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

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