Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata.

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Experimental vaccination in rabbits using the peptide RmS-17 antigen reduces the performance of a Mexican Rhipicephalus microplus tick strain.

The tick vector Rhipicephalus microplus is considered one of the main problems in cattle production in tropical and subtropical regions. Anti-tick vaccines may form an alternative tick control method to the use of acaricides, and tick salivary proteins, such as Serpins, may be valuable as target antigens for developing anti-tick vaccines. In this study, we synthesized a recombinant peptide derived from Serpin RmS-17 protein using an Escherichia coli expression system and characterized the efficacy of the peptide RmS-17 for the control of R. microplus females infesting rabbits. Twelve New Zealand white rabbits were assigned to three experimental groups and vaccinated with three subcutaneous doses of the peptide RmS-17, recombinant R. microplus Bm86 antigen, and adjuvant/saline alone. The tick challenge was conducted with 120 R. microplus adults (60 females and 60 males) per animal, with the ticks placed inside a cotton sleeve glued to the back of the rabbit. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, the reproductive performance of R. microplus was determined. The results showed that experimental vaccination in rabbits using the peptide RmS-17 antigen had a vaccine efficacy of 79% based on reductions in adult tick number, oviposition, and egg fertility compared to control animals. The peptide RmS-17 vaccinated rabbits developed a strong humoral immune response expressed by high anti-pRmS-17 IgG levels, and the Western blot analysis confirmed that it is immunogenic. The efficacy for the Bm86 vaccine was 62%, which is within the range of efficacy reported previously for Bm86 vaccine. The negative correlation between antibody levels and reduction in tick number strongly suggests that the effect of the vaccine was the result of the antibody response in vaccinated rabbits. In conclusion, this is the first study to evaluate the efficacy of the peptide RmS-17 against R. microplus tick infestation and show it to be immunogenic and protective in a rabbit model.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

Immunomolecular Characterization of MIC-1, a Novel Antigen in Babesia bigemina, Which Contains Conserved and Immunodominant B-Cell Epitopes that Induce Neutralizing Antibodies.

Babesia bigemina is one of the most prevalent species causing bovine babesiosis around the world. Antigens involved in host cell invasion are vaccine targets for this disease but are largely unknown in this species. The invasion process of Babesia spp. into erythrocytes involves membrane proteins from the apical complex. A protein stored in the micronemes, called Micronemal Protein 1 (MIC-1), contains a sialic acid binding domain that participates in the invasion process of host cells and is a vaccine candidate in other apicomplexan parasites. It is not known if there is a homologous gene for mic-1 in B. bigemina. Therefore, the aim of this study was to characterize the mic-1 gene homologue in Babesia bigemina. A gene was found with a microneme adhesive repeat (MAR) domain in the predicted amino acid sequence. Transcription was determined by reverse transcription polymerase chain reaction (RT-PCR). Subsequently, antibodies against peptides containing conserved B-cell epitopes were used to confirm the expression of MIC-1 in intraerythrocytic merozoites. The presence of anti MIC-1 antibodies in cattle naturally infected with B. bigemina was determined and up to 97.4% of the cattle sera (113 out of 116) identified MIC-1 using enzyme-linked immunosorbent assay (ELISA) methods. Finally, antibodies against MIC-1 were able to block 70% merozoite invasion in-vitro.