Two project grants from the NC3Rs have aided Dr Keith Redhead in the development of an in vitro assay to replace animal tests for clostrisial vaccine antigens.
Principal Investigator: Keith Redhead, Project Manager, Research and Development
Organisation: MSD Animal Health (previously Intervet)
Awards: £111,903, in 2006, over 36 months; £26,988, in 2009, over 12 months
Title: Developing in vitro assays to replace animal tests for clostridial vaccine antigens
Read more about Dr Redhead's research.
Clostridial bacteria cause a range of diseases in livestock
Bacteria belonging to the Clostridium genus are found widely in soil and in the faeces of animals. Some species of Clostridium are responsible for a wide range of animal diseases and, as such, they represent a major risk to the livestock industry. Clostridial diseases include tetanus, enterotoxaemia in lambs and goats, Braxy in young sheep and Black disease in cattle. These diseases are controlled via a range of vaccines which are some of the most common veterinary treatments.
Clostridial vaccines contain antigens consisting of the toxoid form of the toxin. This is a weakened form of the toxin which when administered to the animal stimulates antibody production against the toxin itself. During the manufacturing process, each batch of antigen is tested for the amount of toxin and toxoid to ensure the safety and quality of the final vaccine and to satisfy regulatory requirements.
Thousands of mice are used in tests on antigens for clostridial vaccines
Mice are used to quantify clostridial toxins and toxoids during vaccine manufacture. The amount of biologically active toxin is assessed in a minimum lethal dose test (MLD) and a L+ test, both of which involve substantial suffering and often death. The amount of toxiod present is measured by the total combining power (TCP) test, which can also involve the death of the mice. The three tests are classified as causing substantial suffering under the Animals (Scientific Procedures) Act 1986. Each test uses a minimum of ten mice for each antigen and more than 200 mice can be required for the full antigen testing of some multicomponent vaccines.
New in vitro assays to replace the use of mice in clostridial vaccine testing
With NC3Rs funding, Dr Keith Redhead, Intervet, has developed in vitro assays to replace the MLD, L+ and TCP tests. A range of cell lines were screened for Clostridial antigens. Two cell lines, VERO and MDCK were found to be sensitive to Clostridium septicum alpha toxin and Clostridium perfringens type D epsilon toxin respectively. These cell lines have been used to develop in vitro MLD, L+ and TCP assays, which have subsequently been validated with data from in vivo studies.
The assays are more sensitive and accurate than the mouse tests. For example, the in vitro MLD assay is up to ten-times as sensitive as the mouse assay and the L+ and TCP assays are four-times as sensitive. They are also quicker to perform, with results available in 24 hours rather than five days.
Cell lines with appropriate sensitivities to other clostridial toxins, including Clostridium perfringens type B/C beta toxin and to Clostridium novyi alpha toxin, have also been identified.
Regulatory acceptance will replace mouse use internationally
The new assays are currently used within Intervet for work on the optimisation of clostridial vaccine manufacture, reducing mouse use by several hundred animals a year. Regulatory acceptance of the assays is, however, required for the full potential of the assays to be realised in terms of replacing animal use. This could have a significant impact on numbers, replacing around 8,500 mice in Intervet alone and potentially thousands more worldwide at other veterinary vaccine manufacturers.
This case study was published in a review of our research portfolio in September 2011.