Search

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

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

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

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

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

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

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

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

The Babesia bigemina rap-1 gene locus contains five tandemly arranged copies of rap-1a genes. However, the size of the locus, as defined by conserved, unrelated orfs at the 5' and 3' ends, suggests that additional genes may be present. In this study, we identified all additional genes in the locus and characterized their pattern of expression in merozoites. The rap-1a genes are separated by 3.38-kbp intergenic (IG) regions, each of which contains an identical copy of a related gene designated rap-1b. One additional copy of rap-1b and one copy of another related gene designated rap-1c is present in the 3' end of the locus. Common sequence features that define the Babesia rap-1 family are present in rap-1b and rap-1c, but otherwise these genes average only 27% identity to rap-1a. Homologues of the rap-1b and rap-1c genes identified in diverse B. bigemina strains have a high degree of predicted amino acid sequence conservation (averaging >90%), with the largest number of changes in the carboxyl end of RAP-1c. We tested whether all rap-1 genes in the locus are co-transcribed in merozoites using RT-PCR, Northern blots, and quantitative real-time PCR. Rap-1a genes produce the most abundant transcripts of the family, while rap-1b transcripts are the least abundant despite the large number of gene copies. Similar patterns of transcription were observed whether merozoites were obtained from in vitro cultures or in vivo infection. Immunoblot analysis of merozoites revealed the expected RAP-1a expression but failed to detect expressed RAP-1b and RAP-1c, indicating that expression of the rap-1 genes is regulated both at the transcriptional and translational levels.

The search for vaccine candidates against bovine babesiosis caused by Babesia bovis is greatly focused on the identification of merozoite surface-exposed antigens that are widely conserved, functionally relevant and immunodominant in cattle protected against B. bovis infections. We have recently identified msa-2c, a member of the B. bovis variable merozoite surface antigen (VMSA) gene family, which in contrast to other members, appears to be highly conserved among geographically distant B. bovis strains. In this study, we further investigated the potential of the msa-2c gene product as diagnostic and vaccine candidate for bovine babesiosis. RT-PCR studies demonstrated that MSA-2c is transcribed in merozoites of the Argentine R1A strain. In addition, antibodies against R1A recombinant MSA-2c reacted in immunoblots with a single protein of approximately 30kDa in B. bovis merozoite extracts from both R1A and Australian "S" strains, demonstrating translation of this protein in these two strains and conservation of B-cell epitopes between them. These antibodies reacted with the cell surface of R1A merozoites in fixed immunofluorescence assays, indicating the surface localization of MSA-2c. This localization was confirmed by live immunofluorescence studies in two different strains, R1A and S2P. These results also demonstrate the conservation of MSA-2c surface-exposed B-cell epitopes between these two strains. Sera from cattle either naturally or experimentally infected with Argentine strains of B. bovis specifically recognized rMSA-2c in immunoblots, reinforcing the idea that B-cell epitopes in rMSA-2c are widely conserved among field strains of B. bovis. Furthermore, our results show that these B-cell epitopes are highly immunogenic, suggesting that MSA-2c may be a useful diagnostic tool for the detection of bovine babesiosis by B. bovis. Experimental vaccination of five bovines with rMSA-2c resulted in elicitation of high specific anti-rMSA-2c IgG titers, with similar amounts of IgG(1) and IgG(2) produced. Importantly, bovine anti-rMSA-2c antibodies were able to neutralize in vitro bovine erythrocyte invasion by R1A merozoites suggesting a significant functional role for MSA-2c. Taken together these results postulate MSA-2c as a candidate for the development of novel tools for improved control of bovine babesiosis.

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

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

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.