ABSTRACT
Recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Babesia bigemina infection by using a full-length B. bigemina rhoptry-associated protein 1 (rRAP-1) and the truncated C-terminal RAP-1 (rRAP-1/CT). While the rRAP-1 showed cross reactivity between B. bigemina- and Babesia bovis-infected bovine sera, the rRAP-1/CT was highly specific to B. bigemina-infected bovine sera and proved useful in the detection of sequential sera collected from an experimentally infected cow during the acute and latent infection. The high yield of soluble rRAP-1/CT and its diagnostic specificity demonstrate its potential in the diagnosis of B. bigemina infection. Its usefulness for epidemiological investigation is currently being evaluated.
Subject(s)
Antigens, Protozoan , Babesia/immunology , Babesiosis/veterinary , Cattle Diseases/diagnosis , Cattle Diseases/parasitology , Protozoan Proteins/immunology , Animals , Babesiosis/diagnosis , Babesiosis/parasitology , Cattle , Cross Reactions , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Fluorescent Antibody Technique, Indirect/methods , Fluorescent Antibody Technique, Indirect/veterinary , Gene Expression , Mice , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sensitivity and SpecificityABSTRACT
In this study, we characterized a novel Babesia bovis cDNA clone obtained by immunoscreening the cDNA expression phage library with B. bovis-infected bovine serum. The genetic analyses showed that it contained an open reading frame of 993 bp, which was considered to encode B. bovis L-lactate dehydrogenase (BbLDH: E.C. 1.1.1.27) because of the strikingly high amino acid identities of its gene product to the LDHs of Plasmodium falciparum and Toxoplasma gondii. Immunological analyses with the anti-recombinant BbLDH mouse serum showed that 36 kDa of the native BbLDH was expressed not only in the cytoplasm of intra- and extraerythrocytic parasites but also along the membrane of infected erythrocytes. The kinetic properties of recombinant BbLDH proved a certain enzymatic activity of LDH, and the activity was significantly inhibited by the addition of gossypol, a competitive inhibitor of protozoan LDHs. Moreover, 100 microM of the gossypol irretrievably arrested the in vitro growth of B. bovis. The results demonstrated that BbLDH provides a suitable drug target for the design of novel babesial chemotherapeutics.
Subject(s)
Babesia bovis/drug effects , Babesia bovis/enzymology , Babesiosis/veterinary , Cattle Diseases/drug therapy , L-Lactate Dehydrogenase/genetics , Amino Acid Sequence , Animals , Antibodies, Protozoan , Babesia bovis/genetics , Babesia bovis/growth & development , Babesiosis/drug therapy , Babesiosis/parasitology , Cattle , Cattle Diseases/parasitology , Cloning, Molecular , DNA, Complementary/genetics , DNA, Protozoan/genetics , Enzyme Inhibitors/pharmacology , Female , Genes, Protozoan , Gossypol/pharmacology , In Vitro Techniques , L-Lactate Dehydrogenase/antagonists & inhibitors , L-Lactate Dehydrogenase/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence DataABSTRACT
An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1-rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)-by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.
Subject(s)
Babesia bovis/immunology , Babesiosis/veterinary , Cattle Diseases/diagnosis , Gene Deletion , Protozoan Proteins/immunology , Recombinant Proteins/immunology , Animals , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Babesiosis/parasitology , Baculoviridae/genetics , Baculoviridae/metabolism , Cattle , Cattle Diseases/parasitology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Sensitivity and SpecificityABSTRACT
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.
