J Med Microbiol 55 (2006), 703-707; DOI: 10.1099/jmm.0.46251-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000
F. Leoni
,
C. Amar,
G. Nichols,
S. Pedraza-Díaz
and
J. McLauchlin
Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK
Correspondence
J. McLauchlin
jim.mclauchlin{at}hpa.org.uk
Received 19 July 2005
Accepted 5 February 2006
The characterization of Cryptosporidium using DNA extracted from whole faecal samples collected from 2414 humans with diarrhoea in England between 1985 and 2000 where cryptosporidial oocysts were detected using conventional methods is described. Characterization was achieved by PCR/RFLP and DNA sequencing of fragments of the Cryptosporidium oocyst wall protein and the 18S rDNA genes. Cryptosporidium parvum was detected in 56.1 % of cases, Cryptosporidium hominis in 41.7 % and a mixture of C. parvum and C. hominis in 0.9 %. In the remainder of cases, Cryptosporidium meleagridis (0.9 %), Cryptosporidium felis (0.2 %), Cryptosporidium andersoni (0.1 %), Cryptosporidium canis (0.04 %), Cryptosporidium suis (0.04 %) and the Cryptosporidium cervine type (0.04 %) were detected.
Abbreviations: COWP, Cryptosporidium oocyst wall protein gene.
Present address: Istituto Zooprofilattico Sperimentale Umbria e Marche, Sezione di Ancona, via Cupa di Posatora 3, 60100 Ancona, Italy. 
Present address: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040-Madrid, Spain.
The GenBank/EMBL/DDBJ accession numbers for the sequences described in this study are DQ116568, DQ116569, DQ116570, DQ116571 and DQ116572.
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INTRODUCTION
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Cryptosporidium is a genus of protozoan parasites that infect a wide range of vertebrates including humans. Human cryptosporidiosis is one of the most common causes of diarrhoeal disease due to enteric protozoa, and results in significant morbidity and mortality in both the developing and developed world. Transmission is through the faecal–oral route, following direct or indirect contact with Cryptosporidium oocysts via person-to-person, zoonotic, waterborne, foodborne or airborne contact (Meinhardt et al., 1996; Fayer, 2004).
Molecular biology has provided powerful new tools for characterizing Cryptosporidium and has revealed significant variation within the genus (Xiao et al., 2004). Cryptosporidium is now recognized as containing assemblages of species which are genotypically heterogeneous but morphologically largely identical (Xiao et al., 2004). In addition to the named species, genotypes are also recognized, some of which have a greater degree of genetic diversity than is found between the named species. However, there is not yet sufficient additional information available for these genotypes to be further classified with species status (Xiao et al., 2004). It is likely that some of these genotypes will be named as separate species in the future. There are marked differences in the host ranges of different Cryptosporidium species and genotypes, and there is growing evidence for differences in parasite development, growth rates, drug sensitivity and disease presentation in humans (Hunter et al., 2004a, b). Because of these inter-species and inter-genotypic differences, molecular epidemiological analysis is essential to understand the transmission of these parasites and will be invaluable in the public health investigation of cryptosporidiosis including the targeting of the most appropriate interventions.
We previously reported different Cryptosporidium species associated with human cryptosporidiosis in England (Pedraza-Díaz et al., 2001a, b; McLauchlin et al., 2000). In this report, we extend these studies to characterize Cryptosporidium species present in more than 2400 faecal samples from humans with diarrhoea in England between 1985 and 2000.
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METHODS
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Samples of whole faeces were collected from 2414 patients with diarrhoea in England between 1985 and 2000 where hospital laboratories had performed routine microbiological analyses and detected Cryptosporidium oocysts using conventional microscopy techniques (Arrowood, 1997). Among the 2414 cases, 589 occurred in seven drinking water borne outbreaks, 51 were associated with seven swimming pool outbreaks and 102 occurred within 37 family outbreaks. The remaining 1672 cases (69 % of all cases) were apparently sporadic. The distribution of Cryptosporidium parvum and Cryptosporidium hominis within the swimming pool and drinking water associated outbreaks has been published elsewhere (McLauchlin et al., 2000; Pedraza-Díaz et al., 2001a).
DNA was extracted from whole faeces by a modification of the ‘Boom’ method using mechanical disruption in the presence of zirconium beads and guanidinium thiocyanate (McLauchlin et al., 1999). Where amplification was not detected, DNA was further purified using polyvinylpyrrolidone treatment as described previously (Lawson et al., 1997).
PCR was applied to DNA samples for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene (Spano et al., 1997; Pedraza-Díaz et al., 2001a), and selected samples were further tested by analysis of a fragment of the 18S rRNA gene (Johnson et al., 1995; Bornay-Llinares et al., 1999). Post-PCR analyses were performed either by RFLP analysis of the COWP gene fragment using RsaI, or by cloning the PCR products into the TOPO plasmid vector with the TOPO-TA cloning kit (Invitrogen) and sequencing in both directions using an ABI377 automated sequencer and BigDye terminator chemistry with M13 primers, or using a CEQ 2000 Dye Terminator Cycle Sequencing (DTCS) Quick Start kit and a CEQ 2000XL automated capillary sequencer (Beckman Coulter) with Ptag primers. Sequence analysis was performed either using the Genetics Computer Group (GCG) program package (University of Wisconsin) and CLUSTALW, or with GeneBuilder, CLUSTAL, Bionumerics version 2.5 (Applied Maths, Hortrijk, Belgium) and BioEdit Sequence Alignment Editor version 5.0.0.
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RESULTS AND DISCUSSION
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DNA extracts from 2414 samples were initially screened by PCR/RFLP analysis of the COWP gene fragment. Selected samples, including those showing no or unusual products, were further analysed by PCR amplification and sequencing of the 18S rDNA fragment as well as, where possible, sequencing of the COWP fragment. Using PCR/RFLP analysis of the COWP fragment alone, C. parvum and C. hominis were detected in the majority of the cases (98.6 %), and Cryptosporidium meleagridis in a further 22 (0.9 %) samples (Table 1
). The COWP fragment was not amplified from the remaining 12 samples.
To investigate the reliability of RFLP analysis of the COWP fragment for identification of C. parvum, C. hominis and C. meleagridis, the COWP and 18S rRNA gene fragments amplified from 71 samples were sequenced. Identification was confirmed for 22 C. hominis and 30 C. parvum samples and no sequence variation from previously deposited sequences was detected within either of the two gene fragments (Table 2).
The COWP gene fragment was also characterized by sequencing from 19 of the samples where C. meleagridis had been identified. Eighteen of the 19 COWP fragments showed no DNA sequence variation from previously deposited C. meleagridis sequences at this locus. Sequence analysis of the 18S rDNA fragment was not performed for six of the C. meleagridis samples. The identification as C. meleagridis was further confirmed in 12 of these samples by an absence of sequence variation at the 18S rDNA locus as compared to previously deposited sequences (Table 2
). Analysis of DNA from the final sample where C. meleagridis was detected by PCR/RFLP analysis showed 99 % identity (504/506 bp) with two mismatches to the sequence of the COWP fragment (AF266266) and 99 % identity (391/392 bp) with one mismatch to the sequence of the 18S fragment (AF112574) previously designated type 2A (Glaberman et al., 2001).
Analysis of the 18S rDNA fragment amplified from the 12 samples from which the COWP gene fragment could not be amplified revealed the presence of: Cryptosporidium felis (six samples), 100 % identical (313/313 bp) to AF087577 (Bornay-Llinares et al., 1999); a Cryptosporidium more similar to Cryptosporidium andersoni than to Cryptosporidium muris (three samples), with 99 % identity (389/390 bp with 1 gap) to C. andersoni AF093496 (Xiao et al., 1999a) and 98 % identity (383/390 bp with 2 gaps) to C. muris AF093498 (Xiao et al., 1999a); Cryptosporidium canis in one sample (Pedraza-Díaz et al., 2001b); Cryptosporidium suis in one sample, 99 % identical (392/394 bp) to AF115377 (Xiao et al., 1999b); and Cryptosporidium cervine type in one sample, 100 % identical (390/390 bp) to isolate 563 AF297512 (Perz & Le Blancq, 2001).
The ages of the patients from whom the least common Cryptosporidium species/genotypes were recovered are shown in Table 3; none were associated with outbreaks. Two of the patients from whom C. felis was recovered were immunocompromised (Pedraza-Díaz et al., 2001b); however, none of the other patients were known to be immunocompromised, and no other enteric pathogens were detected by routine microbiological procedures. Two patients had a history of recent foreign travel: one of the patients with C. andersoni had recently returned from the Canary Islands and the patient with C. canis had returned from Africa.
This study summarizes analyses of a large series of cases of cryptosporidiosis. Data on confirmation by sequencing of identification of Cryptosporidium species by RFLP analyses of the COWP gene fragment validates this as a strategy suitable for national surveillance of human infections since the method accurately identified the most common species associated with human infection. However, we would not recommend this alone for analyses of samples from other sources (e.g. faeces of animals, water or food) since Cryptosporidium species with additional variation in the COWP gene are likely to be present and this RFLP method may give misleading results.
The results of this study confirm the importance of C. parvum and C. hominis (previously named C. parvum genotypes 2 and 1 or calf and human types, respectively) as the major causes of cryptosporidiosis in the general population. Similar data have been reported for other European countries including the Czech Republic (Hajdusek et al., 2004), Denmark (Enemark et al., 2002), France (Guyot et al., 2001), the Netherlands (Homan et al., 1999), Northern Ireland (Lowery et al., 2001), Switzerland (Glaeser et al., 2004; Fretz et al., 2003) and Scotland (Mallon et al., 2003) albeit that these other studies were on a much smaller scale and included a maximum of 135 patients (Mallon et al., 2003). Chalmers et al. (2002) reported on 3100 isolates that were collected in England and Wales after collection of the isolates described here. The majority of their isolates were either C. parvum or C. hominis (the numbers of these were not stated), 21 were C. meleagridis and two could not be identified (Chalmers et al., 2002).
The presence of a Cryptosporidium species other than C. parvum and C. hominis in 1.4 % of patients also confirms our previous report (Pedraza-Díaz et al., 2001b). C. canis, C. felis and C. meleagridis have been reported in immunocompetent patients elsewhere (Xiao et al., 2001). Infection with C. suis (Xiao et al., 2002; Cama et al., 2003) and a Cryptosporidium parasite similar to C. andersoni (Guyot et al., 2001) has been reported in human patients with HIV. The finding here of a heterogeneous group of Cryptosporidium species in humans without immunocompromising illness suggests that ‘unusual’ species may play a role in human infections and highlights the need to improve detection methods for a better understanding of the disease in humans.
The 18S rDNA fragment of the Cryptosporidium cervine type found in this study was identical to that initially described as genotype 3, which was amplified from a white-tailed deer in a wildlife survey in Lower New York State (Perz & Le Blancq, 2001) and from storm water samples collected from a stream in the watershed area of New York State (Xiao et al., 2000). Analysis of the 18S rDNA sequence showed two deletions from the group of isolates described as genotype 3 above as compared with the cervine type previously reported in human infections (Ong et al., 2002), which was identical to the cervine type genotype 3 isolate 524 (AF297511) from a white-tailed deer (Perz & Le Blancq, 2001). Furthermore, the sequences from the human samples reported as the ‘cervine genotype’ had 100 % sequence identity in the 18S rDNA gene fragment to that from an isolate subsequently described from captive lemurs (da Silva et al., 2003). Both isolates from humans and captive lemurs amplified an N-terminal portion of the COWP gene with primers Cry15/Cry9 and had 100 % sequence homology (Ong et al., 2002; da Silva et al., 2003). The isolate described here as the cervine type did not amplify the same fragment of the COWP gene using the nested PCR procedure (Pedraza-Díaz et al., 2001a). It is likely that the human cases previously reported as the Cryptosporidium cervine type were the same as that described as the lemur type, and differed slightly to the Cryptosporidium species from a human case reported here.
In summary, we report here the distribution of Cryptosporidium species amongst 2414 patients with diarrhoea in England between 1985 and 2000. Although C. parvum and C. hominis were identified in the majority of patients, other species were detected in a small proportion of cases including the immunocompetent. This report provides valuable baseline data for identification of changes in the distribution of Cryptosporidium species.
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INTRODUCTION
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