Guy Robinson

Cryptosporidium sp. rabbit genotype, a newly identified human pathogen.

Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or C. hominis, but pathogenicity of some unusual Cryptosporidium species/genotypes is uncertain (1). In July 2008, an outbreak caused by Cryptosporidium rabbit genotype was linked to consumption of tap water in Northamptonshire, England (2). On June 23 and 24, Cryptosporidium oocysts were detected by operational monitoring of treated water at a surface water treatment works. A precautionary boil-water notice was implemented on June 25.

Unusual Cryptosporidium Genotypes in Human Cases of Diarrhea

Several Cryptosporidium spp. are known to infect humans, but most cases of illness are caused by Cryptosporidium hominis or C. parvum. During a long-term genotyping in the United Kingdom, we identified 3 unusual Cryptosporidium genotypes (skunk, horse, and rabbit) in human patients with diarrhea.

The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000-2008.

Routine typing of 14 469 isolates from human cryptosporidiosis cases between 2000 and 2008 revealed that 7439 (51·4%) were Cryptosporidium (C.) hominis, 6372 (44·0%) C. parvum, 51 (0·4%) both C. hominis and C. parvum, 443 (3·1%) were not typable and 164 (1·1%) were other Cryptosporidium species or genotypes. Of the latter, 109 were C. meleagridis, 38 C. felis, 11 C. ubiquitum, one C. canis, two horse, two novel and one skunk genotype. C. hominis monkey genotype and C. cuniculus were identified in a separate study. Patients with unusual infections were older than those with C. hominis (P<0·01) or C. parvum (P<0·01) and were more likely to be immunocompromised (Fishers exact P<0·01). Forty-one percent of unusual cases had travelled abroad, mainly to the Indian subcontinent. Significant risk factors in those with unusual species were travel abroad (C. meleagridis, P<0·01), being immunocompromised (C. felis, Fishers exact P=0·02), and contact with cats (C. felis, Fishers exact P=0·02).

Detection and Differentiation of Cryptosporidium spp. in Human Clinical Samples by Use of Real-Time PCR

ABSTRACT Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.

Cryptosporidium species and subtype analysis from dairy calves in Spain

Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006-2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76.8%) and 166 calves (57.8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80.3% of the 61 farms), followed by subtype IIaA16G3R1 (14.7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1-3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.

Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007–2011 ☆

A novel Cryptosporidium genotype was identified, among travellers with gastro-intestinal symptoms returning to Great Britain from the Indian subcontinent, for which we propose the name Cryptosporidium viatorum n. sp. The epidemiology of these cases was distinctly different from those with Cryptosporidium parvum and Cryptosporidium hominis. Of the 10 cases identified involving C. viatorum, most were in the first quarter of the year. One occurred in 2007, one in 2008, three in 2010 and five to end March 2011. The median age was 19 years but most were in the 20-29 years age group and seven were male. The symptoms included diarrhoea, abdominal pain, nausea, vomiting and fever. Compared with cases due to C. hominis and C. parvum, vomiting was reported less often, although the duration of gastro-intestinal symptoms was longer. The cases of C. viatorum were all travellers to the Indian subcontinent, whereas cases of C. hominis and C. parvum were more likely to have travelled elsewhere. Cryptosporidium viatorum isolates had indistinguishable sequences at each of the 70 kDa heat shock protein (HSP70), actin and ssrRNA loci which did not match any published previously and, although phylogenetically most similar to Cryptosporidium fayeri, they were distinct (<98% similarity) at the ssrRNA, HSP70 and actin genes. Morphologically, oocysts were typical of predominantly human-infecting species. Cryptosporidium viatorum n. sp. is proposed and work is warranted to investigate further the public health significance and occurrence elsewhere of this emerging parasite.

Re-description of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (Apicomplexa: Cryptosporidiidae): Morphology, biology and phylogeny

To provide re-description of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (synonymous with rabbit genotype), a species closely related to Cryptosporidium hominis, the morphology, natural and experimental host specificity, and genetic characterisation were investigated. The morphology and diagnostic characteristics are typical of other intestinal species of Cryptosporidium, albeit with slightly larger oocysts (5.55-6.40×5.02-5.92 μm; mean 5.98×5.38 μm; length:width=1.1; n=50). Natural hosts appear to be European rabbits (Oryctolagus cuniculus) and humans (Homo sapiens). Experimental infections have been established in weanling rabbits (O. cuniculus), immunosuppressed Mongolian gerbils (Meriones unguiculatus) and immunosuppressed adult Porton strain mice (Mus musculus), but not in neonatal mice. Patterns of infection measured by oocyst shedding are significantly different compared with C. hominis, particularly in rabbits. Histological examination reveals endogenous stages in the brush border of the epithelium of the small intestinal villi, but clinical signs are absent. Inoculation of human HCT-8 cells results in discrete clusters of endogenous stages. A close relationship with C. hominis is inferred from molecular analyses at the ssrRNA, 70 kDa heat shock protein (HSP70), actin, Cryptosporidium oocyst wall protein (COWP), 60 kDa glycoprotein (GP60) genes and a region encoding a product of unknown function (LIB13). Sequences contained limited, consistent polymorphisms at the ssrRNA, HSP70 and actin genes, were identical at the COWP and LIB13 genes and demonstrated two unique families at the GP60 gene. Although genetically closely related, there are significant biological differences between C. cuniculus and C. hominis that support these protozoa being separate species. This is based on the current understanding of these organisms and relies on the assumption that mating between these species would not normally occur. If this is subsequently demonstrated their categorisation may need to be re-addressed.

Sporadic human cryptosporidiosis caused by Cryptosporidium cuniculus, United Kingdom, 2007-2008.

To investigate sporadic human cryptosporidiosis trends in the United Kingdom, we tested 3,030 Cryptosporidium spp.–positive fecal samples, submitted for routine typing in 2007–2008, for C. cuniculus. C. cuniculus prevalence was 1.2%; cases were mostly indigenous and occurred across all age groups. Most occurred during August–October and may be linked to exposure opportunities.

Detection of Cryptosporidium species and sources of contamination with Cryptosporidium hominis during a waterborne outbreak in north west Wales.

As part of investigations into the cause of a waterborne outbreak of Cryptosporidium hominis infection linked to a mains water supply, surface waters and wastewater treatment plants were tested for Cryptosporidium spp. Oocyst counts in base flow surface water samples ranged from nil to 29 per 10 l. Oocyst counts in effluent from a community wastewater treatment plant were up to 63 fold higher and breakout from one septic tank five logs higher. There were no peak (storm) flow events during the investigation. C. hominis, four named genotypes (cervine, muskrat II, rat, W19) and six new small subunit ribosomal RNA gene sequences were identified. Four of the new sequences were closely related to Cryptosporidium muskrat genotype I, one was closely related to the fox genotype and one to Cryptosporidium canis. C. hominis was found extensively in the catchment, but only at sites contaminated by wastewater, and in the treated water supply to the affected area. All were gp60 subtype IbA10G2, the outbreak subtype. Multiple routes of contamination of the reservoir were identified, resulting in persistent detection of low numbers of oocysts in the final water. This work demonstrates the utility of genotyping Cryptosporidium isolates in environmental samples during outbreak investigations.

A whole water catchment approach to investigating the origin and distribution of Cryptosporidium species.

Aims:  Investigating the distribution and origin of Cryptosporidium species in a water catchment affected by destocking and restocking of livestock as a result of a foot and mouth disease epidemic.