Molecular identification and antigenic characterization of a merozoite surface antigen and a secreted antigen of Babesia canis (BcMSA1 and BcSA1)

Abstract

Background: Babesia canis is an apicomplexan tick-transmitted hemoprotozoan responsible for causing canine babesiosis in Europe and west Asia. Despite its importance, there is no known rapid diagnostic kit detection of B. canis infection in dogs. The present study identified two novel antigens of B. canis and used the recombinant antigens to establish a rapid, specific and sensitive serodiagnostic technique for detection of B. canis infection.

Methods: A complementary DNA (cDNA) expression library was constructed from the mRNA of B. canis and immunoscreened using B. canis-infected dog sera. The cDNAs encoding a merozoite surface antigen and a secreted antigen protein were identified and designated as BcMSA1 and BcSA1, respectively. The recombinant BcMSA1 and BcSA1 (rBcMSA1 and rBcSA1) expressed in Escherichia coli were purified and injected into mice for production of anti-sera. The native proteins were characterized by Western blot analysis and immunofluorescence. Furthermore, indirect enzyme-linked immunosorbent assays (iELISA) and rapid immunochromatographic tests (ICT) based on rBcMSA1 or rBcSA1 were established and evaluated to test specific antibodies in consecutive plasma samples from two B. canis-infected dogs.

Results: Antiserum raised against rBcMSA1 and rBcSA1 recognized the 39 kDa and 44 kDa native proteins by Western blot analysis, respectively. In addition, immunofluorescence and confocal microscopic observations revealed that BcMSA1 was found on the surface of parasites. However, BcSA1 localized in the matrix of the merozoites. The ELISA and ICT based on rBcMSA1 or rBcSA1 could detect specific antibodies in consecutive plasma samples from two B. canis-infected dogs. They showed no cross-reactions against the serum samples collected from dogs experimentally infected with closely related parasites.

Conclusion: Taken together, the current results indicated that the rBcMSA1 and rBcSA1 are promising serodiagnostic antigens for developing iELISA and ICT to detect B. canis infection. To our knowledge, this study is the first to report BcMSA1 and BcSA1 as potential antigenic proteins for serodiagnosis of B. canis infection in dogs.

Keywords: Babesia canis; BcMSA1; BcSA1; Canine babesiosis; ELISA; Immunochromatographic tests.

Fig. 1

The hypothetical transmembrane helix of BcMSA1 in the membrane using the SOSUI bioinformatic tool. a Structure of the genomic BcMSA1 gene. White bold lines indicate exons, and black rectangles indicate introns. The numeric numbers indicate the number of nucleotides; b BcMSA1 was predicted to have two hypothetical transmembrane helixes located at the N and C terminal; c Nucleotide sequence and predicted amino acid sequences of the genomic BcSA1 gene

Fig. 2

Southern blot analysis of genomic DNA of Babesia canis. a Genomic DNA was digested with different restriction enzymes, BamHI (lane 1), SalI (lane 2), PacI (lane 3), and KpnI (lane 4) and NaeI (lane 5), and hybridized with specific probe of the BcMSA1 gene; b Genomic DNA was digested with different restriction enzymes, BamHI (lane 1), SacI (lane 2), NheI (lane 3), and KpnI (lane 4), and hybridized with a specific probe of the BcSA1 gene

Fig. 3

SDS-PAGE and immunoblot analysis of recombinant and native BcMSA1 and BcSA1. a The 10 % SDS-PAGE stained with Coomassie blue: recombinant BcMSA1 fused with GST (lane 1) and GST (lane 2). Western blot analysis of recombinant protein: recombinant BcMSA1 (lane 3) and GST (lane 4) probed with B. canis-infected dog serum; b Western blot analysis of native BcMSA1. B. canis-infected erythrocyte lysate (lane 1) and normal canine erythrocyte lysate (lane 2) probed with anti-rBcMSA1 mouse serum; c The 10 % SDS-PAGE stained with Coomassie blue: recombinant BcSA1 fused with GST (lane 1), recombinant BcSA1 fused without GST (lane 2) and GST (lane 3). Western blot analysis of recombinant protein: recombinant BcMSA1 (lane 4), recombinant BcSA1 fused without GST (lane 5) and GST (lane 6) probed with B. canis-infected dog serum; d Western blot analysis of native BcMSA1. The plasma from a dog infected with B. canis (lane 1) and a normal dog (lane 2) probed with anti-rBcMSA1 mouse serum

Fig. 4

Localization of BcMSA1 and BcSA1 at inner and outer surface of erythrocyte stages of B. canis by immunofluorescence staining and laser confocal microscopy. a Observation of the native BcMSA1 in thin blood smears of B. canis-infected RBC stained with mice anti-rBcMSA1 serum and pre-immune serum; b Observation of the native BcSA1 in thin blood smears of B. canis-infected RBC stained with mice anti-rBcSA1 serum and pre-immune serum: (i) Phase-contrast images; (ii) Propidium iodide staining; (iii) Immunofluorescent staining; (iv) Merged image

Fig. 5

Evaluation of rBcMSA1 and rBcSA1 as diagnostic antigens by ELISA system. a, b Antibody responses of sequential serum samples from two non-splenectomized dogs experimentally infected with B. canis to rBcMSA1 and rBcSA1 antigens in iELISA; c, d The reactivity of rBcMSA1 and rBcSA1 with B. canis and closely related parasite-infected dog sera. Abbreviations: Bc, B. canis-infected dogs sera; SPF, SPF dog sera; Br, B. rossi-infected dogs sera; Bv, B. vogeli-infected dogs sera; Bg, B. gibsoni-infected dogs sera; Nc, N. caninum-infected dogs sera; N, serum samples from non-endemic area in Osaka, Japan

Fig. 6

Evaluation of ICT based on rBcMSA1 and rBcSA1. a Cross-reactivity of ICT using rBcMSA1 with closely related parasite-infected canine sera: lane 1, B. rossi-infected dog serum; lane 2, B. vogeli-infected dog serum; lane 3, B. gibsoni-infected dog serum; lane 4, L. infantum-infected dog serum; lane 5, B. canis-infected dog serum; lane 6, a SPF dog serum; b Specific antibody responses to rBcMSA1 in sequential serum samples from a non-splenectomized dog experimentally infected with B. canis; c Cross-reactivity of rBcSA1 with closely related parasite-infected canine sera: lane 1, SPF dog serum; lane 2, B. canis-infected dog serum; lane 3, B. rossi-infected dog serum; lane 4, B. vogeli-infected dog serum; lane 5, B. gibsoni-infected dog serum; lane 6, L. infantum-infected dog serum; d Specific antibody responses to rBcSA1 in sequential serum samples from a non-splenectomized dog experimentally infected with B. canis