information expected to be derived frorn the research.)

The California coastal ecosystem is highly impacted by fecal pollution from sewage outfalls, agricultural runoff, and urban stormwater runoff. In the past, water quality monitoring has focused on toxins and bacterial coliform counts. However, this methodology does not address the public health threat of zoonotic protozoal parasites or allow for the identification of pollution sources.

Cryptosporidium species are pathogenic protozoal parasites that are shed by both humans and animals, are endemic in the California livestock populations, have a low infective dose, and have environmentally stable oocysts that can be spread via contaminated water. The diarrheal disease cryptosporidiosis is especially threatening to the immunocompromised population such as AIDs patients. Cryptosporidium has been documented in wildlife along the California coast but no studies have yet investigated the magnitude and sources of this pathogen in the marine environment.

Bivalves, such as clams and mussels, concentrate Cryptosporidium oocysts, thus acting as indicators of fecal pollution. Bivalves can be efficiently tested for Cryptosporidium using a Real-Time PCR technique that we recently developed. The assay can detect the DNA of a single organism and gives a quantitative result. This method can also be used to differentiate between Cryptosporidium species and genotypes from animal and human sources.

Our preliminary studies show that Cryptosporidium DNA is detectable in wild bivalves collected off the California coast. We propose a two year study: the first year will sample bivalves at 20 sites along the coast to map out the hot spots of Cryptosporidium contamination. The second year will target the 10 most highly contaminated sites for further investigation into the probable sources of contamination, as indicated by the Cryptosporidium genotypes and the possible pollution sources located near the collection sites. The results of this study will be very valuable in watershed management and minimizing the impact of fecal pollutants on our coastal ecosystems.

Cryptosporidium spp. are protozoan parasites that cause gastrointestinal disease in humans and animals, and are spread directly by fecal-oral transmission or indirectly by fecal contamination of water and food supplies. In California, there is evidence of significant fecal pathogen pollution flowing from terrestrial to aquatic ecosystems along the coast. To develop ecosystem monitoring and management strategies, we tested the hypothesis that Cryptosporidium spp. are detectable in nearshore filter feeding bivalves and in terrestrial storm runoff collected from fecal-impacted coastal ecosystems in California. The goals of this dissertation research were 1) to evaluate innovative detection methods for Cryptosporidium spp. in bivalve and water samples, 2) to evaluate bivalves such as mussels and clams as bioindicators of fecal contamination with Cryptosporidium spp. in marine, estuarine, and freshwater ecosystems, and 3) to evaluate farm management solutions to reduce the load of Cryptosporidium discharged into downstream waterways from coastal dairies. First, innovative molecular and immunologic methods were evaluated for detection of Cryptosporidium spp. in marine mussels (Mytilus californianus) and freshwater clams (Corbicula fluminea) using tissue spiking and tank exposure experiments. The most analytically sensitive detection method was immunomagnetic separation in combination with immunofluorescent detection of oocysts in mussel digestive tissues. Second, field studies tested 4800 mussels and 600 clams from marine, estuarine, and riverine ecosystems along the California coast forCryptosporidium spp. Genotypes detected in bivalves included the anthropozoonotic C. parvum genotype 2, as well as the host specific C. andersoni and C. felis that are shed by cattle and cats, respectively. In addition, 2 novel Cryptosporidium genotypes were detected in mussels. Third, a 3-year study evaluated the distribution of Cryptosporidium on coastal dairies and the efficacy of various farm management practices to reduce the load of Cryptosporidium oocysts in storm runoff. Calves were found to be important loading sources of Cryptosporidium on the farms, and vegetative buffers were found to effectively reduce the load of Cryptosporidium oocysts in storm runoff. In conclusion, Cryptosporidium spp. were detected in terrestrial runoff and nearshore bivalves, suggesting that Cryptosporidium spp. will be useful bioindicators of fecal contamination in ecosystem monitoring and management programs.

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are useful for monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) methodologies, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a log10 improvement when samples were first processed by IMS. The most sensitive method for the detection of C. parvum in tank-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive.

The wild mussel study was conducted to evaluate estuarine and nearshore marine Mytilus spp. as bioindicators of fecal contamination in coastal ecosystems of California. Hemolymph samples from 4680 mussels were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Cryptosporidium genotypes detected in hemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event.

The freshwater clam study evaluated Corbicula spp. as bioindicators of fecal protozoan contamination using three approaches: 1) clam tissue spiking experiments to compare several detection techniques, 2) clam tank exposure experiments to evaluate clams that that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions, and 3) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. The spiking and tank experiments showed that DFA, IMS in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. In the tank experiment, oocyst dose and collection time were significant predictors for detection of C. parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS in combination with DFA analysis of clam digestive glands, with significant variation by sampling year and season. These bivalve studies have shown that clams and mussels can be used to monitor water quality in California and suggests that humans and animals ingesting fecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.