia bovis merozoite surface antigen 1 (MSA-1), a member of the variable merozoite surface antigen (VMSA) family, is an immunodominant glycoprotein which elicits antibodies that inhibit erythrocyte invasion. While antigenic polymorphism is a general feature of vmsa genes, the molecular basis and extent ofmsa-1 sequence polymorphism have not been well characterized. In this study we defined the msa-1 locus in the biologically cloned Mexico Mo7 strain of B. bovis and identified the sequence differences between MSA-1 antigenically dissimilar strains. We then determined whether sequences conserved between distinct msa-1 alleles would induce cross-reactive CD4+ T lymphocytes or inhibitory antibodies. Themsa-1 locus in Mo7 contains a single msa-1 gene flanked by transcribed genes with no sequence homology to members of the VMSA gene family. Argentina B. bovis strains R1A and S2P have msa-1 genes with amino acid sequences that are 98.8% identical to each other, and antibodies against S2P MSA-1 cross-react with native R1A MSA-1. In contrast, identity between the Argentina and Mexico Mo7 msa-1 alleles is only 52%, with no continuous stretch of identity longer than 16 amino acids. Despite limited sequence conservation, antibodies against R1A MSA-1 were able to inhibit invasion of erythrocytes by Mo7 merozoites. The results indicate that inhibition-sensitive epitopes are conserved despite significant sequence divergence between Mexico and Argentina strain alleles and support a conserved functional role for polymorphic MSA-1 in erythrocyte invasion.

Prospects for recombinant vaccines against Babesia bovis and related parasites

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.

The Babesia bovis Merozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins

ABSTRACT Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus. The four genes, designated msa-2a1 (which corresponds to the originally described msa-2 gene), msa-2a2, msa-2b, and msa-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-2a1 and -2a2 are 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-2a2 and -2b. In contrast, msa-2c is most closely related to the previously described babr 0.8 gene in Australia strains of B. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain of B. bovis with 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 the msa-2 locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

Comparative genomic analysis and phylogenetic position of Theileria equi

BackgroundTransmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites.ResultsThe known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp.ConclusionsThe EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1 *

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge.

ABSTRACT Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen for Babesia bovis and Babesia bigemina infections of cattle. The 60-kDa B. bovis RAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain of B. bovis RAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune to B. bovis. The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4+-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulent B. bovis challenge.

One Health approach to identify research needs in bovine and human babesioses: workshop report

BackgroundBabesia 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.ResultsThe 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.ConclusionsThe 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.