The development and implementation of a method using blue mussels (Mytilus spp.) as biosentinels of Cryptosporidium spp. and Toxoplasma gondii contamination in marine aquatic environments.

Surveillance monitoring for microbial water quality typically involves collecting single discrete grab samples for analyzing only one contaminant. While informative, current approaches suffer from poor recoveries and only provide a limited snapshot of the microbial contaminants only at the time of collection. To overcome these limitations, bivalves have been proposed as effective biosentinels of water quality particularly for their ability to efficiently concentrate and retain microbial contaminants for long periods of time. In this study, we examined the use of indigenous blue mussels (Mytilus spp.) as biosentinels to monitor for the presence of Toxoplasma gondii and Cryptosporidium water. An efficient method to extract oocyst DNA from various mussel tissues followed by PCR-based detection of these pathogens was developed, which resulted in the detection down to 10 oocysts. This method was then used to conduct a small survey in Point Lobos and Morro Bay, California to determine prevalence T. gondii and Cryptosporidium. Results revealed that mussels from Morro Bay were contaminated with T. gondii (33 %), while mussels from Point Lobos were contaminated with T. gondii (54 %) and Cryptosporidium (26.9 %) oocysts. Phylogenetic analysis using the SSU rRNA gene identified two novel Cryptosporidium parvum-like genotypes. Overall, this study demonstrated the application of using native California Mytilus spp. as biosentinels for pathogen contamination along the central California shorelines. More importantly, T. gondii and Cryptosporidium were found at higher prevalence rates in Morro Bay and in Point Lobos, an area not previously reported to be contaminated with these pathogens.

Five Hundred Microsatellite Loci for Peromyscus.

Mice of the genus Peromyscus, including several endangered subspecies, occur throughout North America and have been important models for conservation research. We describe 526 primer pairs that amplify microsatellite DNA loci for P. maniculatus bairdii, 467 of which also amplify in P. polionotus subgriseus. For 12 of these loci, we report diversity data from a natural population. These markers will be an important resource for future genomic studies of Peromyscus evolution and mammalian conservation.

Novel microsatellite markers for the saltmarsh sharp-tailed sparrow, Ammodramus caudacutus (Aves: Passeriformes).

We have developed eight high-quality microsatellite DNA loci for the saltmarsh sharp-tailed sparrow and one additional locus with evidence of null alleles. In a sample of 250-350 individuals, the average number of alleles per locus was 14.7 and average observed heterozygosity was 0.80. These loci were tested in three additional species of emberizid sparrows, indicating that more than half of the loci could be useful in other sparrows.

Microsatellite loci for the invasive colonial hydrozoan Cordylophora caspia.

Cordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has become a common invader of both fresh and brackish water ecosystems of North America and Europe. We describe 11 polymorphic microsatellite loci for this species. Preliminary analyses indicate that population substructure may contribute to departures from Hardy-Weinberg equilibrium. In addition, new loci failed to consistently amplify Cordylophora samples known to be genetically distant from those utilized in this study, indicating the presence of cryptic diversity within the taxon.

Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water.

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

Isolating microsatellite DNA loci.

A series of techniques are presented to construct genomic DNA libraries highly enriched for microsatellite DNA loci. The individual techniques used here derive from several published protocols but have been optimized and tested in our research laboratories as well as in classroom settings at the University of South Carolina and University of Georgia, with students achieving nearly 100% success. Reducing the number of manipulations involved has been a key to success, decreasing both the failure rate and the time necessary to isolate loci of interest. In our lab during the past 3 years alone, these protocols have been successfully used to isolate microsatellite DNA loci from at least 55 species representing three kingdoms. These protocols have made it possible to reduce the time to identify candidate loci for primer development from most eukaryotic species to as little as 1 week.