The use of PrP transgenic Drosophila to replace and reduce mice in the bioassay of mammalian prions
Lead Research Organisation:
University of Cambridge
Department Name: Veterinary Medicine
Abstract
Prion diseases include BSE of cattle, chronic wasting disease of cervids, scrapie of sheep and CJD of humans. These conditions are infectious and can spread between individuals of the same or different species. Animal prion diseases can be transmitted to humans and are therefore a threat to public health, evidenced by the outbreak of classical BSE in cattle followed by the emergence of variant CJD (vCJD) in humans. The pathogen that causes these transmissible diseases is an aggregated rogue form of a normal protein found in neurons and is referred to as a prion. Transmission of BSE to humans is believed to have occurred by dietary intake of BSE-contaminated food. Strict controls now protect humans from BSE, including removal of cattle tissues most likely to contain infectious prions when the animal is slaughtered. These cattle tissues are called specified risk material (SRM) and do not enter the human food chain.
The only reliable way to detect BSE prion infectivity is by bioassay in experimental animals, which have traditionally been rodents, such as mice. The mouse prion bioassay involves injecting suspected BSE-infected samples into experimental mice and waiting to see if these animals develop prion disease. These assays are slow and cumbersome since the incubation time for prion disease in mice may be 1-2 years before clinical signs become evident and a predetermined end-point is reached. Collectively, BSE prion infectivity studies have utilised large numbers of mice over a long time period, and have subjected a high proportion of these animals to experience terminal clinical signs of experimental neurological disease. Since the emergence of classical BSE, the increased surveillance for the condition has led to identification of novel forms of the disease, which are a new threat to the human food chain as we do not know which tissues in affected cattle contain these new forms of BSE prion infectivity. It is essential therefore, to verify if the current SRM control measures are sufficient to prevent the new forms of BSE prions from entering the human food chain. Many more mice will be used in proposed food safety research programmes in order to measure prion infectivity levels in an extensive range of samples from cattle infected with new forms of BSE. In their entirety, these mouse prion bioassays will use more than 100,000 mice. It is vital to apply the 3Rs principles to this intended research programme by reducing and replacing, where possible, this large number of experimental mice with a prion bioassay that uses a less sentient host.
In our laboratory we model prion disease in the fruit fly Drosophila melanogaster because they are relatively easy and economical to work with, and are a widely accepted ethical alternative to higher organisms including mice. We have developed a Drosophila-based prion bioassay that can detect BSE prion infectivity in order to provide an alternative to mice for the bioassay of bovine prions, and reduce the use of mice, and other vertebrate animal species, to measure prion infectivity in general. To do so, we have introduced a gene into the flies that allows them to produce the protein that aggregates in the brain of animals with prion disease. We already know that this mammalian protein will aggregate and cause prion disease in transgenic Drosophila when flies are fed infectious prions. What is equally significant is that the response by the transgenic flies to prion-infected material is evident within a few weeks following exposure to prion material. We can now develop a faster, more versatile and more sensitive bioassay to detect BSE prion infectivity than currently exists. We aim to transfer this know-how to the APHA, a main user and internationally recognised reference laboratory of the mouse prion bioassay. Our Skills and Knowledge Transfer to the APHA will provide the best opportunity to ensure world-wide impact of our new Drosophila-based prion bioassay.
The only reliable way to detect BSE prion infectivity is by bioassay in experimental animals, which have traditionally been rodents, such as mice. The mouse prion bioassay involves injecting suspected BSE-infected samples into experimental mice and waiting to see if these animals develop prion disease. These assays are slow and cumbersome since the incubation time for prion disease in mice may be 1-2 years before clinical signs become evident and a predetermined end-point is reached. Collectively, BSE prion infectivity studies have utilised large numbers of mice over a long time period, and have subjected a high proportion of these animals to experience terminal clinical signs of experimental neurological disease. Since the emergence of classical BSE, the increased surveillance for the condition has led to identification of novel forms of the disease, which are a new threat to the human food chain as we do not know which tissues in affected cattle contain these new forms of BSE prion infectivity. It is essential therefore, to verify if the current SRM control measures are sufficient to prevent the new forms of BSE prions from entering the human food chain. Many more mice will be used in proposed food safety research programmes in order to measure prion infectivity levels in an extensive range of samples from cattle infected with new forms of BSE. In their entirety, these mouse prion bioassays will use more than 100,000 mice. It is vital to apply the 3Rs principles to this intended research programme by reducing and replacing, where possible, this large number of experimental mice with a prion bioassay that uses a less sentient host.
In our laboratory we model prion disease in the fruit fly Drosophila melanogaster because they are relatively easy and economical to work with, and are a widely accepted ethical alternative to higher organisms including mice. We have developed a Drosophila-based prion bioassay that can detect BSE prion infectivity in order to provide an alternative to mice for the bioassay of bovine prions, and reduce the use of mice, and other vertebrate animal species, to measure prion infectivity in general. To do so, we have introduced a gene into the flies that allows them to produce the protein that aggregates in the brain of animals with prion disease. We already know that this mammalian protein will aggregate and cause prion disease in transgenic Drosophila when flies are fed infectious prions. What is equally significant is that the response by the transgenic flies to prion-infected material is evident within a few weeks following exposure to prion material. We can now develop a faster, more versatile and more sensitive bioassay to detect BSE prion infectivity than currently exists. We aim to transfer this know-how to the APHA, a main user and internationally recognised reference laboratory of the mouse prion bioassay. Our Skills and Knowledge Transfer to the APHA will provide the best opportunity to ensure world-wide impact of our new Drosophila-based prion bioassay.
Technical Summary
The only method available to measure prion infectivity is by bioassay in an appropriate experimental animal, usually mice. We have developed an alternative prion bioassay whereby mice are replaced by the less sentient host Drosophila. To do so, we have generated PrP transgenic Drosophila and have shown that these flies are susceptible to mammalian prion infection, evidenced by a transmissible neurotoxic phenotype associated with accumulation of PK-resistant PrPSc. In this Skills and Knowledge Transfer to the APHA, we will validate the sensitivity of bovine PrP transgenic Drosophila for their bioassay for classical and atypical BSE prion infectivity. Prion inocula will consist of brain material from terminal clinical cases of cattle infected with classical BSE, or H-type or L-type atypical BSE. Prion-free bovine brain tissue will be used as control material. Prion inoculation of Drosophila will be at the larval stage while assessment of their phenotypic response by locomotor assay, detection of PrPSc by in vitro PMCA and IHC analysis of fly brains will be performed on adult flies. Specifically, we will: (a) Inoculate bovine PrP transgenic Drosophila with dilutions of either classical BSE- or atypical BSE-infected bovine brain homogenate to determine the sensitivity of these flies to bovine prions. The sensitivity of our Drosophila bioassay will be compared with existing data for these inocula obtained from titration in mice; (b) Detect PrPSc in BSE-exposed PrP transgenic Drosophila by in vitro PMCA & IHC. This will confirm the specificity of our novel fly-based prion bioassay; (c) Instruct the APHA on how to maintain and prion infect PrP transgenic Drosophila in an appropriate fly laboratory.
Planned Impact
Highlight:
Our Skills and Knowledge Transfer will demonstrate that PrP transgenic Drosophila can be used to bioassay classical and atypical BSE prion infectivity. This will provide proof-of-principle that this novel host can bioassay classical BSE and atypical BSE prions and therefore provide a new tractable approach to the assessment of human food safety with respect to animal prion diseases. The relative facile nature of transgenesis in Drosophila means that different PrP transgenic Drosophila can readily be generated for the purpose of using such flies to rapidly bioassay prion infectivity from virtually any mammalian species. This is of particular relevance with the emergence of cervid prion disease in Europe and its unknown zoonotic potential. Successful completion of this Skills and Knowledge Transfer will lead to more Drosophila studies that will allow future training of young scientists so that the next generation of prion researchers can be nurtured by experienced prion biologists. This is an essential component of any grant to allow the transfer of knowledge to future scientists.
Beneficiaries:
Animal Health Regulatory Authorities and the Food Industry:
The Animal and Plant Health Agency (APHA), Defra and the Food Standard Agency (FSA) rely heavily on animal prion bioassays to maintain surveillance of animal prion diseases, their control and their potential affect on the human population in the UK, and on a wider basis. Our Drosophila based prion bioassay will provide a novel rapid and sensitive bioassay that will make a significant contribution to a more thorough assessment of the risk that new animal prion diseases have upon the human food chain.
Pharmaceutical Industry:
Our prion bioassay will provide a tractable system to screen for chemical interventions effective against prion-induced neurotoxicity and other protein misfolding neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. It can also be used to screen animals in the production of animal products for human health and as a blood prion assay to test export breeding stock.
Government:
Our Drosophila-based prion bioassay will impact upon the Department of Health CJD Oversight Committee's need of an independent confirmatory bioassay for prion infectivity to support pre-clinical biochemical tests for vCJD prions. It will also impact on the UK House of Commons Science and Technology Select Committee enquiry into the vCJD recommendation for the development of a pre-clinical test for detection of prion infectivity and disease-specific prion protein in classical BSE-affected cattle, the animal reservoir of vCJD.
Medical and Veterinary Profession:
Development of a tractable and rapid prion diagnostic test to screen organ donors for transplantation and as a confirmatory blood and / or urine test for human prion disease will impact upon human health care. Our outputs will also impact upon the veterinary profession through development of new versatile diagnostic tests to screen the prion status of farmed animals and improve the health status of herds of cattle or flocks of sheep.
3Rs Potential:
Successful completion of our Skills and Knowledge Transfer will allow us to inform, the European (Prion) Strain Typing Expert Group (STEG) at the APHA, and EFSA, that our Drosophila-based prion bioassay is a suitable pre-screen or confirmatory test that will reduce the reliance on the mouse prion bioassay in the proposed cattle pathogenesis study for atypical BSE. In addition, the successful demonstration that PrP transgenic Drosophila can bioassay BSE-infected bovine samples will impact on sentient experimental animals (including primates, cattle, deer, sheep and rodents) used to analyse other species forms of prion infectivity, such as CWD of cervids and vCJD in humans.
Our Skills and Knowledge Transfer will demonstrate that PrP transgenic Drosophila can be used to bioassay classical and atypical BSE prion infectivity. This will provide proof-of-principle that this novel host can bioassay classical BSE and atypical BSE prions and therefore provide a new tractable approach to the assessment of human food safety with respect to animal prion diseases. The relative facile nature of transgenesis in Drosophila means that different PrP transgenic Drosophila can readily be generated for the purpose of using such flies to rapidly bioassay prion infectivity from virtually any mammalian species. This is of particular relevance with the emergence of cervid prion disease in Europe and its unknown zoonotic potential. Successful completion of this Skills and Knowledge Transfer will lead to more Drosophila studies that will allow future training of young scientists so that the next generation of prion researchers can be nurtured by experienced prion biologists. This is an essential component of any grant to allow the transfer of knowledge to future scientists.
Beneficiaries:
Animal Health Regulatory Authorities and the Food Industry:
The Animal and Plant Health Agency (APHA), Defra and the Food Standard Agency (FSA) rely heavily on animal prion bioassays to maintain surveillance of animal prion diseases, their control and their potential affect on the human population in the UK, and on a wider basis. Our Drosophila based prion bioassay will provide a novel rapid and sensitive bioassay that will make a significant contribution to a more thorough assessment of the risk that new animal prion diseases have upon the human food chain.
Pharmaceutical Industry:
Our prion bioassay will provide a tractable system to screen for chemical interventions effective against prion-induced neurotoxicity and other protein misfolding neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. It can also be used to screen animals in the production of animal products for human health and as a blood prion assay to test export breeding stock.
Government:
Our Drosophila-based prion bioassay will impact upon the Department of Health CJD Oversight Committee's need of an independent confirmatory bioassay for prion infectivity to support pre-clinical biochemical tests for vCJD prions. It will also impact on the UK House of Commons Science and Technology Select Committee enquiry into the vCJD recommendation for the development of a pre-clinical test for detection of prion infectivity and disease-specific prion protein in classical BSE-affected cattle, the animal reservoir of vCJD.
Medical and Veterinary Profession:
Development of a tractable and rapid prion diagnostic test to screen organ donors for transplantation and as a confirmatory blood and / or urine test for human prion disease will impact upon human health care. Our outputs will also impact upon the veterinary profession through development of new versatile diagnostic tests to screen the prion status of farmed animals and improve the health status of herds of cattle or flocks of sheep.
3Rs Potential:
Successful completion of our Skills and Knowledge Transfer will allow us to inform, the European (Prion) Strain Typing Expert Group (STEG) at the APHA, and EFSA, that our Drosophila-based prion bioassay is a suitable pre-screen or confirmatory test that will reduce the reliance on the mouse prion bioassay in the proposed cattle pathogenesis study for atypical BSE. In addition, the successful demonstration that PrP transgenic Drosophila can bioassay BSE-infected bovine samples will impact on sentient experimental animals (including primates, cattle, deer, sheep and rodents) used to analyse other species forms of prion infectivity, such as CWD of cervids and vCJD in humans.