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

Abstract

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.

Keywords: Babesia bigemina; bovine babesiosis; micronemal proteins; sialic acid binding domain.

Figure 1

Babesia bigemina micronemal protein 1 gene: (a) mic-1 gene is localized in chromosome V as a hypothetical gene of 1658 bp (Accession number: XM_012914667). This gene contains 5 introns shown in the mRNA sequence as black bars. The product of mic-1 is a protein of 445 amino acids with a predicted signal peptide that is cleaved in amino acid 23 and a microneme adhesive repeat (MAR) domain located from amino acid 85 to amino acid 172; (b) Selected MIC-1 peptides containing predicted B-cell epitopes; (c) Multiple alignment of the MAR domain of MIC-1 in 8 different isolates of B. bigemina indicating in grey the localization of the three selected peptides.

Figure 2

Micronemal Protein 1 is expressed in merozoites of Babesia bigemina (B. bigemina): (a) mic-1 is a gene transcribed in blood stages of B. bigemina. Lane 1: Molecular weight marker. Lane 2: B. bigemina mRNA. Lane 3: B. bigemina mRNA without reverse transcriptase; (b) Intraerythrocytic merozoites of B. bigemina were incubated with sera from rabbits immunized with each MIC-1 peptide or with its corresponding pre-immunization serum. 100× objective; (c) Western blot analysis of MIC-1: Lane 1: infected red blood cells lysate incubated with pre-immune serum. Lane 2: Infected red blood cells lysate incubated with post-immune serum. Molecular markers on the left.

Figure 3

Effect of antibodies against Babesia bigemina MIC-1 to block parasite invasion in-vitro. Percentage of parasitemia inhibition in cultures supplemented with antibodies against each MIC-1 peptide. The percentage of infected erythrocytes was determined in B. bigemina cultures incubated with rabbit serum immunized with each anti-MIC-1 peptide. Serum from a rabbit immunized only with adjuvant (CS) was used as control. All data are expressed in mean percentage, the asterisk indicates the values that are significantly different from the control cultures incubated with pre-immunization serum (p < 0.05).