Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands☆
Introduction
Cryptosporidium is a globally distributed protozoan parasite which has been found in both vertebrates (e.g., humans, cattle, and birds) and invertebrates (e.g., clams) (Xiao and Ryan, 2004, Caccio et al., 2005). In most cases, including those in humans, Cryptosporidium causes an enteric infection leading to gastrointestinal (GI) problems such as severe diarrhoea. In birds, Cryptosporidium may also infect the respiratory tract causing respiratory problems (Morgan et al., 2001). Due to the relatively uniform appearance and parasitic life-cycle of different Cryptosporidium strains, classical morphological, and phenotypical classification are limited in distinguishing the various species and genotypes found in humans and animals. Molecular genetic techniques are able to distinguish the different species and genotypes and have shown that some are very host-specific while others have a broad host range. For instance, Cryptosporidium baileyi and Cryptosporidium meleagridis are mainly associated with birds; Cryptosporidium canis with dogs; Cryptosporidium felis with cats and Cryptosporidium molnari with fish; however Cryptosporidium parvum has a much broader host range and is found in cattle, sheep, goats, deer, raccoon dogs, horses and humans (for more details see reviews Xiao and Ryan, 2004, Caccio et al., 2005). In humans, the species mainly found to cause disease are Cryptosporidium hominis (also found in monkeys) and C. parvum. Other species (e.g., C. meleagridis, Cryptosporidium andersoni, C. canis, and C. felis) are only found in humans sporadically (Caccio et al., 2002).
The incidence of gastroenteritis and the role of a broad range of pathogens in The Netherlands has been studied before (Mank, T.G., 1997. Intestinal protozoa and diarhoea in general practice. Academic thesis, Vrije Universiteit Amsterdam, The Netherlands. ISBN9 056690856; de Wit et al., 2001a, de Wit et al., 2001b, de Wit et al., 2001c, de Wit et al., 2001d). In these studies Cryptosporidium was found in about 2–3% of gastroenteritis cases, compared with a prevalence of about 0.2% in the general population. In these studies the Cryptosporidium isolates were not further genetically analysed. Several studies have reported molecular detection of Cryptosporidium in The Netherlands in humans (Verweij et al., 2004), water (Medema and Schijven, 2001) and animals (Homan et al., 1999b, Huetink et al., 2001, Schets et al., 2007). C. hominis (or genotype 1) was found in humans and C. parvum (or genotype 2) in animals and sporadically in humans (Homan et al., 1999b). Microsatellite 1 analysis showed the presence of C. parvum C1 and C3 in dairy cows (Huetink et al., 2001). Detailed molecular epidemiological information on zoonotic potential and transmission routes in The Netherlands is, however, limited.
We sought to study the zoonotic potential of Cryptosporidium in The Netherlands by studying the prevalence and genetic diversity of Cryptosporidium in humans and farm animals. Faecal samples from humans and farm animals with cryptosporidiosis were genotyped for several loci and linked to epidemiological information. Several different methods and genetic loci have been used to genotype Cryptosporidium (Xiao and Ryan, 2004, Caccio et al., 2005, Chalmers et al., 2005, Thompson et al., 2005). In the present study we chose to use PCR and DNA sequencing of a broad spectrum of markers to try to include both conserved and polymorphic markers. The following markers were tested: the 18S ribosomal RNA gene (18S) (Morgan et al., 2001), the Cryptosporidium outer wall protein gene (COWP) (Pedraza-Diaz et al., 2000), the heat shock protein 70 gene (HSP70) (Morgan et al., 2001), the 60 kDa glycoprotein gene (GP60) (Strong et al., 2000, O’Connor et al., 2002), and the two microsatellite markers ML1 and ML2 (Caccio et al., 2000, Caccio et al., 2001). The sub-types found for these markers were compared with each other and correlated with the available epidemiological information such as age, recent travel history, gender, region, and seasonality.
Section snippets
Origin of the samples and DNA isolation
Between 2003 and 2005 patients consulting their general practitioner with GI complaints (mostly diarrhoea and abdominal pain) indicating a parasitic infection were tested in collaboration with five clinical diagnostic laboratories for the presence of Cryptosporidium in their stool. Ninety-one of these Cryptosporidium-positive (modified Ziehl–Neelsen stain; Henriksen and Pohlenz, 1981) faecal samples were anonymized and send to the National Institute for Public Health and the Environment (RIVM)
Genotyping of human Cryptosporidium isolates
The 91 human isolates were genotyped for all six markers used in this study. Table 2 shows the genotypes identified for the human Cryptosporidium isolates. Sixty-four isolates were typed as C. hominis, 17 as C. parvum, nine as mixed C. hominis/C. parvum and one as C. felis. For COWP, all human C. parvum sequences analysed were identical to the C. parvum reference strain (Abrahamsen et al., 2004). The C. hominis isolates were identical to C. hominis AF266265 from GenBank and showed a single A to
Discussion
Ninety-one Cryptosporidium-positive human faecal samples were genotyped for 18S, COWP, HSP70, GP60, ML1, and ML2. With the exception of one C. felis isolate (∼1%), which is normally found in cats and sporadically in humans (Pieniazek et al., 1999, Leoni et al., 2006), all human infections were caused by C. hominis and/or C. parvum, similar to findings in other countries (Enemark et al., 2002, Hunter et al., 2004, Caccio et al., 2005, Chalmers et al., 2005). There was a good correlation between
Acknowledgements
We thank the diagnostic laboratories of Enschede, Goes, Utrecht and Veldhoven for providing us with human patient samples. This study was financially supported by the Dutch Food and Consumer Product Safety Authority (VWA).
References (48)
- et al.
Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum
Int. J. Parasitol.
(2001) - et al.
Unravelling Cryptosporidium and Giardia epidemiology
Trends Parasitol.
(2005) - et al.
Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species
Int. J. Parasitol.
(2005) - et al.
Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations
Vet. Parasitol.
(2007) - et al.
Experimental evidence for genetic recombination in the opportunistic pathogen Cryptosporidium parvum
Mol. Biochem. Parasitol.
(2002) - et al.
Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm
Vet. Parasitol.
(2001) - et al.
Modelling the sewage discharge and dispersion of Cryptosporidium and Giardia in surface water
Water Res.
(2001) - et al.
Molecular and phylogenetic characterisation of Cryptosporidium from birds
Int. J. Parasitol.
(2001) - et al.
Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates
Mol. Biochem. Parasitol.
(2002) - et al.
The identification and characterisation of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis
FEMS Microbiol. Lett.
(2000)
Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, The Netherlands
Int. J. Food Microbiol.
Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice
Exp. Parasitol.
Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom
Vet. Parasitol.
Cryptosporidium and cryptosporidiosis
Adv. Parasitol.
Complete genome sequence of the apicomplexan, Cryptosporidium parvum
Science
Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species
Infect. Immun.
A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum
Parasitology
Human infection with Cryptosporidium felis: case report and literature review
Emerg. Infect. Dis.
Gastroenteritis in sentinel general practices, The Netherlands
Emerg. Infect. Dis.
Etiology of gastroenteritis in sentinel general practices in The Netherlands
Clin. Infect. Dis.
Sensor, a population-based cohort study on gastroenteritis in The Netherlands: incidence and etiology
Am. J. Epidemiol.
A comparison of gastroenteritis in a general practice-based study and a community-based study
Epidemiol. Infect.
Molecular characterization of Danish Cryptosporidium parvum isolates
Parasitology
Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin
J. Clin. Microbiol.
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