Cryptosporidium In Bivalves as Indicators of Fecal Pollution in the California Coastal Ecosystem

(Funded 2002-2003)

Principal Investigators:
Patricia A. Conrad
School of Veterinary Medicine
UC Davis
(530) 752-7210
paconrad@ucdavis.edu

Edward R. Atwill
School of Veterinary Medicine
UC Davis
(559) 688-1731
ratwill@ucdavis.edu

Ian A. Gardner
School of Veterinary Medicine
UC Davis
(530) 752-6992
iagardner@ucdavis.edu

Executive Summary:
The freshwater, estuarine, and nearshore marine waters of California provide critical resources for drinking water, food production, industry, recreation, and wildlife habitat. Though these water resources are highly utilized, they are only minimally monitored for the presence of human pathogens. The coastal ecosystem is highly impacted by fecal pollution from sewage effluent, agricultural wastewater, and municipal/urban runoff. Water quality monitoring has historically focused on trace metals, pesticides, and bacterial coliforms. However, this monitoring does not address the public health threat of zoonotic protozoal parasites such as Cryptosporidium in the aquatic environment.

Cryptosporidium is a widespread fecal pathogen that has caused many waterborne epidemics of diarrheal disease in people. For example, the 1993 Milwaukee outbreak infected over 400,000 people and was traced to Cryptosporidium oocysts in the public water supply. The Center for Disease Control has reported thousands of cases of cryptosporidiosis in the US this year, including hundreds of cases in California. Infected humans and animals can shed the infective egg, or oocyst, of Cryptosporidium in their feces, thus serving as a source of infection for others. Humans have been known to shed over a billion oocysts, and cattle over 60 billion oocysts during the initial phase of Cryptosporidium infection. The diarrheal disease is often self-limiting but can be life threatening to immunocompromised individuals such as AIDs patients. Cryptosporidium was recently documented for the first time in marine mammals along the California coast, and our preliminary research has also detected Cryptosporidium in bivalves collected along the California coast. No studies have been conducted to investigate the magnitude and sources of Cryptosporidium contamination in California waters.

Bivalves, including clams and mussels, have been shown to concentrate Cryptosporidium oocysts, thus acting as indicators of fecal pollution in aquatic ecosystems. They filter large amounts of water, up to 2.5L/hr/bivalve, and have been shown to concentrate protozoal parasites from the surrounding water. Bivalves such as Mytilus and Corbicula are plentiful in salt and freshwater coastal California ecosystems, respectively, and can be efficiently tested for Cryptosporidium using a Real-Time TaqMan PCR technique developed in our laboratory. This assay can detect DNA of a single organism, provides a quantitative result of how much DNA or oocysts are present in the sample, and can differentiate between various Cryptosporidium species.

Many species of Cryptosporidium exist, with variable pathogenicity to humans and animals. Cryptosporidium oocysts have a very low infective dose, are environmentally stable, are endemic in California livestock populations, and are most commonly transmitted in water contaminated with human or animal feces. The most common pathogenic species, C. parvum, has been molecularly categorized into two primary genotypes: genotype 1 has only been recovered from humans, while genotype 2 has been recovered from both humans and animals. This information is important to consider when investigating environmental sources of fecal pollution. Genotype 1 is most likely to be found in association with point sources of human sewage contamination, such as urban sewage outfalls, while genotype 2 is most likely to be found in association with sources of livestock fecal contamination, such as agricultural runoff. The TaqMan PCR assay can distinguish between these two genotypes.

We have detected Cryptosporidium DNA in our preliminary study of wild bivalves from coastal California. To characterize the severity and extent of this fecal parasite contamination, we propose a two year study: in the first year we will sample bivalves at 18 sites along the California coast, including freshwater, estuarine, and nearshore marine sites located either 1) within 500 m of livestock runoff, 2) within 500 m of human sewage runoff, or 3) more than 3 km from both livestock and human sewage runoff. The second year will target at least 4 highly contaminated sites for further investigation of potential sources in the vicinity of bivalve collection sites. We will evaluate bivalves as water quality indicators of fecal pollution by testing bivalves collected on days 1, 2, 7, and 14 of large storms for Cryptosporidium levels. The results of this study will be highly significant for optimal watershed management as well as to minimize the impact of fecal pollutants in our coastal ecosystems.