Title

The Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for the Measurement of Erysipelothrix rhusiopathiae Specific Antibody Levels In the serum of Atlantic Bottlenose Dolphins (Tursiops truncatus)

Date of Award

2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Bobby L. Middlebrooks

Advisor Department

Biological Sciences

Abstract

An isolate of Erysipelothrix rhusiopathiae implicated in the death of a Pacific bottlenose dolphin was obtained for use in the development of an enzyme linked immunosorbent assay (ELISA). After verification of its identity by a battery of morphological and biochemical tests, the bacterium was used to prepare reagents for an ELISA. The ELISA was developed using surface antigens extracted from the E. rhusiopathiae as the ELISA capture antigen and an indicator system consisting of biotin labeled rabbit anti-Atlantic bottlenose dolphin IgG, alkaline phosphatase labeled avidin and p -nitrophenyl phosphate as the chromogenic substrate. The ELISA was used to evaluate anti-E. rhusiopathiae titers in a series of serum samples from captive Atlantic bottlenose dolphins. Titers were compared with direct agglutination results using whole E. rhusiopathiae cells. No direct correlation was seen between the results of the two assays except that higher titers were seen for animals housed in open ocean pens (as opposed to closed water systems). Assays performed using mouse and rabbit antisera showed that the extracted E. rhusiopathiae antigens were successful in detecting immunoglobulins specific for several isolates of Erysipelothrix rhusiopathiae and even an isolate of E. tonsillarum . It was also shown that the capture antigen was specific for capturing anti-Erysipelothrix antibodies and not antibodies produced against other closely related bacteria. A second ELISA was developed using a single antigen instead of a mixed antigen as capture antigen. An E. rhusiopathiae specific antigen with a molecular weight of approximately 64,000 Daltons was chosen and purified from the antigen extract by affinity chromatography. Results of this ELISA showed a smaller percent deviation within the assay and titers comparable to those obtained using the extracted antigen mixture. When compared to results from latex agglutinations, a qualitative correlation was seen with each assay indicating positive and negative antibody levels in the serum samples. In the absence of known positive sera, a quantitative comparison of the ELISA and agglutination assays was less clear since titer definitions had to be very stringent to avoid false positive results.