Prion diseases are infectious and able to spread between individuals of both the same and different species. Animal prion diseases, such as Bovine Spongiform Encephalopathy (BSE), can be transmitted to humans and so are considered a threat to public health. Transmission from cattle to humans is thought to occur through ingesting BSE-contaminated food and strict controls are now in place to protect humans from BSE. Prion infectivity is typically measured by intracerebral inoculation with test material of an appropriate experimental animal, predominantly mice. These mice are then euthanised when clinical signs of terminal prion disease are visible, including weight loss, kyphosis, ataxia, and tremors, with the end point being mono- or bilateral hind limb paralysis. The Drosophila prion bioassay was developed as a replacement technique, funded previously by an NC3Rs Project Grant, and can detect BSE prion infectivity. The flies are transgenic containing the prion protein (PrP) gene and are susceptible to mammalian prion infection, demonstrated using the scrapie prion that causes transmissible spongiform encephalopathy (TSE) in sheep.
Why we funded it
This Skills and Knowledge Transfer Grant aims to transfer a Drosophila prion bioassay as a replacement to the mouse prion bioassay for use at the Animal and Plant Health Agency (APHA).
The APHA is the leading prion surveillance institute in the UK and following further validation work this invertebrate prion bioassay could replace the use of an estimated 750 mice per year. Increased surveillance after the emergence of BSE in cattle has also identified atypical forms of bovine prion disease. However, their zoonotic potential is unknown and it is not known whether the controls in place to prevent classical BSE entering the human food chain can also prevent atypical BSE. This will likely need to be established using prion bioassays.1 The introduction of the Drosophila prion bioassay to research groups worldwide has the potential to replace the use of approximately 25% of mice as an initial estimate, equating to a replacement of up to 5,000 mice annually.
The bovine PrP transgenic Drosophila will first be validated for their sensitivity as a bioassay for classical and atypical BSE prion infectivity. Drosophila will be inoculated at the larval stage and the neurotoxicity of the inoculum assessed in adult flies. Inocula will consist of brain material from terminal cases of cattle infected with BSE, both classical BSE and atypical BSE samples. This material has already been assessed for prion infectivity in mice by the APHA, and so will allow for a direct comparison of assay sensitivity between the mouse and Drosophila bioassay. Prion-free bovine tissue will be used as a control. Neurotoxicity will be assessed by examining the locomotor ability of the flies, where neurotoxic samples will negatively impact the locomotion of the flies. The presence of disease-associated PrP will be confirmed in the brain using immunohistochemistry. After these validation studies the Drosophila prion bioassay will be transferred to the APHA.
1 Anon. (2014). Atypical BSE study protocol EFSA Journal. 12(7):3798 [55 pp]
Thackray AM et al. (2021). A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila. Journal of Biological Chemistry, in press. doi: 10.1016/j.jbc.2021.100878