Cryptosporidium in upland water biota, United Kingdom (2012-2015)

Sample preparation methods included a newly developed means of testing whole aquatic invertebrates by grinding them up in Liquid Nitrogen and extracting DNA using a commercially available kit (Gentra PureGene), Qiagen stool and tissue DNA kits for the fish and mammal samples. Nested Image result for PCR (Polymerase chain reaction) PCR targeting Cryptosporidium small subunit rDNA was used to screen the samples for the presence of Cryptosporidium followed by identification of the species using sequencing or species-specific real-time PCRs. Where Cryptosporidium hominis or Cryptosporidium parvum were found, isolates were subtyped by sequencing part of a 60 kilodaltons (kDa) glycoprotein gene. Biota samples were collected by Cardiff University staff and transported to the Cryptosporidium Reference Unit, Swansea for testing. DNA from biofilm samples was extracted at Cardiff University. Invertebrates were pooled up to a maximum of 5 larvae per sample and ground in liquid nitrogen before DNA extracted using the Gentra PureGene DNA extraction kit (Qiagen). Fish gut and gill samples (collected from fish culled for food-web studies) were processed by extracting DNA using the QIAamp DNA Mini kit tissue protocol (Qiagen). Faecal samples from fish, collected by gently massaging the gut just above the anus and mammals, collected from the ground in the riparian zone on ad hoc basis had DNA extracted using QIAamp Fast DNA Stool Mini kit. Samples were screened for any Cryptosporidium DNA using a published nested polymerase chain reaction (PCR) targeting the small subunit rhibosomal DNA (rDNA). The presence of DNA from the two main Cryptosporidium species that cause human cryptosporidiosis (Cryptosporidium parvum and Cryptosporidium hominis) was also screened for using species-specific real-time PCRs targeting the Lib13 and A135 genes. Any C. hominis and C. parvum positive samples were subtyped by sequencing part of the 60kDa glycoprotein gene. The prevalence of Cryptosporidium was very low, which could indicate that the water in these upland rivers is not overly contaminated. However, caution must be taken as no water samples were tested as part of this project and the low prevalence may also be linked to the location of the sampling, the sample sizes, the analytical sensitivity of the testing method and/or the potential for the host species tested to actually carry or be infected by Cryptosporidium species.

Results were entered into Excel spreadsheets and exported to comma separated value files for ingestion into the Environmental Information Data Centre.

This dataset is included in the following collections

The DURESS project - Diversity of Upland Rivers for Ecosystem Service Sustainability