High-throughput in vitro culture system for Cryptosporidium oocysts: replacing animals in research

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Cryptosporidium is a water-borne pathogen infecting farm animals and humans. It poses a major threat to animal health and welfare, and to public health, because there is no proper treatment and no immediate prospect of vaccine development. Focal infections in new-born calves within the dairy and beef industries are widespread; infected animals experience severe diarrhoea, lose condition and in extreme cases can die of the disease. Cryptosporidium is also the second most important diarrhoeal pathogen of small children in sub-Saharan Africa, and even in developed countries human outbreaks can involve tens of thousands of people. The disease has remained under the radar of charity organisations, research institutions and pharma companies, but increasing attention has focussed substantially more funding to develop drugs to eliminate Cryptosporidium as a human pathogen. As maintenance of the pathogen depends on cultures kept in calves or mice, recent increase in funding will dramatically increase animal use in this research field.Cryptosporidium is a single-celled microbe, related to the pathogen which causes malaria. It infects the gut lining of mammals. It multiplies within the cells of the gut, killing them and releasing into the gut lumen large numbers of resistant infective stages (oocysts), which are passed out in faeces and remain dormant in the environment until eaten by an animal such as a new-born calf. The accelerating pace of research into the disease requires an increasing supply of Cryptosporidium oocysts for experimentation, a supply that is met entirely from infections of neonatal calves, because to date there has never been an effective culture method which does not use live animals. A promising new method of rearing Cryptosporidium without using calves was developed and published in 2016 by Prof Nigel Yarlett. This method grows parasite stages in mammalian gut cells grown in culture using new technology. The method claims to generate up to 108 Cryptosporidium oocysts per day. If this can be adapted into a routine methodology, the output of oocysts would meet the needs of the research community without using calves or other live animals.This proposal seeks funds to visit the laboratory of Prof Yarlett in New York to learn the new methodology for producing large numbers of infective stages, and to return this technology to Cardiff University where a facility to produce oocysts without animal use will be built. Our long-term ambition is to replace calves for the production of oocysts, focussing first on replacing supply in our own laboratory in Cardiff, and then making this supply available to other European Cryptosporidium laboratories. Critically, before Yarlett's method can be adopted as the method of choice for Cryptosporidium oocyst supply, it is imperative that we (a) demonstrate the reliability of the method for the growth of different Cryptosporidium species and variants, including the human-infecting forms, and (b) assure the genetic stability of parasites grown in this system for many generations, relative to parasites reared in calves. Thus, we need to show that this new method is better than using animals both in terms of quantity and quality of infective stages produced.The work will be disseminated to interested research units in the UK and Europe explaining the method, and demonstrating advantages compared to the animal model. Training will be offered to relevant Society members, and data comparing Cryptosporidium derived from the two systems (in vitro culture vs. calves) will be made publicly available through scientific publication and deposition on line.