Abstract
BackgroundBabesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis.ResultsThe B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas.ConclusionsThe data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.
Highlights
Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods
In Plasmodium falciparum, the “tight junction” is known as the “moving junction”, and it was described as a specific and irreversible interaction between two proteins: the apical membrane antigen-1 (AMA-1) located on the merozoite surface and the rhoptry neck protein 2 (RON2), which is integrated to the red blood cells (RBC) membrane after its secretion from the rhoptries in a complex formed with other RON proteins [7,8,9]
Employing eight different pairs of primers, which were designed based using the BBOV_I001630 reference sequence, it was possible to obtain the full sequence of ron2 in four isolates of B. bovis: Chiapas, Colima, Nayarit and Veracruz
Summary
Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. The intraerythrocytic protozoan Babesia bovis is the major causal agent of bovine babesiosis, which is one of the most important veterinary diseases transmitted by arthropods. B. bovis belongs to the phylum Apicomplexa, which includes Plasmodium spp., and Toxoplasma gondii, two examples of pathogens within this phylum with medical importance. The parasites of this phylum are characterized by apical organelles such as rhoptries, micronemes and spherical bodies. B. bovis merozoites invade red blood cells (RBC), while secreting proteins from the apical organelles and forming close junctions between the membrane of the parasite and the RBC membrane. The purpose of the present study was to identify a B. bovis homolog gene of RON2 and define its pattern of expression and functional relevance
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