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 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. The 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. The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Transmission of Anaplasma marginale by unfed Rhipicephalus microplus tick larvae under experimental conditions

Rhipicephalus microplus economically impacts cattle production in tropical and subtropical countries. Application of acaricides constitutes the major control method; however, inadequate use has increased resistant tick populations, resulting in environmental and cattle product contamination. Anti-tick vaccines based on the Bm86 antigen are an environmentally friendly, safe, and economically sustainable alternative for controlling R. microplus infestations. Nevertheless, variable efficacy has been experienced against different geographic tick strains. Herein, we evaluated the efficacy of a conserved polypeptide Bm86 derived from a Mexican R. microplus strain previously characterized. Twelve cows were assigned to three experimental groups and immunized with three doses of the polypeptide Bm86 (pBm86), adjuvant/saline alone, and Bm86 antigen (control +), respectively. Specific IgG antibody levels were measured by ELISA and confirmed by Western blot. In addition, the reproductive performance of naturally infested R. microplus was also determined. The more affected parameter was the adult female tick number, with a reduction of 44% by the pBm86 compared to the controls (p < 0.05), showing a vaccine efficacy of 58%. Anti-pBm86 IgG antibodies were immunogenic and capable of recognizing the native Bm86 protein in the eggs, larvae, and guts of R. microplus. The negative correlation between antibody levels and the reduction of naturally tick-infested cattle suggested that the effect of the polypeptide Bm86 was attributed to the antibody response in immunized cattle. In conclusion, the polypeptide Bm86 showed a specific immune response in cattle and conferred protection against R. microplus in a Mexican tropical region. These findings support further experiments with this antigen to demonstrate its effectiveness as a regional vaccine.

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.