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Verification of "Organic Meat production" by detection of permitted and not-permitted uses of veterinary medicines.
Project Code: Q01047
Central Science Laboratory
1. In the production of “Organic” meat, one of the controlled processes is the use of veterinary drugs. Strict standards are in place as to when and how such drugs may be used. Therefore the overall aim of this project was to determine whether it was possible to distinguish between a single therapeutic dose of antibiotics (permitted under the standards) and both multiple therapeutic dosing and prophylactic dosing (not permitted). This comprised an evaluation of (i) pigs that were treated with oxytetracycline, (ii) chickens dosed with two different tetracycline antibiotics (oxytetracycline and chlortetracycline) and iii) chickens does with a sulphonamide (sulphadiazine).
The four main scientific objectives for the project were:
· To determine the deposition patterns for tetracyclines in poultry and porcine bone under controlled dosing conditions;
· To determine the deposition patterns for sulphonamide antibiotics (sulphadiazine) in poultry tissues under controlled dosing conditions;
· To produce validated method(s) for the verification that correct veterinary medical procedures have been used during organic meat production.
· To carry out an exercise to assess method performance on an extended range of test samples (“field validation”), of organic and non-organic chicken and pork.
Tetracyclines – initial study
2. Deposition patterns for oxytetracycline and chlortetracycline in poultry bones, and oxytetracycline in porcine bones, under conditions of single therapeutic, double therapeutic and long-term prophylactic dosing have been determined.
3. The developed methodology uses bone sectioning and examination under ultraviolet illumination (either direct observation or fluorescent microscopy). This allows samples from animals that have been treated with different dosing regimes (a single therapeutic dose, two successive therapeutic doses and long-term, low-level “prophylactic” dosing) to be
assessed for compliance with organic farming regulations. The method was subjected to blind checks of unknown samples by a second operator and was shown to be applicable to a variety of species and a selection of tetracycline drugs.
Tetracyclines – semi-quantitative analyses via imaging software
4. The initial method for tetracyclines relied on expert interpretation of the visual images. In order to design a more objective test further research was undertaken to investigate the use of image processing software.
5. Three software packages were investigated. Image-Pro Plus was selected as the most suitable. This software allowed the conversion of the existing body of digital photographs and photomicrographs (generated previously) to be converted to intensity plots. These intensity plots provided characteristic numerical response ranges for each dosing regime in the two species examined. Statistical assessment of the numerical data so derived allowed criteria for each dosing regime to be defined, thus providing a semiquantitative result.
6. Good discrimination could be achieved between un-dosed (control) samples and samples dosed under different treatment regimes (single and double therapeutic and Prophylactic) for chicken. This semi-quantitative approach was found to be less robust for porcine samples.
7. For sulphadiazine, two visualisation approaches, a histochemical method based on diazotisation; and an immunohistochemical method based on a fluorescent labelled monoclonal antibody, were investigated.
8. Both the histochemical and immunohistochemical methods were able to distinguish untreated control samples and dosed samples in bone sections. The immunohistochemical method was also able to distinguish untreated control samples and dosed samples in feathers and toenail (claw) sections. Neither method could distinguish untreated control samples and dosed samples in tendon sections.
9. None of the methods were able to determine different deposition patterns for sulphonamide antibiotics in poultry tissues arising from different dosing regimes (i.e. prophylactic versus therapeutic).
Field validation of tetracycline methods
10. None of the samples of organic chicken (n=19) and organic pork (n=13) examined in the “field validation” showed any evidence (either visual or semi-quantitative) of tetracycline exposure.
11. Of the non-organic samples, three samples of chicken (n=13) and one sample of pork (n=14) showed evidence of tetracycline exposure. Visual and semi-quantitative assessment of the chicken samples indicated that they had undergone single therapeutic dosing. Visual and semi-quantitative assessment of the pig sample was unable to unequivocally establish the dosing regime, which could have been either multiple therapeutic or prophylactic.
12. The field validation did not find any samples that were non-compliant with the Regulations governing the use of drugs in organic meat production. Data from all of the field validation samples were consistent with data collected in earlier phases of the project. This indicates that the methodology is fit for the purpose of assessing samples for tetracycline antibiotic use for compliance with the Regulations governing organic meat production.
13. Since the techniques have been shown to be capable of determining whether or not exposure to tetracyclines or sulphonamides has occurred, and in the case of tetracyclines, the dosing regime, it has been proposed to use the methods to assess the extent of unprescribed use of veterinary antibiotics, and compliance with the conditions of issued veterinary prescriptions. From January 2006, new European Union animal feed regulations will forbid the use of growth-promoting additives. One possible consequence of this actionis that commonly used antibiotics such as tetracyclines will be used prophylactically. This practice is illegal. Another added benefit of the methodology developed under this project is that it could be used to address this problem.
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