Genome characteristics of B. bovis, T. parva and P. falciparum .
Genomics approaches in veterinary research have been a very useful tool to identify candidates with potential to be used in prevention of animal diseases. In Babesia, genome information analysis has elucidated a wide variety of protein families and some members are described in this chapter. Here, we present some of the most recent studies about B. bovis and B. bigemina genomes where some proteins have been identified with potential to prevent infections by these parasites.
- bovine babesiosis
Bovine babesiosis is a tick-transmitted disease caused by apicomplexan parasites of the genus Babesia. This disease is caused by Babesia bovis and B. bigemina in the Americas including Mexico, where it is distributed in tropical and subtropical regions, occupying 51.5% of the national territory [1, 2]. This disease was reported for the first time by Viktor Babes in Rumania in 1888. However, it was until 1893 when Smith and Kilborne demonstrated that the disease is transmitted to cattle by infected ticks [3, 4]. In Mexico, the first report of bovine babesiosis occurred in 1905; however, it is believed that it was first introduced to the American continent by the Spaniards during the conquest. To date, measures used to control bovine babesiosis include vector control, an early diagnosis, treatment of sick animals and vaccination. The negative, severe impact that cattle fever tick and babesiosis have in the cattle industry in Mexico and the world has not diminished due mainly to a lack of commercially available, safe and effective vaccines. Vaccines based on approaches using genomics and bioinformatics are a promissory solution to this problem . It has been shown that experimental vaccines based on recombinant antigens have been developed successfully in apicomplexan parasites like Plasmodium, Toxoplasma and Theileria [6, 7, 8]. With the completion of the B. bovis genome  and the partial sequencing of the Babesia bigemina genome (
2.1. Babesia bovis genome
Although Babesia bovis, B. bigemina and B. divergens are causative agents of bovine babesiosis, B. bovis is regarded as the most important and has a bigger impact in the livestock industry due to its virulence and high mortality rate. For this reason, the B. bovis genome was the first to be sequenced. This was done by Washigton State University in collaboration with the Agricultural Research Service and The Institute for Genomic Research (TIGR) in the USA. The genome sequence was obtained from the T2Bo strain, a virulent strain isolated from a clinical case in Texas, USA.
The genome of Babesia bovis has a length of 8.2 Mbp, contains 3671 genes and consists of four chromosomes, three of them are acrocentric: chromosome 1, has a length of 1.25 Mpb, is the smallest and contains a gap, which is estimated to be 150 Kpb long. Chromosome 2 is fully sequenced and contains 1.73 Mbp in length. Chromosome 3 is also fully sequenced and is 2.59 Mpb in length. Finally, chromosome 4, which is the only submetacentric one, it is partially sequenced because it contains an assembly gap that has not been solved and is 2.62 Mbp in length. The structural features of the B. bovis genome are similar to those of Theileria parva but have major differences with Plasmodium falciparum (Table 1), despite the fact that B. bovis and P. falciparum share similar clinical and pathological features .
B. bovis contains two extrachromosomal genomes: a lineal mitochondrial genome of 6 Kbp and an apicoplast genome that is circular and consists of 33 kpb with 32 genes and 25 sequences of tRNA. The apicoplast, which is an organelle conserved in the phylum apicomplexa, is a nonphotosynthetic plastid essential for survival . The apicoplast genome was first sequenced in 1974 from Plasmodium lophurae and was thought that it could be mitochondrial DNA. In 1994 it was finally related to plastids DNA when a gen coding for a protein of 470 amino acids in length was identified and it contained a 50% identity with a protein only described in the plastome of red algae . It is believed that the plastid is derived from a secondary endosymbiosis from red algae like in dinoflagellates . Furthermore, Chromera velia has a plastid originated from red algae that is closely related with the apicoplast .
3. Babesia multigenic families
3.1. Variant erythrocyte surface antigen-1
Even though the apicoplast is an apicomplexa organelle, they share a complex of organelles that is characteristic of the apicomplexa: the apical complex. This complex is composed of spherical body, rhoptries and micronemes; in this organelle, different proteins involved in the life cycle are generated, and some of these are secreted to the media or directed to the membrane [16, 17, 18]. In the erythrocyte stage, some Babesia parasites, including B. bovis and bigemina, can invade the host erythrocytes in a directly way without a pre-erythrocyte stage. Antigenically, the surface of infected erythrocytes is different between Babesia strains and the parasitized erythrocytes present in an infected bovine could fluctuate widely over time, in a process called antigenic variation [19, 20, 21]. This process on the infected erythrocytes is carried out by the antigenically variant protein named the variant erythrocyte surface antigen-1 (VESA1) that is constituted by two subunits (a and b) and encoded in a multigene family; the genes ves-1 involved in this family are related too in cytoadherence and are distributed in the four chromosomes of Babesia [19, 22, 23]. Although more of 350 genes ves-1α and more of 80 genes ves-1β were previously reported, in B. bovis genome only 119 were evidenced .
The family of genes ves-1 is associated across all four chromosomes with another multigene family of proteins that are smaller in size than the ves-1 genes (Figure 1), due to small open reading frames (SmORFs). This family is the second largest in the B. bovis genome and comprises 44 genes without significant sequence identity to any protein or gene sequence available in databases. Of 44 genes, 42 are codified in a single exon, but from these 44 proteins that are extracellular just one does not have a signal peptide . Sequence analysis in the T2Bo and Mo7 strains demonstrated that the repertoire varies between strains and has multiple semi-conserved and variable blocks; this family comprises two major branches called SmORFs A and B, and these branches are defined by a large hypervariable insertion in 20 genes . Although the function of these proteins is unknown, it is believed that it could play a functional role in VESA1 biology or contribute to the antigenic variation and immune evasion as a consequence [9, 24].
In American strains, the variable merozoite surface antigen (VMSA) family contains the proteins MSA-1, MSA-2a1, MSA-2a2, MSA-2b and MSA-2c, while in Australian strains only tree genes were found: msa-1, msa-2c and msa-2a/b . The genes that conform this family reside on chromosome 1, and the four copies of msa2 gene are arranged tandemly in a head-to-tail fashion as long as msa1 gene is located 5 kbp upstream from the msa2 genes . These five proteins have a conserved GPI domain and are involved in the first attachment to the erythrocyte. However, the exposed epitopes are not conserved between these proteins of this family and between different strains around the world [18, 26, 27]. Even though these proteins are variable, some studies have been shown that msa-2c gene is the most conserved of this family. Monoclonal antibodies against this protein can recognize strains from different geographic regions, and polyclonal antibodies have an effect on the invasion process, suggesting its utility as recombinant vaccine antigen or in diagnostic tests [18, 26, 28, 29, 30, 31, 32, 33]. These results have not been observed in the other MSA proteins; the MSA-1 protein is immunogenic and avoids the invasion process in vitro, but the immunogenic response is not protective . It could be due to the fact that msa-1 gene has an important allelic variation in strains from the nearby geographical regions. This variation suggests that the antibodies generated could not have a cross-reaction between different strains [34, 35].
The spherical body protein (SBP) constitutes another family in B. bovis, and these proteins that are located in the spherical body of the apical complex are known as SBP1, SBP2, SBP3 and SBP4. In the invasion process, SBP2 is released from the spherical bodies to the cytoplasmic membrane of the erythrocyte . Twelve truncated copies and just one complete copy of sbp2 gene were identified, showing a conserved 3′ region in these copies [9, 37]. The complete copy and one truncated are located in the chromosome 4, the other truncated copies are located in the chromosome 3, and some of these truncated copies are expressed in erythrocytic stages of B. bovis .
A novel family of genes that codify proteins with similarities to 6cys family of Plasmodium were identified in B. bovis genome. This family contains six genes (6cys-A, B, C, D, E and F), and these genes are located in tandem in the chromosome 2 except for 6cys-F that is located in a distal region. To identify this family was employed the sequence of the P. falciparum PFS230 protein as a query that has a higher homology with Bbo-6cys-E gene. Antibodies against this protein have an inhibitory effect on the invasion process, suggesting its importance in control methods against B. bovis infection .
Inhibitors of cysteine proteases have been shown to hamper intraerythrocytic replication of B. bovis, and four papain-like cysteine proteases are found in B. bovis genome. The bovipain-2, which is the orthologous gene of P. falciparum falcipain-2 that is involved in hemoglobin digestion , is located in chromosome 4 by an ORF of 1.3 kb without introns, the characterization of this protein shown a molecular weight of 42 kDa, and a transmembrane region and is highly conserved between B. bovis strains of North and South America . The bovipain-2 could be employed as a vaccine or as a target of drugs in the babesiosis control.
4. Vaccine antigens
4.1. RON proteins and AMA-1
In Toxoplasma, the characterization of the invasion process allowed the identification of a complex of proteins generated in the rhoptry neck, called rhoptry neck proteins (RONs), this family of proteins consists of RON2, RON4, RON5 and RON8 that are related to AMA-1 in the formation of the moving junction (MJ) in the invasion process . The RON complex is inserted into the host cell; meanwhile, AMA-1 is released to the parasite membrane; this process is described in P. falciparum, where the specific interaction between the host membrane and the parasite membrane is mediated by AMA-1 and RON2; the disruption of this interaction avoids the invasion process [42, 43]. RON2 was identified in B. divergens and B. microti, has a full-length sequence of 4053 bp that codifies to a protein of 170 kDa and has apical localization; antibodies against this protein are inhibitors of parasite invasion like in other apicomplexan parasites; The B. divergens RON2 protein has a closely related sequence in B. bovis identified by BLASTp [42, 44, 45]. The firts protein described to participate in the invasion process as part of the MJ in apicomplexan parasites was AMA-1. This protein is stored in the micronemes and secreted to the apical end during the invasion process. In Babesia bovis, AMA-1 contains 606 amino acids and it is codified by a 1818 bp-long gene, without introns. AMA-1 is a type I transmembrane protein with a N-terminal ectodomain, which is divided into three subdomains containing 14 cysteins [18, 46, 47].
One of the most studied proteins of the rhoptries identified in B. bovis is the rhoptry-associated protein 1 (RAP-1). The gene is constituted by only one exon and has a length of 1698 bp with two copies separated by a noncodifying sequence of 1 kbp in B. bovis . However in B. bigemina, rap-1 is represented by three genes: rap-1a, rap-1b, and rap-1c, arranged in tandem, as explained latter . The members of this family have a signal peptide, four cysteine residues and a 14 aa motive and, moreover, contain immunogenic epitopes B and T that elicit a Th1 humoral response in the host that avoids the attachment of the parasite to the erythrocyte; its structure is conserved between different isolates and is expressed in the sporozoite [37, 50, 51, 52, 53, 54, 55].
In B. bovis was described a gene that codifies to a protein like to the T. gondii protein 1 of the micronemes (MIC1). This gene is located in the chromosome 3 and is highly conserved between strains of B. bovis. Its function is involved in the cito-adherence process through the binding to sialic acid, and antibodies against the recombinant protein and synthetic peptides designed on antigenic regions of B. bovis MIC-1 avoid the invasion process on the in vitro culture of the parasites .
The heat shock protein 20 (HSP-20) is a protein of 20.2 kDa associated with other small proteins related to heat shock in mammals and plants . The hsp-20 gene consists of 686 bp with an intron of 153 bp that makes a polypeptide of 177 aa . Antibodies against this protein recognize both B. bovis and B. bigemina, suggesting that HSP-20 contains conserved epitopes in these species [59, 60].
5. Babesia bigemina
During the first decade of twenty-first century, the sequencing and description of B. bovis genome  have helped to find genes that play important roles during its life cycle, currently. The Sanger Institute is leading the sequencing project of B. bigemina genome, which is estimated in 10 Mb size distributed in four chromosomes . The advances in genomics of both Babesia species that affects cattle in America are allowing researcher to find, analyze, compare and predict proteins involved directly in pathogenesis and its life cycle. For more than five decades, researchers have been trying to develop a vaccine against piroplasmosis; before 1980, several studies were carried out to immunize cattle in an effective way. The first attempts were directed in animals that were infected on purpose and healed from babesiosis as a strategy to avoid nondesirable infections . Some studies were focused on trying to find a way to reduce the virulence of high infective B. bigemina strains through inoculum passages in several animals , and some even tried to immunize calves in utero . As we know now, the development of a of an effective and low-cost vaccine is more complex than initially thought. Nowadays some countries produce vaccines against bovine babesiosis. The Queensland Government in Australia offers a vaccine made of parasitized bovine blood ; this live attenuated parasite vaccine must be stored at −196°C and during its production is necessary a batch of splenectomized calves that must remain in quarantine three times before the first procedures of manufacturing . It is evident that piroplasmosis vaccination involves long periods of production, surgery, and maintenance of animal infected blood donors and thorough procedures to achieve high standards of bioethical considerations.
Currently, research is focused on developing vaccines that avoid complex production procedures and the use of live animals; new technologies have arrived bringing opportunities to develop a vaccine using high throughput production. For this, certain obstacles must be solved before an effective vaccine is produced.
In vaccination, developing gene polymorphisms and antigenic variation is one of the first problems that researchers must cope with, and the selection of most suitable antigen candidates is a crucial step. With gene databases, the analysis of sequence variations has been made easier to find differences in distant geographical strains. In this sense, several studies have been carried out to find whether some proteins are conserved and how auspicious its election as vaccine candidate would be.
Antigenic variation is used by microorganisms as an evasion mechanism of the immune response, and in Babesia, the vesa family is the most studied group of genes used to “escape” from the host immune system. As described above for B. bovis, the vesa family is composed of two multicopy genes, ves1α and ves1β, which are distributed within the four chromosomes, and it is estimated that there are 72 and 43 copies of them, respectively. Both genes are located in opposite transcription directions and are governed by a bidirectional promoter followed by small sequences that seem to be incomplete fragments of the same recombined gene. The mechanism proposed for the multiple versions of the protein product of ves1α and ves1β has to do with the fact that along the genome are ves pseudogenes that act like reiterative donors of divergent sequences during several rounds of DNA replications, while the genes that are being transcribed are located in a locus of active transcription, which means that during this multigenic conversion segment event new versions of VESA proteins of B. bovis are being generated [68, 69].
Even though in B. bigemina antigenic variation as vesa family in B. bovis is not described, there is information about important genetic differences between strains from diverse geographical locations. On the next lines, we are going to review in general terms some of the genes that are promissory vaccine candidates in B. bigemina.
The apical merozoite antigen (AMA-1) is a protein that has been related to the tight junction complex formation; during this step in the red blood cell invasion, the protein interacts directly with the Rhoptry proteins to anchor both membranes; this process is well studied in the apicomplexan parasite T. gondii . In Babesia species, AMA-1 as been described as a low-diversity protein; in B. divergens, ama-1 was sequenced from nine isolates from France, in which only two punctual mutations were observed compared with the reference strain , in B. bovis, the analysis of Sri Lankan strains showed that ama-1 is a conserved member with about 95% of identity; the msa genes of this strains were mapped and showed variability. As mentioned previously, msa family is highly polymorphic. This last statement proves that the isolates analyzed are different because of their genotype, and among them, there is a highly conserved ama-1 gene . The B. bigemina ama-1 seems to be conserved as well, and Italian strains have a conserved sequence among them and considerable differences in comparison with Australian reference strains; however, when these are compared with Mexican and Argentinian reference strains, the sequence matches in a 99% of identity . The conservation level makes AMA-1 an excellent target for vaccine development.
The Rhoptry Associated Proteins are part of a multigenic family composed in B. bigemina of five genes arranged in tandem designated as rap-1a; between them, there are two other genes designated as rap-1b which is present in the same number of copies as rap-1a and at the end of the locus a single copy gene called rap-1c. All rap-1 family members are co-transcribed in merozoites, and some members seem to be conserved in geographical strains . It has been demonstrated in B. bovis that specific antibodies are capable of reducing sporozoite invasion to red blood cells in vitro ; in B. bigemina, antibodies against RAP-1 reduced parasitemia in comparison with an ovalbumin control in calves inoculated with iRBC .
The product of the gp45 gene is a glicosylphosphatidylinositol-anchored protein of 45 KDa, which has been related to the adhesion step during the invasion process to red blood cells; it is postulated that play the same role of msa family in B. bovis . Some studies proved that immunization of calves with the purified GP45 reduced significantly the parasitemia when challenged with a Mexican B. bigemina isolate . At this point, GP45 seemed to be an ideal vaccine candidate, however, several years later, other studies demostrated that the gene is not present among all B. bigemina strains. Southern Blot analysis revealed that Puerto Rico and St. Croix isolates do not possess gp45 sequence and also that in Texcoco strain the upstream sequence has polymorphisms and consequently there are nonfunctional promoters. As a result of this, there is a lack of transcription . This last information put in doubt if GP45 would be a good immunological target, not because of its neutralization efficiency but because of that this would not be a good candidate if the purpose of the vaccine is to have a broad-spectrum protection that includes several strains from distant geographical locations.
Profilin is a protein that participates in cytoskeleton ensemble ; in Toxoplasma gondii, due to its characteristic gliding motility where the cytoskeleton takes part of, profilin has been involved as an important protein to invade host cells and its antigenicity has been proved for its recognition by Toll receptors . There is new evidence that profilin is present in B. bigemina, B. bovis and B. microti, and more interesting is that sera from infected cattle with B. bovis and B. bigemina are capable to cross-react with recombinant profilin from both species and even with B. microti; the recombinant cattle babesial profilin is capable of conferring immunity in mice against B. microti . Even though there is no information about protective activity in cattle of profilin immunization against B. bigemina and B. bovis, profilin seems to be another promissory target to work on to achieve an effective vaccine.
The genes mentioned are examples of genes with low variability that can be used as a target to prevent babesiosis by B. bigemina; unfortunately, for vaccines developers the variation of sequences and gene products does not follow a high conservation rule. In this sense, experimental strategies have been built up to find a more suitable way to neutralize Babesia infection. Taking advantage of the information available on databases and the sequence analysis is possible to track the most appropriate targets. Alignment tools allow researchers to display the protein sequences from several distant geographical strain similarities among their proteins and find the most suitable candidates.
6. New strategies against apicomplexan parasites
There are several studies on Babesia proteins, which have an important role on the parasite’s life cycle and their immunogenicity. However, even though the protein role on the parasite development has been described in detail, currently there is not a single protein proposed as vaccine candidate against B. bovis or B. bigemina that generates an immunological protective response as effective as the one produced by the live, attenuated vaccines; the reason of it might be with the fact that one single antigen used as immunogen is not enough to display a strong immunological response. In an attempt to achieve protection against microorganisms, new strategies have been raising and one of them is the design of multiepitopic vaccines; in these novel strategies, Plasmodium genus and Toxoplasma gondii are some of the microorganisms within the apicomplexan parasites where these methodologies are being applied.
Researchers have been developing vaccines using more than one antigen. Such is the case of P. falciparum Chimeric Protein 2.9 (PfCP-2.9) composed of two blood-stage antigens, the carboxyl-terminal region of the protein known as Apical Membrane Merozoite Surface Protein 1 (MSP1–19) and domain III of the Apical Membrane Antigen 1 (AMA-1 III). The PfCP-2.9 resulted in being highly immunogenic in rabbits and in primates and is capable of producing an antibody titer 18-fold higher than both antigens administered in a mixture. The neutralization assays demonstrated that the fusion protein reduces substantially the parasite growth .
One concern of last decades is that scientists are predicting that some vector-borne diseases will increase as a consequence of expansion of vector habitats due to global warming . In addition to the fact that vectors are acquiring resistance to pesticides that have been using as a mean of eradication and control, today there are reports that malaria vector exhibits multiresistance to diverse chemical families used for its control leaving any suitable choice to reduce mosquito population [81, 83]. As a novel alternative to cope with this situation, multistage vaccines have been designed in an attempt to interrupt the life cycle in both vertebrate and invertebrate hosts. Using antigens from blood stages as the glutamate-rich protein (GLURP) that had been recognized as a natural antigen in acquiring immunity against malaria  and by the usage of sexual stage antigens Pfp48/45 that are involved in gamete fusion during sexual reproduction within the mosquito vector , the central objective of this alternative multiepitope vaccine is to confer immunity in the people that is at risk to acquire the infection and to reduce in long term the infection in the vector avoiding transmission . The usage of more than two antigens is also an opportunity to confer immunity against parasites and to ensure the success to block the infection. In T. gondii, an alternative vaccine prototype was designed as a chimeric protein with six predicted epitopes from surface proteins all of them bound in a single polypeptide sequence. This synthetic protein proved to be a good immunogen, and to stimulate CD8+ T cells from seropositive patients in comparison with a mixture of the same antigens and tachyzoite lysates, also the survival percentage in murine models infected with parasites increased substantially in the immunized individuals . The list of new vaccine candidates against Plasmodium and T. gondii still grows. The information that is generated in these two well-studied models serves as a starting point to extrapolate the strategies and propose new ones in the research of vaccines against apicomplexan parasites. This last section is a very narrow landscape of a long list of options that have been generated against other parasites that for sure are helping scientists to find the effective vaccines against cattle babesiosis.