Date of Award

Fall 2001

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Aaron B Margolin

Abstract

The Great Bay Estuarine system is being impacted by point source and nonpoint source pollution that affect shellfish quality. Many outbreaks of human gastroenteritis are potentially caused by the consumption of raw or undercooked oysters in which the protozoan pathogens, Giardia lamblia and Cryptosporidium parvum are present. The development of a rapid and sensitive technique to detect infectious protozoa in oysters is a necessity.

Immunofluorescence has been employed as a technique for the detection of these organisms in drinking water. Cross reactivity with algae and the production of false positive results limits the usefulness of this technique in environmental studies. Molecular approaches, such as the polymerase chain reaction, are often overlooked in environmental studies. This is due to obstacles such as enzymatic inhibitors that are intrinsic in the environment as well as the small sample size that can be analyzed.

The goal of this study was to develop a multiplex polymerase chain reaction for the simultaneous detection of Giardia lamblia and Cryptosporidium parvum in oysters. An immunomagnetic separation technique was utilized to capture the Giardia cysts and Cryptosporidium oocysts from oyster homogenate. This method resulted in the removal of enzymatic inhibitors and increased detection sensitivity. Primers designed to specifically hybridize to Giardia lamblia DNA and Cryptosporidium parvum DNA were also modified at the 5' end by the addition of a 20-mer universal primer sequence, stabilizing hybridization kinetics. The development of a multiplex PCR assay achieved the specificity necessary to target the human pathogens and produce results more quickly then detection via immunofluorescence. A subsequent nested PCR assay prevented any potential false positive results.

To determine the applicability of this method to field studies, three sites were sampled in the Great Bay Estuarine system using a sample size of 25 oysters. Two of the selected field sampling sites were classified as approved for shellfish harvesting based on most-probable-number water analysis and one site was closed to shellfish harvesting, due to high fecal coliform counts (>70 fecal coliforms/100 ml water). Each site sample was processed and analyzed using the multiplex PCR and nested PCR procedure. The presence of Cryptosporidium parvum and/or Giardia lamblia was detected at each field sampling site.

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