Background The Thrombospondin-Related Anonymous Protein (TRAP) has been described as a potential vaccine candidate in several apicomplexan parasites. However, this protein and members of this family have not been characterized yet in Babesia bigemina, one of the most prevalent species causing bovine babesiosis.Methods The Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein family (TRP) families were identified in Babesia and Theileria through the search of the TSP-1 adhesive domain, the hallmark motif of TRAP proteins. Structural modelling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 and specific antisera were produced, and used in Western blot analysis and indirect fluorescent antibody test (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by ELISA and invasion assays.Results TRAP family has 3 members in B. bigemina (BbiTRAP-1-3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1) and cytoplasmic C-terminus domains along with transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site (MIDAS) for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lack the vWFA domain,and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units is present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed expression of BbiTRAP-1 by blood stage parasites. Partial recognition by a panel of sera from B. bigemina infected cattle in ELISA using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti- recombinant BbiTRAP-1 antibodies were capable of neutralize merozoite invasion in vitro. Conclusions We identified the TRAP and TRP gene families in several Babesia and Theileria species, and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in future vaccine candidates against B. bigemina.

BackgroundThe thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate in several apicomplexan parasites. However, this protein and members of this family have not been characterized yet in Babesia bigemina, one of the most prevalent species causing bovine babesiosis.MethodsThe Babesia bigemina TRAP-1 (BbiTRAP-1) gene of 3186 bp was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through the search of the TSP-1 adhesive domain, the hallmark motif in both proteins. Structural modelling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at ~107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody test (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by ELISA and invasion assays.ResultsTRAP family has 3 members in B. bigemina (BbiTRAP-1-3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1) and cytoplasmic C-terminus domains along with transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site (MIDAS) for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units is present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed expression of BbiTRAP-1 by blood stage parasites. Partial recognition by a panel of sera from B. bigemina infected cattle in ELISA using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were capable of neutralizing merozoite invasion in vitro. ConclusionsWe identified the TRAP and TRP gene families in several Babesia and Theileria species, and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in future vaccine candidates against B. bigemina.

Background The thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate in several apicomplexan parasites. However, this protein and members of this family have not been characterized yet in Babesia bigemina, one of the most prevalent species causing bovine babesiosis.Methods The Babesia bigemina TRAP-1 (BbiTRAP-1) gene of 3186 bp was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through the search of the TSP-1 adhesive domain, the hallmark motif in both proteins. Structural modelling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at ~107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody test (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by ELISA and invasion assays.Results TRAP family has 3 members in B. bigemina (BbiTRAP-1-3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1) and cytoplasmic C-terminus domains along with transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site (MIDAS) for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units is present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed expression of BbiTRAP-1 by blood stage parasites. Partial recognition by a panel of sera from B. bigemina infected cattle in ELISA using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were capable of neutralizing merozoite invasion in vitro. Conclusions We identified the TRAP and TRP gene families in several Babesia and Theileria species, and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in future vaccine candidates against B. bigemina.

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