Additional file 5: Figure S1. Intraerythrocytic Babesia bovis merozoites express RON2. Merozoites were incubated with bovine antiserum against RON2 ( a- c), bovine pre-immune serum ( d-f), or bovine antiserum against B. Bovis ( g- i), then with an Alexa Fluor-488 conjugated with protein G (green fluorescence) and DAPI for DNA staining (blue fluorescence). The smears were analyzed by confocal microscopy using the following channels: individual channel for Alexa Fluor-488 ( a, d and g), individual channel for DAPI ( b, e and h) or merged channels for Alexa Fluor-488 and DAPI ( c, f and i).

Scale-bars: 10 μm. (TIF 17380 kb).

Results The B. Bovis ron2 gene has a similar synteny with the orthologous gene in the B.

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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. 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.

Background 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. 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. The proteins related to these organelles are implicated in the invasion and egression of host target cells [–]. Importantly, most apicomplexan parasites share four basic steps in the invasion process: (i) attachment to the target host cell; (ii) parasite reorientation to align the apical organelles in close contact with the membrane surface of the target cell; (iii) target cell surface membrane invagination, involving several molecular interactions between protozoan ligands and host receptors so as to make tight junctions; and (iv) parasite internalization, a process that also occurs continuously in the blood of Babesia infected bovines.

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. Once inside the RBC, the parasite multiplies by binary fission in two merozoites, which, upon egression from their original host erythrocyte, go to invade other RBCs to perpetuate this cycle of asexual replication [–]. In Plasmodium falciparum, the “tight junction” is also 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 RBC membrane after its secretion from the rhoptries in a complex formed with other RON proteins [–]. The disruption of AMA-1-RON2 interaction ceases the merozoite invasion, making these proteins vaccine candidates []. Expression of the neutralization-sensitive AMA-1 was already reported in B. Bovis; however, there are no previous reports describing the conservation and functional role of RON2 in this parasite despite being recently described in B. Pareto dr setup rw exe keygens. Divergens and B.