Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

John B. Malone

Abstract

A monoclonal antibody (MAb)-based western blot analysis (WB) was developed for detection of a 26-28 kD coproantigen in Fasciola hepatica infected cattle. Using WB the coproantigen was detected in feces from experimentally infected calves with 22 flukes or more. Using the biotin-streptavidin modification of WB, antigen was detected in the feces from experimentally-infected calves with 10 flukes or more as early as 6 weeks post-infection. The MAbs did not react with the ES of Paramphistomum sp. or Moniezia sp, but showed slight cross-reactivity with ES from F. gigantica, and there was moderate cross-reactivity with Fascioloides magna ES. Differential staining of purified 26-28 kD coproantigen on SDS-PAGE, under reducing and non-reducing conditions, indicated that the coproantigen was a monomeric, highly glycosylated glycoprotein. Alkaline treatment of the purified coproantigen resulted in an 8 KD protein core which still contains the epitope recognized by the MAbs. No protease activity was associated with the 26-28 kD coproantigen. The coproantigen was cleaved by trypsin without altering the reactive epitope, but was resistant to pepsin treatment. The coproantigen was stable under several different storage conditions. Indirect immunofluorescence on tissue sections of adult flukes indicated that coproantigen was present on gut cells and tegument. A capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the 26-28 kD coproantigen. Monoclonal antibody M2 DS/DSF10 was used to capture the antigen from the feces and hyper-immune rabbit ant-26-28 kD glycoprotein followed by peroxidase labeled goat anti-rabbit immunoglobulin was used for detection. The assay was able to detect down to 300 pg coproantigen/ml. Feces from 27 experimentally-infected calves with known numbers of flukes were used to evaluate the test. Pre-infection feces from the same animals were used as negative controls. Coproantigen was detected in feces of calves with more than 10 flukes. In conclusion, both the coproantigen capture ELISA and WB were both more sensitive than microscopic examination for the diagnosis of Fasciola infection and both could detect pre-patent infections. Moreover, the ELISA for the detection of coproantigen was easier to perform.

ISBN

9780591034981

Pages

102

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