BackgroundWhole 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.ResultsThe 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.ConclusionThis represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.
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.
Arundell, K., Dunn, A., Alexander, J. L., Shearman, R., Archer, N., Ironside, J. (2015). Enemy release and genetic founder effects in invasive killer shrimp populations of Great Britain. Biological Invasions, 17 (5), 1439-1451. Document embargo until 04/11/2015The predatory ?killer shrimp? Dikerogammarus villosus invaded Britain from mainland Europe in 2010. Originating in the Ponto-Caspian region, this invader has caused significant degradation of European freshwater ecosystems by predating and competitively excluding native invertebrate species. In contrast to continental Europe, in which invasions occurred through the migration of large numbers of individuals along rivers and canals, the invasion of Great Britain must have involved long distance dispersal across the sea. This makes the loss of genetic diversity and of debilitating parasites more likely. Analysis of nuclear microsatellite loci and mitochondrial DNA sequences of D. villosus samples from the four known populations in Britain reveal loss of rare alleles, in comparison to reference populations from the west coast of continental Europe. Screening of the British D. villosus populations by PCR detected no microsporidian parasites, in contrast with continental populations of D. villosus and native amphipod populations, most of which are infected with microsporidia. These findings suggest that the initial colonisation of Great Britain and subsequent long distance dispersal within Britain were associated with genetic founder effects and enemy release due to loss of parasites. Such effects are also likely to occur during future long-distance dispersal events of D. villosus to Ireland or North America.authorsversionPeer reviewe
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