Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium
Introduction
Cryptosporidium is an apicomplexan parasite of humans and many other vertebrate species world-wide (O'Donoghue, 1995). In humans, the parasite infects the microvilillus border of the gastrointestinal epithelium causing acute self-limiting diarrhoea in immunocompetent individuals, and a chronic life-threatening disease in immunocompromised patients. Over 20 different species of Cryptosporidium have been named based on host occurrence, but only 11 are currently considered to be valid by most researchers (Fayer et al., 2000, Fayer et al., 2001, Thompson, 2002).
Two species of Cryptosporidium have been described in cattle. Cryptosporidium parvum infects the intestine, produces small-type oocysts (5.0×4.5 μm), mainly in young calves and is responsible for considerable economic losses in the cattle industry and water-borne outbreaks of diarrhoeal disease in human populations (O'Donoghue, 1995). Cryptosporidium andersoni is a recently renamed species that infects the abomasum of cattle (Lindsay et al., 2000). This species was originally referred to as Cryptosporidium muris in cattle. However, C. andersoni produces large-type oocysts (7.4×5.6 μm) lacking the lateral flattening characteristic of C. muris found in rodents (Lindsay et al., 2000). In addition, oocysts of C. andersoni are not infective to outbred, inbred, immunocompetent or immunodeficient mice, rats, rabbits or guinea pigs (Anderson, 1991, Koudela et al., 1998, Sréter et al., 2000). Recent molecular analysis of the rDNA (18S and ITS1 regions), heat-shock protein 70 (HSP-70) and small subunit rRNA genes confirmed that C. andersoni is genetically distinct from C. muris (Morgan et al., 2000).
Cryptosporidium was originally classified as a coccidian based on its possession of similar life cycle features (Levine, 1988). However, Cryptosporidium demonstrates several peculiarities that separate it from any other coccidian. These include: the location of Cryptosporidium within the host cell where the endogenous developmental stages are confined to the apical surfaces of epithelial cells (intracellular but extracytoplasmic); the attachment of the parasite to the host cell where a multi-membranous attachment or feeder organelle is formed at the base of the parasitophourous vacuole to facilitate the uptake of nutrients from the host cell; the presence of two morpho-functional types of oocyst, thick-walled and thin-walled, with the latter responsible for the initiation of the auto-infective cycle in the infected host; the small size of the oocyst (7.4×5.6 μm for C. muris and 5.0×4.5 μm for C. parvum) which lacks morphological structures such as sporocyst, micropyle, and polar granules; and finally the insensitivity of Cryptosporidium to all anti-coccidial agents tested so far (O'Donoghue, 1995, Fayer et al., 1997, Carreno et al., 1998).
These unique biological and morphological characteristics of Cryptosporidium have been complemented further by the results of molecular characterisation studies, which consistently group Cryptosporidium as a clade separate from the coccidian taxa (Relman et al., 1996, Barta, 1997, Morrison and Ellis, 1997, Carreno et al., 1998, Lopez et al., 1999). Furthermore, a recent study by Carreno et al (1999) based on SSrRNA sequencing showed that the gregarines and Cryptosporidium formed a clade separate from the other major apicomplexan clade containing the coccidia. Despite the molecular similarities between Cryptosporidium and the gregarines, Carreno et al. (1999) highlighted differences in the developmental cycles between gregarines and Cryptosporidium, namely: the lack of stages of syzygy and other extracellular trophozoite/gamont stages from the life cycle of Cryptosporidium.
In the present study, we describe the complete development of all life cycle stages of C. andersoni in the HCT-8 cell line and the presence of extracellular gamont-like stages in the life cycle of Cryptosporidium with syzygy obvious in some of these stages. Confirmation of the presence of such novel stages in the Cryptosporidium life cycle argue against its present classification within the coccidia and confirm its affinity to the gregarines as proposed by Carreno et al. (1999).
Section snippets
Source and purification of oocysts
Oocysts of C. andersoni (isolate 356) were obtained from a 6-year-old Charlais Cross steer, from Calgary, Canada. This steer has been passing C. andersoni oocysts for over 5 years. Cryptosporidium andersoni oocysts were purified from cattle faeces by two rounds of ficoll centrifugation, bleach-treated (5% bleach for 20 min at room temperature), washed and stored at 5°C in 10 ml phosphate-buffered saline (PBS)/15 μm antibiotics containing ampicillin (100 mg/ml) and lincomycin (4 mg/ml).
The C. parvum
Cryptosporidium andersoni infectivity to mice
Cryptosporidium andersoni oocysts purified from cattle faeces were not infective to 7–8 day old ARC/Swiss mice.
Excystation of Cryptosporidium oocysts and development in HCT-8 cells
The excystation process of sporozoites from C. andersoni oocysts was the same as observed for C. parvum (Hijjawi et al., 2001) except some oocysts excysted immediately after acid treatment. Fig. 1a–c shows the sequential stages of excystation of C. andersoni oocysts.
The in vitro proliferation and development of C. parvum (cattle genotype) followed the same pattern described by Hijjawi
Infectivity of C. andersoni oocysts to mice
The fact that C. andersoni oocysts purified from cattle faeces were not infective to 7–8 day-old ARC/Swiss mice is consistent with the previous findings of other authors who failed to infect mice with oocysts of C. andersoni (Anderson, 1991, Koudela et al., 1998, Lindsay et al., 2000).
Excystation of C. andersoni oocysts and development in HCT-8 cells
The excystation process of sporozoites from C. andersoni oocysts was the same as that observed for C. parvum (Hijjawi et al., 2001) except that some oocysts excysted immediately after acid treatment which could
Acknowledgements
We would like to thank K. Heel for her excellent assistance and expertise in the laser microdissection technique and A. Estcourt for her contribution and support with the molecular work. The financial support provided by Murdoch University and the Australian Research Council are gratefully acknowledged.
References (30)
Investigating phylogenetic relationships within the Apicomplexa using sequence data: the search for homology
Methods
(1997)- et al.
Cross-reaction of an anti-Cryptosporidium monoclonal antibody with sporocysts of Monocystis species
Vet. Parasitol.
(1998) - et al.
Epidemiology of Cryptosporidium: transmission, detection and identification
Int. J. Parasitol. (special issue)
(2000) - et al.
Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture
Int. J. Parasitol.
(2001) - et al.
Analysis of segmental renal gene expression by laser capture microdissection
Kidney Int.
(2000) - et al.
Infectivity of Cryptosporidium muris isolated from cattle
Vet. Parasitol.
(1998) Presidential address: rediscovering parasites using molecular tools – toward revising the taxonomy of Echinococcus, Giardia, and Cryptosporidium
Int. J. Parasitol.
(2002)Experimental infection in mice of Cryptosporidium muris isolated from a camel
J. Protozool.
(1991)- et al.
Cryptosporidium is more closely related to gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences
Parasitol. Res.
(1999) - et al.
Phylogenetic analysis of coccidia based on the 18S rDNA sequence comparison indicates that Isospora is more closely related to Toxoplasma and Neospora
J. Eukaryot. Microbiol.
(1998)
Laser capture microscopy
Mol. Pathol.
A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice
J. Protozool.
The general biology of Cryptosporidium
Cryptosporidium canis n. sp. from domestic dogs
J. Parasitol.
Laser microdissection and optical tweezers in research
Today's Life Sci.
Cited by (79)
In vitro cultivation methods for coccidian parasite research
2023, International Journal for ParasitologyTaxonomy and molecular epidemiology of Cryptosporidium and Giardia – a 50 year perspective (1971–2021)
2021, International Journal for ParasitologyPast and future trends of Cryptosporidium in vitro research
2019, Experimental ParasitologyResponse of cell lines to actual and simulated inoculation with Cryptosporidium proliferans
2018, European Journal of ProtistologyIt's official – Cryptosporidium is a gregarine: What are the implications for the water industry?
2016, Water ResearchCitation Excerpt :However, it has long been speculated that Cryptosporidium represents a ‘missing link’ between the more primitive gregarine parasites and coccidians. The similarities between Cryptosporidium and gregarines have been supported by extensive microscopic, molecular, genomic and biochemical data (Pohlenz et al., 1978; Bull et al., 1998; Carreno et al., 1999; Beyer et al., 2000; Hijjawi et al., 2002, 2004; Leander et al., 2003a; Rosales et al., 2005; Barta and Thompson, 2006; Butaeva et al., 2006; Valigurová et al., 2007; Boxell et al., 2008; Karanis et al., 2008; Zhang et al., 2009; Borowski et al., 2008, 2010; Hijjawi, 2010; Hijjawi et al., 2010; Templeton et al., 2010; Karanis and Aldeyarbi, 2011; Boxell, 2012; Koh et al., 2013, 2014; Huang et al., 2014; Clode et al., 2015; Valigurová et al., 2015; Aldeyarbi and Karanis, 2016a, 2016b; 2016c; Edwinson et al., 2016; Paziewska-Harris et al., 2016), which have served as the basis for the formal transfer of Cryptosporidium from subclass Coccidia, class Coccidiomorphea to a new subclass, Cryptogregaria, within class Gregarinomorphea (Cavalier-Smith, 2014). The genus Cryptosporidium is currently the sole member of Cryptogregaria and is described as comprising epicellular parasites of vertebrates possessing a gregarine-like feeder organelle but lacking an apicoplast (Cavalier-Smith, 2014).
Cryptosporidium — What is it?
2016, Food and Waterborne Parasitology