Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples.

BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Microsporidian parasites feminise hosts without paramyxean co-infection: support for convergent evolution of parasitic feminisation.

Feminisation of amphipod crustaceans is associated with the presence of at least three microsporidian parasites and one paramyxean parasite, suggesting that the ability to feminise has evolved multiple times in parasites of amphipods. Co-infection by a paramyxean with one of the putative microsporidian feminisers, Dictyocoela duebenum, has inspired the alternative hypothesis that all feminisation of amphipods is caused by paramyxea and that all microsporidian associations with feminisation are due to co-infection with paramyxea (Short et al., 2012). In a population of the amphipod Gammarus duebeni, breeding experiments demonstrate that the microsporidia D. duebenum and Nosema granulosis are associated with feminisation in the absence of paramyxea. Co-infection of the two microsporidia is no more frequent than expected at random and each parasite is associated with feminisation in the absence of the other. These findings support the original hypothesis that the ability to feminise amphipods has evolved in microsporidia on multiple occasions. Additionally, the occurrence of a non-feminising strain of D. duebenum in Gammarus pulex suggests that different strains vary in their feminising ability, even within microsporidian species. The presence or absence of feminising ability in a particular microsporidian strain should not therefore be generalised to the species as a whole.