Investigating the public health significance of Cryptosporidium in the environment

Abstract

The high-resolution molecular characterisation of the Cryptosporidium species and subtypes biquitous in environmental samples can provide important information regarding their potential public health significance. The purpose of this study was to develop, evaluate and apply sensitive screening and DNA recovery methods to environmental Cryptosporidium for molecular characterisation. A systemic literature review as undertaken to identify methods of subtyping Cryptosporidium species recovered from environmental samples. Prior to molecular characterisation, the recovery and detection of oocysts from water is by immunomagnetic separation and immunofluorescence microscopy. However, this method is not currently suited for screening large numbers of faecal samples. A commercially available faecal parasite concentrator protocol was modified and evaluated for the enhanced detection of Cryptosporidium oocysts. Once recovered from samples, DNA must be released from the oocyst bound sporozoites before molecular methods can be applied. Commonly used oocyst disruption methods were identified and using samples containing high numbers of oocysts, evaluated by microscopy and a SYBR Green real-time polymerase chain reaction (PCR) developed from the internal stage of a previously published nested small subunit ribosomal DNA PCR. The eight best methods were then evaluated with low numbers of oocysts in the presence of immunomagnetic beads to replicate field samples, using the published nested PCR and SYBR Green real-time PCR. The enhanced screening and optimal DAN recovery combined with microsatellite multilocus fragment analysis was applied in a study investigating the species and subtypes of Cryptosporidium recovered from water, non-clinical farmed and wild animal faeces as well as clinical human and cattle samples from within a single water catchment. Several human pathogenic Cryptosporidium subtypes were identified in addition to the unprecedented finding of Cryptosporidium as the predominant species in the catchment surface water. This study demonstrated the potential application of the developed methodology in the public health investigation of environmental Cryptosporidium