Abstract
The neglected intestinal protozoan parasite, Cryptosporidium spp. is considered one of the main etiologic factors for diarrheal illnesses and death in children under five years of age. Belonging to the phylum Apicomplexa (akin to Plasmodium spp. and Toxoplasma spp.), Cryptosporidium spp. infects the intestine of a wide range of vertebrate hosts and is an important cause of morbidity in livestock. Despite its importance, basic and applied research into Cryptosporidium spp. has been stymied by the inability to continuously culture Cryptosporidium spp. in vitro. Consequently, there is no vaccine nor effective treatment against the acute gastroenteritis caused by Cryptosporidium spp. (cryptosporidiosis). As in vitro models are key to the preclinical screening and development of new therapeutics targeting cryptosporidiosis, there has been a concerted effort to develop an in vitro culture system for this intracellular parasite. One promising protocol, utilising a cell-free (axenic) culture system claims to support the continuous growth of Cryptosporidium spp. for a period of 7 days. The findings reported by this group suggest the development of meronts, leading to the sexual life cycle, completing with production of de novo oocysts. Unfortunately, this host-cell-free model only show qualitative results using electron microscopy techniques with no quantitative data and has not been applied by other groups (Hijjawi et al., 2004). Hence the aim of this project is to investigate and optimise this host-cell-free model using a variety of specialised growth conditions and quantitative analytical techniques such as real time qPCR and fluorescent microscopy. In parallel, we have also investigated the asexual cycle of Cryptosporidium using a commonly used host-cell model. Our data show that Cryptosporidium parasites preferentially choose uninfected host cells during the early life-cycle stages with a transition into the sexual phase at 3 and 72 hours post infection, respectively. We then show for the first time that a net parasitic growth was observed and sustained using horse-derived serum for up to eight days. The clear growth of Cryptosporidium within our improved axenic model opens a new frontier into the parasite’s biology and drug discovery