ABSTRACT
Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Protozoan/blood , Antigens, Protozoan/analysis , Babesia/immunology , Babesiosis/diagnosis , Horse Diseases/diagnosis , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antigens, Protozoan/immunology , Antigens, Protozoan/isolation & purification , Babesia/growth & development , Babesia/isolation & purification , Babesiosis/parasitology , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Erythrocytes/parasitology , Fluorescent Antibody Technique, Indirect , Horse Diseases/parasitology , Horses , Immune Sera , Immunoblotting , Microscopy, Confocal , Microscopy, Immunoelectron , Species SpecificityABSTRACT
Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.
Subject(s)
Babesia/growth & development , Babesiosis/parasitology , Erythrocytes/parasitology , Horse Diseases/parasitology , Horses/parasitology , Parasitemia , Animals , Babesia/isolation & purification , Cells, Cultured , Culture Media , In Vitro Techniques , Parasitology/methodsABSTRACT
Antigen for the indirect fluorescent antibody test (IFAT) was routinely prepared from infected erythrocytes from horses experimentally infected with Babesia equi and Babesia caballi. With the successful establishment of in vitro cultures of B. equi and B. caballi, it is now possible to employ culture-derived antigens in this test. In this study, in vitro-propagated B. equi- and B. caballi-infected erythrocytes were used as antigen in the IFAT. Various modifications to an established protocol had to be implemented to allow repeatable results. Cultures with 3-4% parasitized erythrocytes were found to be most suitable. As cross-reactions of control sera on heterologous antigen were observed at serum dilutions of up to 1/40, a reciprocal titre of 80 was considered to be positive. In positive samples, specific fluorescence of Babesia parasites and/or erythrocyte membranes was observed. Fifteen sera from Babesia-free horses from Japan all tested negative in the IFAT. One hundred and ten field-horse sera from Central Mongolia were investigated in this study. The results indicate that both B. equi and B. caballi are endemic in horses in Central Mongolia, with 88.2% and 84.5% of horses being seropositive to B. equi and B. caballi, respectively.
