Development of fluorescent in situ hybridization for Cryptosporidium detection reveals zoonotic and anthroponotic transmission of sporadic cryptosporidiosis in Sydney.

Cryptosporidium is the most common non-viral cause of diarrhea worldwide. Of the 5 described species that contribute to the majority of human infections, C. parvum is of major interest due to its zoonotic potential. A species-specific fluorescence in situ hybridisation probe was designed to the variable region in the small subunit of the 18S rRNA of C. parvum and labeled with Cy3. Probe specificity was validated against a panel of 7 other Cryptosporidium spp. before it was applied to 33 human faecal samples positive for cryptosporidiosis which were obtained during the period from 2006-2007. Results were compared to PCR-RFLP targeting the 18S rDNA. FISH results revealed that 19 of the 33 isolates analysed were identified as C. parvum. Correlation of PCR-RFLP and FISH was statistically significant (P<0.05), resulting in a calculated correlation coefficient of 0.994. In this study, species identification by FISH and PCR-RFLP provided preliminary evidence to support both anthroponotic and zoonotic transmission of sporadic cases of cryptosporidiosis in the Sydney basin. In conclusion, FISH using a C. parvum-specific probe provided an alternative tool for accurate identification of zoonotic Cryptosporidium which will be applied in the future to both epidemiological and outbreak investigations.

Effects of tributyltin and other metals on the phenoloxidase activating system of the tunicate, Styela plicata.

Toxic metals, such as tributyltin (TBT), contribute substantially to anthropogenic pollution in many estuarine environments. Animals that live in those environments, particularly invertebrate filter feeders like tunicates, are likely to be exposed to substantial metal contamination. This study investigates the effects of TBT and other metals on the phenoloxidase activity of the estuarine tunicate, Styela plicata, in an effort to identify a biochemical marker of metal pollution. Hemocytes harvested from S. plicata that were exposed to tributyltin or copper in aquaria had significantly enhanced phenoloxidase activities relative to non-exposed controls. This enhanced phenoloxidase activity could be explained by an increased frequency of morula cells, which contain high levels of phenoloxidase's proenzyme, prophenoloxidase. Unlike those from tunicates exposed to metals in aquaria, the phenoloxidase activities of hemocytes incubated with tributyltin in vitro were significantly reduced when compared with hemocytes cultured without tributyltin. The ability of tributyltin to decrease phenoloxidase activity in tissue culture may reflect its known inhibitory effects on calcium-dependent signaling systems such as those involved in the exocytosis of prophenoloxidase from morula cells.

Phylogenetic structure of unusual aquatic microbial formations in Nullarbor caves, Australia.

The nature of unusual aquatic microbial formations in flooded passages of cave systems in the Nullarbor region of Australia was investigated using electron microscopy and DNA analysis. The caves are located in a semiarid region but intersect the watertable at depths of approximately 100 m below the surface. Throughout submerged portions of the caves divers have noted the presence of unusual microbial formations. These 'microbial mantles' comprise sheets or tongues of mucoid material in which small crystals are embedded. Examination of the biomass revealed it to be primarily composed of densely packed, unbranched filaments, together with spherical-, rod- and spiral-shaped cells, and microcrystals of calcite in a mucoid matrix. Molecular phylogenetic analysis of the community structure revealed approximately 12% of clones showed high similarity to autotrophic nitrite-oxidizing bacteria (Nitrospira moscoviensis). The remainder of the clones exhibited a high proportion of phylogenetically novel sequence types. Chemical analysis of water samples revealed high levels of sulphate and nitrate together with significant nitrite. The community structure, the presence of nitrite in the water, and the apparent absence of aquatic macrofauna, suggest these microbial structures may represent biochemically novel, chemoautotrophic communities dependent on nitrite oxidation.