Developing Novel Imaging Techniques for Analysis of Non-Fixed Cryptosporidium parvum Oocysts under Physiological Conditions

Cryptosporidium is a waterborne apicomplexan parasite typically infecting the upper gastrointestinal tract of humans and animals. Infection in immunocompetent hosts can cause acute self-limiting diarrhoeal symptoms, although in infants and the immunocompromised, infection can be life threatening. The infective stage of the parasite survives in the environment as a hardy cyst/spore or 'oocyst'. These oocysts have high resistance to disinfectants, enabling them to survive for long periods in various environments whilst remaining infective. The nature and characteristics of these "spores" remain elusive, and further research into oocyst composition is necessary to enable the development of effective water treatment methods and medical prophylaxis, for which options are currently limited. This project describes a novel method for live imaging and peak force quantitative nanomechanical property mapping of Cryptosporidium spp. oocysts using atomic force microscopy (AFM). Applying this method, data on the surface topography and deformation characteristics of Cryptosporidium parvum oocysts has been acquired and analysed to identify physiological characteristics of live oocyst of the species C. parvum, both in air and in a near native liquid environment. Scanning electron microscopy (SEM), field emission scanning electron microscopy (FESEM), and fluorescence microscopy were used for comparison between imaging methods, and previous reports. This work will enable investigations into live Cryptosporidium spp. oocyst structure, composition, and mechanical properties in unprecedented depth. In turn these capacities can be used to inform and advance our understanding of host specificity and excystation control, and to develop new methods for treatment and eradication of the parasite, all areas of vital importance to progress research towards combating this significant disease.