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Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.

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

RESUMO

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2) and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR-RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.

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Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.

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To evaluate the effect of Lactobacillus casei on the effectiveness of the Mexican bovine babesiosis mixed vaccine, 20 bovines were randomly allocated into four groups of five animals (I, II, III, and IV). At day -2 animals in groups I and II were inoculated with saline solution by intramuscular route (i.m.) and animals in groups III and IV were inoculated with L. casei. At day 0 bovines in groups I and III were inoculated i.m. with bovine normal erythrocytes and animals of groups II and IV were inoculated with the babesiosis vaccine. Twenty-four days later each bovine was challenged with Babesia bovis- and B. bigemina-infected erythrocytes. The average rectal temperature in groups I and III was higher (P < 0.05) than that in the vaccinated groups after challenge. The average packed cell volume was lower (P < 0.01) in the control groups than in the vaccinated groups. At day 10 after challenge, the average anti-Babesia antibody level was higher in group IV than in group II. At day 7 after vaccination, the percentage of bovines positive to gamma interferon, as determined by real-time PCR, was 20, 0, 40, and 80 for groups I, II, III, and IV, respectively. All animals in control groups (I and III) were treated against babesiosis to avoid their death because they showed signs of babesiosis. The results indicate that L. casei, inoculated 2 days before the inoculation of the Mexican bivalent bovine babesiosis vaccine, improves the vaccine's efficiency.

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Gamma irradiation on bovine serum and red blood cells (RBC) allows proliferation and growth of in vitro-cultured Babesia sp., and has potential application to inactivate contaminating viruses and bacteria from the substrate. Gamma irradiation with 25 kGy in a source of (60)Co was able to inactivate infectious bovine rinotracheitis (IBR) and bovine viral diarrhea (BVD) viruses in artificially contaminated serum; besides, bacteria were also eliminated. In vitro culture of Babesia bovis (B. bovis) in modified substrate, by adding irradiated serum with (60)Co at 25 kGy was propagated from 24-well culture plates to 225 cm(2) tissue culture flasks, and percentages of parasitized erythrocytes (PPE) from 2.4% to 8.8% were obtained. Infected RBC adapted to Irrad S were transferred to the irradiated substrate in vitro culture system, by using serum irradiated at 25 kGy and RBC from 10 to 70 Gy. The PPE ranged from 3.1 to 11. Culture of Babesia bigemina (B. bigemina) was established with Irrad S (25 kGy); its propagation was achieved in tissue culture flasks reaching PPE from 0.5 to 4.3 with no statistical difference (P > 0.05) when compared to the nonirradiated control culture (1.2-4.8). B. bigemina-infected RBCs were transferred to the modified culture system by adding irradiated serum and RBC (25 kGy and 70 Gy, respectively). PPE obtained in culture flasks were from 0.8 to 4.2. The results indicate that gamma irradiation is a suitable method to inactivate potential viral contamination and eliminate bacteria from bovine serum, to produce a live attenuated vaccine through the in vitro culture.

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