In the European Union, human health concerns have resulted in the antibiotic growth promoters avoparcin, spiramycin, tylosin, virginiamycin and zinc bacitracin being banned from use in animal feeding stuffs. In order to effectively monitor compliance with the ban, the “Feedstuffs-RADIUS” project was funded under the 5 th Framework programme to develop new screening and confirmatory tests.
Antibacterial agents used as feed additives to promote growth in animal husbandry contribute to the ever rising burden of resistant bacteria. Many of these antibacterial growth promoters (AGPs) belong to classes or show cross-resistance with classes which are being looked at as our last defence against multi-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, which can cause severe problems in human medicine. Therefore, the European Union decided in 1999 to ban the use of zinc bacitracin, spiramycin, tylosin, virginiamycin (Council Regulation (EC) No 2821/98 of 17 December 1998 amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feedingstuffs) and avoparcin (Commission Directive 97/6/EC of 30 January 1997 amending Council Directive 70/524/EEC concerning additives in feedingstuffs) in animal feedingstuffs.
Control strategy for feedingstuffs
The “Feedstuffs-RADIUS” project aimed to improve the effectiveness of controls on animal feedingstuffs throughout the EU, thereby benefiting both consumers and animals. During the course of this project a control strategy to allow the detection of the banned antibiotic growth promoters was developed and consisted of two distinct analytical approaches: (i) a multi-antibiotic ELISA procedure for simultaneously screening five banned antimicrobial growth promoters in animal feeds and (ii) a multi-analyte confirmation method for the detection of the banned antibacterial growth promoters in feed. The developed control strategy for the five banned antimicrobial growth promoters shows a good sensitivity, specificity, reliability and simplicity. The project results are of interest not only to the industry but also to control bodies and laboratories, possibly also to regulators.
Commercial screening test
To develop immunoassay based screening tests, the partners generated specific antibodies to all the compounds included within the project (avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin) and then incorporated them into a series of user-friendly rapid screening tests. To achieve this, a simple to perform but reliable and effective sample extraction procedure was developed. The prototype test was then subjected to a rigorous internal validation study followed by an external collaborative trial including 11 European laboratories. The screening tests proved to be operational, commercially interesting and widely applicable for the detection of the presence at or above illegal concentration of 5 antimicrobial growth promoters in ruminant, pig and poultry feed. The rapid test can be used on commercially produced feedstuffs, which are often characterised by a low homogeneity of the mixtures. To ensure reliable results are obtained it has been recommended that several sub samples per batch of feed should be tested to make certain none of the banned antibacterials are present, even in very small quantities.
The multi-AGP ELISA test is now commercialised since 2005 by Euro-Diagnostica, one of the partner of the “Feedstuffs-RADIUS” project. The test has gained significant interest from many potential users.
The partners developed an operational and highly precise method for confirmation of the positive results of the rapid test. This confirmatory procedure is based on liquid chromatography mass spectrometry (LC-MS). The target was set that all five of the compounds would be detected in a single test procedure combining a single sample preparation technique and a single sample injection into the LC-MS. The developed procedure was subjected to an equally rigorous internal validation followed by an external collaborative trial between 11 European laboratories, from 7 different European countries. The method had to be proven to unequivocally detect the presence of all five of the banned compounds in a range of animal feed samples at or below the 20% minimum inclusion rate that the compounds were used at prior their ban. From a qualitative point of view the method performed in an extremely satisfactory manner. No false non-compliant results were reported. All compounds that were present in the medicated samples were correctly detected and confirmed, in accordance with Commission Decision 2002/657/EC (Commission Decision 2002/657/EC concerning the performance of analysis methods and the interpretation of results) , by all participants, resulting in no false compliant results. The confirmatory LC-MS/MS method proved to be able to distinguish between truly contaminated and non contaminated samples and to determine if antibacterial compounds are present due to bona fide prescription, cross-contamination during production or illegal use as growth promoters.
The developer of the LC MS/MS has produced a CD ROM outlining the full details of the method. This CD is available to any laboratory on request free of charge from the project co-ordinator and this will also be supplied by Euro-Diagnostica to any customers purchasing their test kit.
QLK5-CT-2000-00900, Rapid Antibiotic Detection for Illegal and Unlicensed Substances in animal feedingstuffs - "Feedstuffs-RADIUS"
Project coordinator: Pr Chris Elliott
Adress: Institute of Agri-Food and Land Use, Queen's University of Belfast, Stranmillis Road, Stoney Road, Stormont, Belfast, BT9 , UK
Telephone number: +44 28 90976549
Fax number: +44 28 90976513
E-mail: Chris.Elliott @qub.ac.uk
- Council Regulation (EC) No 2821/98 of 17 December 1998 amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feeding stuffs