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Naturally acquired immunity to buffalo fly (Haematobia irritans exigua) infestation was examined in cattle. Animals exposed to flies had serum antibodies to buffalo fly antigens at levels that correlated with the intensity of exposure. Two weeks of intense exposure to buffalo fly induced an increase in peripheral blood eosinophil numbers and a concomitant rise in serum antibody levels in exposed animals. Antigens specific for antibody induced by natural exposure were identified using antisera from exposed cattle to probe Western blots of whole fly homogenate separated using SDS-PAGE. Similar immunoreactive bands were found with buffalo fly saliva. Immunoreactive proteins were partially purified from whole fly homogenates by anion-exchange chromatography. Fractions eluted from columns were screened using Western blots probed with serum from exposed animals. Exposed animals showed immediate hypersensitivity to partially purified antigens and to buffalo fly saliva. Flies which fed on exposed animals with high serum levels of antibody to fly antigens did not show greater mortality than flies fed on unexposed animals.