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

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Abstract

The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72 h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72 h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.

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.

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