EU legislation demands that residues of xenobiotics used in poultry production to prevent and treat disease are monitored. However, there is strong evidence that some coccidiostat residues may be present in meat and the consumer is not being given adequate protection. Vast quantities of such drugs are used to treat and prevent disease in poultry production. Some coccidiostats have been banned from use in the EU due to their carcinogenic properties of (nitroimidazoles). Some coccidiostats are authorised and have been (or are being) assigned Maximum Residue Levels (MRLs). There is strong evidence that both authorised and unauthorised coccidiostats are being abused in the EU. Despite widespread use of these drugs, and the potential for adverse effects, few National Reference Laboratories (NRLs) have the ability to test for their presence in food. A recent survey showed that more than 75% of labs felt that existing methods for coccidiostats required improvement. Only one third of labs were able to measure residues of all the banned nitroimidazole drugs. More than 2/3rds of the labs believe that there is a need to develop methods for more coccidiostats.
The central aim of this project is to address the above issues. Nitroimidazoles, halofuginone, toltrazuril, and nicarbazin have, therefore, been selected as priorities for the analysis method development. High-throughput, multi-residue and user-friendly tests will be developed and validated. The system can be applied for rapid detection of toxic agents throughout the food chain and subsequently also other residues can be measured with the same system. Chemical methods are also going to be developed to confirm the findings of the screening assays. The present consortium comprises 3 NRLs with GLP/EN45000 accreditation, 2 innovative SMEs and a research centre.
During the first year of the project the following results and milestones have been achieved: A high number of different immunogens have been designed and antibodies have been generated in the project using high volume polyclonal production techniques. The resulting antibodies have been characterised by enzyme immunoassays (ELISAs) and selected antibodies have been further evaluated in a time-resolved fluoroimmunoassay (TR-FIA) system. Working antibodies have so far been produced for all except one of the coccidiostatic compounds included in the project. The development of prototype test kits (TR-FIA) has been started for each compound when a promising antibody has already been generated and the respective hapten structure is thus identified. For two of the coccidiostats the microtitre plate tests have been adapted for all-in-one (AIO) dry chemistry principle by using the optimised conditions already obtained. The development of sample preparation methods has been started ahead of planned schedule. The confirmatory methods, based on LC-MS/MS-techniques, have already been optimised for all compounds included in the project as planned. A dedicated web-site has been established for the project according to plan and the methods will be disseminated to end-users in Technology Transfer events.
During the second year of the project, the following results and milestones have been achieved. Antibodies have been developed for all of the analytes and the antibody production is now complete. The development of analyte-specific TR-FIA labels and TR-FIA prototype test kits has also been completed. The adaptation of the tests into dry chemistry all-in-one concept is in progress and it has already been completed for two of the assays. The confirmatory methods have been validated and the standard operating procedures prepared. Two methods have been also tested by another partner laboratory.