Identification of secreted antigen 3 from Babesia gibsoni

Abstract

A cDNA expression library of Babesia gibsoni was screened with the serum collected from a dog experimentally infected with B. gibsoni. A novel antigen sharing homology with secreted antigen 1 of B. gibsoni was isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome of B. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed in Escherichia coli could recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected with B. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.

FIG. 1.

(A) Comparison of the predicted amino acid sequence of BgSA3 with BgSA1 (GenBank accession no. AB246895). Conservation between amino acids is indicated by boxes. (B) Southern blot analysis. The B. gibsoni genomic DNA was digested with SphI (lane 1), KpnI (lane 2), HinfI (lane 3), AccI (lane 4), and HindII (lane 5). There is one cleavage site each for KpnI, HinfI, AccI, and HindII and no SphI cleavage site within the probes. The blot from lane 1 to lane 3 was hybridized with a 350-bp probe, and that from lane 4 to lane 5 was hybridized with a 1,000-bp probe.

FIG. 2.

SDS-PAGE and Western blot analysis of BgSA3. The samples were separated on a 12% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Lanes 1 and 2, purified rBgSA3 and rGST; lane 3, B. gibsoni-infected dog erythrocyte lysate; lane 4, normal dog erythrocyte lysate; and lanes 5 and 6, the same sample with lanes 3 and 4. Lanes 1 to 4 were SDS-PAGE, while lanes 5 and 6 were Western blot analysis probed with an anti-rBgSA3 mouse serum.

FIG. 3.

Observation of an antigen recognized by a mouse anti-rBgSA3 serum in confocal laser micrographs. (Top left) Immunofluorescent staining of B. gibsoni merozoites with a mouse anti-rBgSA3 serum. (Top right) Propidium iodide staining of B. gibsoni merozoite nuclei. (Bottom left) The top left panel overlaid on the top right panel. (Bottom right) The top left and top right panels overlaid on phase-contrast images of B. gibsoni merozoites. The images were derived from a single section.

FIG. 4.

Values of the ELISA with experimentally infected dog sera. Serum samples: 1, sera from B. gibsoni-infected dogs (n = 16); 2, SPF dog sera (n = 27); 3, sera from B. canis canis-infected dogs (n = 3); 4, sera from B. canis rossi-infected dogs (n = 3); 5, sera from B. canis vogeli-infected dogs (n = 2); 6, sera from N. caninum-infected dogs (n = 2); and 7, sera from L. infantum-infected dogs (n = 3). OD, optical density.

FIG. 5.

Detection of antibody against BgSA3 in a dog experimentally infected with B. gibsoni by ELISA using the rBgSA3. OD, optical density.

FIG. 6.

Detection of circulating BgSA3 in plasma from a splenectomized dog (A) and a nonsplenectomized dog (B) experimentally infected with B. gibsoni by a double-antibody sandwich ELISA.