MOLECULAR TOOLS TO EVALUATE THE EPIDEMIOLOGY AND CONTROL OF WATERBORNE ZOONOTIC INFECTIONS

Authors: Higgins, James; Shelton, Daniel; Karns, Jeffrey; BELT, KENNETH - USDA FOREST SERVICE

Submitted to: American Association of Veterinary Parasitologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 5/1/2003
Publication Date: 7/18/2003
Citation: Higgins, J.A., Shelton, D.R., Karns, J.S., Belt, K. 2003. Molecular tools to evaluate the epidemiology and control of waterborne zoonotic infections. American Association of Veterinary Parasitologists Proceedings, July 22, 2003.Denver, Colorado. p. 61.

Interpretive Summary: In the past 15 years in the USA, severe and sometimes life-threatening infections with diarrhea-casusing strains of the bacterium Escherichia coli have been linked to exposure to contaminated water. Sometimes livestock production, or use of manure to fertilize crops grown in proximity to freshwater streams, has been responsible for the deposition of these pathogens into the water. Scientists at the USDA-ARS and the USDA-Forest Service have been working in collaboration with researchers at the Center for Urban Environmental Research and Education at the University of Maryland, Baltimore County, on molecular biology-based methods for screening stream water samples for the presence of enteropathogenic E. coli (EPEC). Nine streams, located in west Baltimore County, Maryland, and representing a variety of ecological locales--forested, semi-urban, agricultural, and urban--have been sampled on a bimonthly or weekly basis since March, 2002. The majority of samples have been found to contain EPEC; the more polluted streams are more likely to harbor this organism. Urban runoff, particularly that associated with leaking septic tanks and failed sewage treatment plant systems, plays a major role in depositing EPEC into streams. Streams associated with forested areas and agricultural production have lower EPEC prevalence rates. In conjunction with examining the stream water samples for the presence of EPEC, the research teams are also screening water from two of the streams for the presence of the protozoan parasite Cryptosporidium parvum. A combination of centrifugation, and molecular biology-based techniques, are being employed to detect the parasite. Since their start in March 2003, the screening assays have detected C. parvum in ~40% of the samples. DNA sequencing analysis of the detected parasites is being used to determine what host animal the parasites originate from; results to date indicate that cattle, or humans, may be implicated in this regard.

Technical Abstract: Since March 2002, we have been testing water samples-- collected from streams associated with a variety of ecosystems--for the presence of pathogenic E. coli (EPEC). Our protocol involves culturing total coliforms from the water samples, extracting DNA from the cultures, and detecting the pathogenicity-related tir gene using PCR. As of February 2003 coliform cultures from over 450 samples have been assayed and over 63% found to contain EPEC by tir gene PCR. DNA sequencing of the ~450 bp amplicon from 30 of these samples yields two clades: a major one, consisting of sequences with a high degree of similarity to that of E. coli strain O157:H7; and a minor clade with similarity to E. coli strains O26, B10, and 95ZG1. Among the myriad PCR assays published for use in detection of Cryptosporidium, we have focused on the use of an RT-PCR targeting the RNA of a viral symbiont present in the nucleus of this protozoan. When fresh oocysts are used as template, the assay is capable of detecting as few as one oocyst. It is feasible to distinguish between strains of C. parvum by exploiting differences in the sequences of their viral symbionts. The viral symbiont RT-PCR assays is being evaluated for its ability to distinguish between aged (and nonviable) oocysts and those that are younger and viable.