High-throughput in vitro culture system for Cryptosporidium oocysts: replacing animals in research
Lead Research Organisation:
Cardiff University
Department Name: School of Biosciences
Abstract
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.
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.
Technical Summary
In vitro culture of Cryptosporidium has previously failed to generate oocysts suitable for research purposes. Supply of the pathogen relies upon maintenance in calves, a usage which has now reached crisis point because of increasing demand.
A novel in vitro methodology, developed by Prof Yarlett (US), can potentially produce large numbers of oocysts, replacing maintenance of Cryptosporidium cultures in animals. The method is based on a hollow fibre bioreactor, seeded with cultured human colon tumour cells. The cells form a monolayer on the outside of the hollow microfibers, fed by culture medium flowing through the inside of the microfibers and diffusing through to the cells. Between the microfibers the cells create a stagnant micro-oxygenic environment within which Cryptosporidium sporozoites, following hatching from the oocyst, survive and invade host cells. This method has advantages over conventional cell culture: the biphasic architecture gives Cryptosporidium a micro-oxygenic environment to inhabit while providing host cells with adequate oxygen and nutrients, and the parasite is able to undergo multiple cycles of host cell invasion, growth, reproduction and release, before oocysts are produced. These steps are limited in other culture systems, leading to reduced or no oocyst production. Yarlett's method can generate 108 oocysts/ml/day for 6+ months; this could replace commercial production in calves, where the normal purchased quantity is 106 oocysts per batch.
This project will fund transfer of the Yarlett system to Cardiff University generating a European hub for Cryptosporidium oocyst supply. We will test the genetic stability of Cryptosporidium maintained in this system for 100 days by resequencing the genome at 20 fold coverage for SNP comparison with the published C. parvum genome from parasites maintained in calves. The culture method will also be tested with a C. hominis isolate from the NHS Cryptosporidium Reference Strain Laboratory.
A novel in vitro methodology, developed by Prof Yarlett (US), can potentially produce large numbers of oocysts, replacing maintenance of Cryptosporidium cultures in animals. The method is based on a hollow fibre bioreactor, seeded with cultured human colon tumour cells. The cells form a monolayer on the outside of the hollow microfibers, fed by culture medium flowing through the inside of the microfibers and diffusing through to the cells. Between the microfibers the cells create a stagnant micro-oxygenic environment within which Cryptosporidium sporozoites, following hatching from the oocyst, survive and invade host cells. This method has advantages over conventional cell culture: the biphasic architecture gives Cryptosporidium a micro-oxygenic environment to inhabit while providing host cells with adequate oxygen and nutrients, and the parasite is able to undergo multiple cycles of host cell invasion, growth, reproduction and release, before oocysts are produced. These steps are limited in other culture systems, leading to reduced or no oocyst production. Yarlett's method can generate 108 oocysts/ml/day for 6+ months; this could replace commercial production in calves, where the normal purchased quantity is 106 oocysts per batch.
This project will fund transfer of the Yarlett system to Cardiff University generating a European hub for Cryptosporidium oocyst supply. We will test the genetic stability of Cryptosporidium maintained in this system for 100 days by resequencing the genome at 20 fold coverage for SNP comparison with the published C. parvum genome from parasites maintained in calves. The culture method will also be tested with a C. hominis isolate from the NHS Cryptosporidium Reference Strain Laboratory.
Planned Impact
The proposed knowledge and technological transfer of a novel in vitro culture system (the Yarlett bioreactor) for Cryptosporidium to Europe will replace animals used for this research.
Cardiff University will be the first institution in Europe to use this alternative approach for producing Cryptosporidium oocysts, and seeks to become a hub replacing oocyst supply from animal sources for European researchers. The method offers an immediate benefit in reducing use of calves for commercial production of Cryptosporidium oocysts, and in providing a source of supply that does not require the use of animals for its production. Although the bioreactor is developed it has to be validated and upscaled for use outside Yarlett's laboratory.
The C. parvum reference strain IOWA is maintained commercially in calves by two companies worldwide; other isolates are maintained by a third company and by the Moredun Institute in the UK. The pathogen undergoes a short patent period (12-20d) in young calves (probably no more than several days old); given the young age of these animals, and the diarrheic pathology of Cryptosporidium, there are substantial welfare implications of this use. Oocysts remain viable for a period of weeks to months, and so regular passaging is needed. Although we lack details, we estimate that larger commercial organisations (Waterborne Inc., US) probably use 50 calves each year just for passaging. The smaller Moredun probably uses 12-15 calves. In the past 12 months, 32 papers have been published utilising commercially supplied oocysts. Given the short lifespan of oocysts, and based on our own usage (5 batches purchased in 2 years) these users would have used freshly passaged cultures, implying bespoke production in calves on-demand. Assuming each paper used 2 batches of oocysts this would translate to a further 60+ calves worldwide used to supply oocysts. We anticipate most of this calf use (over 90%) would be replaced by the Yarlett in vitro culture approach. Reduction of animal use is also possible for a newborn mouse experimental model; over the past 12 months, 11 papers have used >200 newborn mice to test hypotheses about Cryptosporidium basic biology. This is a poor model for the pathogen and so the Gates Foundation (gcgh.grandchallenges.org) has funded (2016) projects to develop infant baboon, rabbit and novel mouse models to replace and modify neonatal mouse models for Cryptosporidium research; expansion of the Yarlett system for supply and experimentation could have a significant impact on animal use in all of these systems.
In the first instance the method will be used to replace commercial oocyst use (calf production) of up to 2 batches a year in the Cable laboratory, ultimately offering a cost-effective alternative for commercial oocyst providers, and replacing the usage of around 200 calves per year. Additionally, we envisage replacement of a proportion (10-20%) of mice used for research on Cryptosporidium (for example for first phases of drug screening). Acceptance of the method by the research community will depend on the genetic stability of oocysts derived from in vitro culture; demonstration of comparable genetic stability to that of oocysts derived from animals will hasten adoption of the method to replace passaging in animals in many research labs. Successful optimization of this method will lead us to expand the approach for the supply of other apicomplexan parasites of medical and veterinary importance such as Eimeria and Cytoisospora for which supply via in vitro culture is currently not possible.
Cardiff University will be the first institution in Europe to use this alternative approach for producing Cryptosporidium oocysts, and seeks to become a hub replacing oocyst supply from animal sources for European researchers. The method offers an immediate benefit in reducing use of calves for commercial production of Cryptosporidium oocysts, and in providing a source of supply that does not require the use of animals for its production. Although the bioreactor is developed it has to be validated and upscaled for use outside Yarlett's laboratory.
The C. parvum reference strain IOWA is maintained commercially in calves by two companies worldwide; other isolates are maintained by a third company and by the Moredun Institute in the UK. The pathogen undergoes a short patent period (12-20d) in young calves (probably no more than several days old); given the young age of these animals, and the diarrheic pathology of Cryptosporidium, there are substantial welfare implications of this use. Oocysts remain viable for a period of weeks to months, and so regular passaging is needed. Although we lack details, we estimate that larger commercial organisations (Waterborne Inc., US) probably use 50 calves each year just for passaging. The smaller Moredun probably uses 12-15 calves. In the past 12 months, 32 papers have been published utilising commercially supplied oocysts. Given the short lifespan of oocysts, and based on our own usage (5 batches purchased in 2 years) these users would have used freshly passaged cultures, implying bespoke production in calves on-demand. Assuming each paper used 2 batches of oocysts this would translate to a further 60+ calves worldwide used to supply oocysts. We anticipate most of this calf use (over 90%) would be replaced by the Yarlett in vitro culture approach. Reduction of animal use is also possible for a newborn mouse experimental model; over the past 12 months, 11 papers have used >200 newborn mice to test hypotheses about Cryptosporidium basic biology. This is a poor model for the pathogen and so the Gates Foundation (gcgh.grandchallenges.org) has funded (2016) projects to develop infant baboon, rabbit and novel mouse models to replace and modify neonatal mouse models for Cryptosporidium research; expansion of the Yarlett system for supply and experimentation could have a significant impact on animal use in all of these systems.
In the first instance the method will be used to replace commercial oocyst use (calf production) of up to 2 batches a year in the Cable laboratory, ultimately offering a cost-effective alternative for commercial oocyst providers, and replacing the usage of around 200 calves per year. Additionally, we envisage replacement of a proportion (10-20%) of mice used for research on Cryptosporidium (for example for first phases of drug screening). Acceptance of the method by the research community will depend on the genetic stability of oocysts derived from in vitro culture; demonstration of comparable genetic stability to that of oocysts derived from animals will hasten adoption of the method to replace passaging in animals in many research labs. Successful optimization of this method will lead us to expand the approach for the supply of other apicomplexan parasites of medical and veterinary importance such as Eimeria and Cytoisospora for which supply via in vitro culture is currently not possible.
