Professor Joanne Cable was awarded funding to bring to Europe, via a transatlantic exchange, a high throughput in vitro culture system for the enteric protozoan parasite Cryptosporidium.
Principal Investigator: Professor Joanne Cable
Organisation: Cardiff University
Award type: Skills and knowledge transfer grant
Start date: 2017
Duration: 2 years
Cryptosporidium is a waterborne pathogen which poses a major threat to farm animals and humans because there is no available drug treatment and no immediate prospect of vaccine development. Together, two species of the parasite, C. parvum and C. hominis, are responsible for more than 200,000 infant deaths attributable to diarrhoea in South Asia and sub-Saharan Africa each year, but even in developed countries outbreaks can occur.
There is an urgent need to understand the host immunological response and how the parasite interacts with the host microbiome, in order to develop effective therapeutics. Until recently the generation of parasites (specifically transmissive stages, termed oocysts) for experimental purposes was dependent on the use of neonatal calf and immunocompromised neonatal mouse infection models, because production of in vitro systems was difficult with poor yields and low infectivity.
In 2016, Professor Nigel Yarlett from Pace University, New York, published methodology for the continuous culture of C. parvum using a hollow fibre bioreactor seeded with human colon tumour-derived HCT-8 cells. The bioreactor provides an environment that mimics the gut by delivering nutrients and oxygen through the lumen of the fibres to the basal layer of host cells growing on the outside of the fibres, while allowing separate redox and nutrient control outside the fibres for parasite development. The system produces a high oocyst yield, replacing the use of animals and avoiding the suffering associated with infection (such as diarrhoea, dehydration, inappetence and lethargy leading to severe weight loss and a high risk of death). Importantly unlike Cryptosporidium parasites generated through in vivo infection models which show batch-to-batch variation in their transcriptomic and proteomic profiles, in vitro production generates genetically identical parasites helping to ensure findings are reproducible from one experiment to another.
3Rs benefits (actual and potential)
Following training in the Yarlett laboratory, Dr Anna Paziewska-Harris, the post-doctoral researcher on the grant has established the bioreactor system at Cardiff University.
Calf-derived oocysts were initially required for primary seeding of the bioreactor, but subsequently oocysts produced in vitro have been used for experiments or for seeding of additional bioreactors. The bioreactor has allowed the Cable laboratory to replace the use of calves for parasite production – saving a minimum of six animals per year. Further savings have been made for experimental work. Because parasite viability decreases when stored outside of the host, each experiment requires a fresh batch of oocysts – without the bioreactor, a recent series of experiments by the Cable laboratory on environmental factors affecting sporozoite viability would have required four calves.
Scientific and technological benefits
The bioreactor produces 106 oocysts per week for over six months, however, through manipulation of nutrient content in both compartments, the Cable laboratory has shown that it can be scaled-up to produce 108 oocysts per ml/week. This is similar to the commercial production in calves where the typical quantity is 108 to 109 oocysts per batch. Anna has also optimised the viability assay for oocysts and the gliding motility assay for sporozoites providing an estimation of the parasites’ condition following the in vitro culture. Together with a newly developed 3D infectivity assay for sporozoites using HCT-8 cell spheroids, the assays constitute a quality control system for assessing the phenotype of parasites produced by the in vitro platform.
Jo and Anna organised a workshop, part funded by the NC3Rs, at the 2018 British Society for Parasitology Spring meeting, to promote the use of the bioreactor for the production of parasites and other applications to advance Cryptosporidium research. This has led to five new collaborations, including with the NHS Cryptosporidium Reference Unit in Wales, with the Cable laboratory acting as a training hub for researchers and organisations. Wide uptake of the bioreactor system could have significant cost saving benefits. For example, more than 20 water companies in the UK each spend around £60k per annum on purchasing Cryptosporidium parasites for positive controls for their daily drinking water screening which is required by law – use of the bioreactor could cut the cost to around £20k per annum.
Data from studies using Cryptosporidium oocysts produced by the in vitro system have been included in successful funding applications totalling £290k to date. This includes a Marie Curie COFUND Fellowship to Anna, and a Global Challenges Research Fund project and a BBSRC-funded PhD studentship to Jo.
The project has been showcased at various public engagement events including the Welsh Eisteddfod festival in August 2018, with a ‘soapbox’ presentation on the 3Rs. Further-a-field, Jo has talked about the bioreactor in Namibia during a launch event for Phoenix Waters, a collaboration between the Universities of Cardiff and Namibia to understand issues relating to water security and quality in sub-Saharan Africa.
This case study was published in our 2019 Research Review.