Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle

Abstract

The gene encoding Babesia bovis rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant B. bovis RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between B. bovis-infected sera and B. bigemina-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to B. bovis.

FIG. 1.

IFAT analysis of mouse anti-B. bovis RAP-1 antibody. Noninfected bovine erythrocytes (A) B. bovis-infected bovine erythrocytes (B), and B. bigemina-infected bovine erythrocytes (C) were reacted with mouse anti-B. bovis RAP-1 antibody.

FIG. 2.

Western blot analysis of B. bovis RAP-1 expressed in High five insect cells by using mouse anti-B. bovis RAP-1 antibody or bovine anti-B. bigemina antibody. High five insect cells (lane 1) and High five insect cells infected with AcRAP-1-Ht (lane 2) were reacted with mouse anti-B. bovis RAP-1 antibody. High five insect cells infected with AcRAP-1-Ht (lane 3) were reacted with bovine anti-B. bigemina antibody.

FIG. 3.

IFAT analysis of B. bovis RAP-1 expressed in High five insect cells by using bovine anti-B. bovis antibody or bovine anti-B. bigemina antibody. High five insect cells (A) and High five insect cells infected with AcRAP-1-Ht (B) were reacted with bovine anti-B. bovis antibody. High five insect cells (C) and High five insect cells infected with AcRAP-1-Ht (D) were reacted with bovine anti-B. bigemina antibody.

FIG. 4.

Value from ELISA with recombinant RAP-1 with experimentally infected bovine sera. Lane 1, B. bovis-infected bovine sera; lane 2, B. bigemina-infected bovine sera; lane 3, noninfected bovine sera.