Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands

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Abstract

The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type 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)

  • F.M. Schets et al.

    Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, The Netherlands

    Int. J. Food Microbiol.

    (2007)
  • L.M. Smith et al.

    Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice

    Exp. Parasitol.

    (2001)
  • A.P. Sturdee et al.

    Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom

    Vet. Parasitol.

    (2003)
  • R.C. Thompson et al.

    Cryptosporidium and cryptosporidiosis

    Adv. Parasitol.

    (2005)
  • M.S. Abrahamsen et al.

    Complete genome sequence of the apicomplexan, Cryptosporidium parvum

    Science

    (2004)
  • D.E. Akiyoshi et al.

    Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species

    Infect. Immun.

    (2002)
  • S. Caccio et al.

    A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum

    Parasitology

    (2000)
  • S. Caccio et al.

    Human infection with Cryptosporidium felis: case report and literature review

    Emerg. Infect. Dis.

    (2002)
  • M.A. de Wit et al.

    Gastroenteritis in sentinel general practices, The Netherlands

    Emerg. Infect. Dis.

    (2001)
  • M.A. de Wit et al.

    Etiology of gastroenteritis in sentinel general practices in The Netherlands

    Clin. Infect. Dis.

    (2001)
  • M.A. de Wit et al.

    Sensor, a population-based cohort study on gastroenteritis in The Netherlands: incidence and etiology

    Am. J. Epidemiol.

    (2001)
  • M.A. de Wit et al.

    A comparison of gastroenteritis in a general practice-based study and a community-based study

    Epidemiol. Infect.

    (2001)
  • H.L. Enemark et al.

    Molecular characterization of Danish Cryptosporidium parvum isolates

    Parasitology

    (2002)
  • D.C. Feltus et al.

    Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin

    J. Clin. Microbiol.

    (2006)
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