ABSTRACT
Bovine polyclonal antisera to Babesia bigemina antigens separated by phenyl-Sepharose chromatography were used to screen a B. bigemina lambda gt11 cDNA expression library. Eleven B. bigemina-specific cDNA clones were studied in detail. DNA sequencing of 2 representative clones identified open reading frames encoding polypeptides representing the carboxy-termini of 2 different proteins. Both polypeptides contained a related central motif of tandem repeats flanked by a highly conserved carboxy-terminal region, but the sequences preceding the repeats were not related. Hybridisation and restriction enzyme analysis of the cDNA clones indicated that they were derived from a family of at least nine related, but not identical genes. Four different members of the gene family have been isolated from a B. bigemina lambda EMBL3 genomic library. The genes are not closely linked and they occur on the largest and smallest B. bigemina chromosomes resolved by pulsed field gel electrophoresis (PFGE). Antibodies raised against the native antigens and purified on recombinant fusion proteins bound to multiple proteins (50-70 kDa) in the original B. bigemina antigenic fractions.
Subject(s)
Antigens, Protozoan/genetics , Babesia/genetics , Genes, Protozoan , Repetitive Sequences, Nucleic Acid , Amino Acid Sequence , Animals , Babesia/immunology , Base Sequence , Blotting, Western , Cloning, Molecular , DNA, Protozoan/chemistry , Gene Expression Regulation , Gene Library , Molecular Sequence Data , Multigene Family , Nucleic Acid Hybridization , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Restriction Mapping , Sequence Analysis, DNAABSTRACT
Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphate-binding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions--one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.
Subject(s)
Antigens, Protozoan/immunology , Babesia/immunology , Babesiosis/prevention & control , Cattle Diseases/prevention & control , Dextran Sulfate/metabolism , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/metabolism , Cattle , Male , Vaccination/veterinaryABSTRACT
A slide enzyme-linked immunosorbent assay (SELISA), a modification of the standard ELISA technique, was developed for detection of Babesia bovis antibodies in bovine sera. Smears of B. bovis-infected blood were used as the source of antigen in the test which was read using a light microscope. Monoclonal antibodies to defined B. bovis antigens were used to demonstrate the cellular specificity of the test. The SELISA was shown to be as sensitive as existing non-enzyme based serological tests for B. bovis. Comparative to the conventional ELISA technique, it was more economical and technically simpler, thus making it an ideal test for field application.