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We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).

This is the first report to characterize the genotypes and subtypes of Cryptosporidium species infecting a geographically isolated population of feral Soay sheep (Ovis aries) on Hirta, St. Kilda, Scotland, during two distinct periods: (i) prior to a population crash and (ii) as host numbers increased. Cryptosporidium DNA was extracted by freeze-thawing of immunomagnetically separated (IMS) bead-oocyst complexes, and species were identified following nested-PCR-restriction fragment length polymorphism (RFLP)/PCR sequencing at two Cryptosporidium 18S rRNA loci. Two hundred fifty-five samples were analyzed, and the prevalent Cryptosporidium species in single infections were identified as C. hominis (11.4% of all samples tested), C. parvum (9%), C. xiaoi (12.5%), and C. ubiquitum (6.7%). Cryptosporidium parvum was also present with other Cryptosporidium species in 27.1% of all samples tested. Cryptosporidium parvum-and C. hominis-positive isolates were genotyped using two nested-PCR assays that amplify the Cryptosporidium glycoprotein 60 gene (GP60). GP60 gene analysis showed the presence of two Cryptosporidium genotypes, namely, C. parvum IIaA19G1R1 and C. hominis IbA10G2. This study reveals a higher diversity of Cryptosporidium species/genotypes than was previously expected. We suggest reasons for the high diversity of Cryptosporidium parasites within this isolated population and discuss the implications for our understanding of cryptosporidiosis.

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