Tests for antibiotic residues

Charm Sciences Inc. (USA). The Screening Test for Antibiotic Residues (STAR) five-plate test was evaluated for the screening of 66 antibiotics in milk [178]. 

Handbook No. 601, How to Perform the Live Animal Swab Test for Antibiotic Residues, United States Department of Agriculture, Food Safety and Inspection Service, Washington, DC (1984). 

A high level of FFAs may cause minor errors in determination of the fat content of milk (e.g., a decrease of 0.01 0.03% fat per 1 meq/100 g increase in FFA), whether by older methods such as Rose-Gottlieb or turbidimetric or infrared methods (van Reusel, 1975 van de Voort et al., 1987). Lipolysis may also lead to false positives in testing for antibiotic residues in milk (Carlsson and Bjorck, 1992). 

In the case of laboratories engaged in testing for antibiotic residues in food, it may be necessary to implement a quality system to meet contractual, statutory, or regulatory requirements, to comply with international regulations or agreements, or to achieve third-party certification or accreditation, which in some instances is mandatory under those international regulations or agreements (see Section 10.3.4). 

ISO/IEC 17025 2005, which provides a framework for quality management and the technical competence of laboratories to carry out tests and calibrations. This is probably the most appropriate standard for regulatory laboratories engaged in testing for antibiotic residues and is discussed in more detail in Section 10.4.3. 

FSIS has developed a series of overnight, inexpensive, easy to perform swab bioassay tests for screening tissues, body fluids, or feed extracts for antibiotic residues. The swab tests are used on the farm, in the slaughter plant, or in the laboratory for their designated purpose. Swab test results indicate whether antimicrobial activity is present in the sample at or above allowable levels or absent. Further testing with more sophisticated tests is required to identify and quantify the antibiotics producing the antimicrobial activity. These are usually done in a laboratory as required. 

LAST is used by farmers, veterinarians, and other interested parties to screen urine from cull dairy cows for antibiotic residues before marketing. If the LAST test is positive, the animal is retained for several days and retested before sale, A negative LAST test allows the farmer to market his cull cow with a high degree of confidence that the edible meat, liver, kidney, etc, at slaughter will be antibiotic residue free or below established tolerances,... 

Edible tissues from STOP-positive animals are retained until tested further by FSIS laboratories. If the laboratory report indicates antibiotic at or above tolerance levels, the viscera and/or the carcass are condemned. If antibiotic levels are below tolerance levels upon laboratory testing, the tissues are released into the food supply. In-plant STOP-negative animals are released without delay into the food chain. STOP may be used to test all food animals and poultry for antibiotic residues. Since STOP began in 1979, the incidence of antibiotic residues in the bovine meat supply has been reduced to approximately one percent. 

From January to June, 1985, a total of 1080 samples from fresh milk, swine liver and muscle, chicken liver and muscle, and hen eggs marketed at three cities located at the middle area of Taiwan were collected and analyzed for antibiotic residues (45). The positive rates found in the screen tests were 22.2% for milk, 21.1% for swine liver, 12.7% for swine muscle, 49.4% for chicken liver, 19.4% for chicken muscle, and 1.1% for hen eggs. 

In Belgium, 0.1% of the slaughtered cattle and swine are screened for antibiotic residues each year. In the analytical strategy applied, meat samples are screened with a modified four-plate test followed by screening with a group-specific ELISA for the identification of the antibiotics and confirmation by specific LC methods. 

More versatile than the growth-inhibition assays and potentially applicable to determining the presence of different antibiotic residues in different matrices are the microbial receptor CHARM I and II test assays (19, 20). The Charm I test, developed exclusively for -lactams in milk, constitutes the first rapid test recognized by The Association of Official Analytical Chemists (AOAC) with a test time of 15 min (19). The speed and sensitivity of this test permitted testing of milk tankers before they unloaded at the processing plant (21). In 1984-1985, the CHARM I test was further developed to test for antibiotics beyond -lactams to include tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, novobiocin, and macrolides. The extended version has been referred to as CHARM II test. 

Shortly after their development in the 1940s, antibiotics were used in veterinary medicine, first to prevent or treat mastitis in cows and later for the treatment of other diseases. Initial concern about antibiotic residues in milk was not a public health issue but came from dairy processors who noticed inhibition of starter cultures used in the production of cheese and yogurt, thus generating a need for screening tests to examine milk for antibiotic residues. " Since inhibition of starter cultures by penicillin in milk was the main problem, the earliest microbial inhibition assays were based on growth inhibition of lactic acid bacteria. Spores of Bacillus species were also utilized spores are easier to handle and far more stable than the vegetative cells. 

TABLE 5.5 Range of Charm II Test Kits and Their Applications Currently Available for Antibiotic Residue Screening from Charm Sciences Inc. 

When choosing to implement a particular screening assay for antibiotic residue analysis, the laboratory must ensure that the performance criterion is fit for the intended purpose and in line with the appropriate legislative requirements within that country or the country for which the testing is undertaken, for example, the country to which the produce is to be exported. 

Notes STOP = swab test on premises, measures antibiotic residues in the kidney FAST = fast antimicrobial screen test, measures antibiotic and sulfonamide residue in kidney and liver SOS = sulfa-on-site, measures for sulfonamide residues. 

The USA monitoring and surveillance programs for detecting antibiotic residues in the domestic and imported meat supply are described. An overview of the field/laboratory tests currently in use is also provided ... 

FSIS currently uses a variety of tests for detecting antibiotic residues in meat among these are field, in-plant, and laboratory screen tests, bioassays, immunoassays, and related biochemical techniques. 

Other tests used by FSIS to detect, identify, and/or quantify antibiotic residues in meat are primarily designed for laboratory use. 

This assay, commonly referred to as the Charm Test, is based upon the affinity of antibiotics for specific sites on the cell wall of microorganisms and the irreversible binding of the antibiotic to these sites. By adding C-labelled or H-labelled antibiotic to a sample of milk, urine or the aqueous extract of tissues together followed by microbial binding sites and measuring the quantity of the labelled antibiotic that binds to the microbial sites, the antibiotic residue can be measured. 

Penicillins form several major metabolites which are excreted in the urine (83,84). These metabolites are usually inactive microbio-logically and they would not be detected by the usual microbiological tests. There are no analytical methods for these metabolites in tissues and, therefore, little is known as to their occurrence and persistence in tissues. There are no methods available for identifying residues of some commonly used B-lactam antibiotics including carbenicillin and ticarcillin. For cephapirin and ampicillin, except for one HPLC method for ampicillin in milk (79) only TLC procedures (72-74,76) with detection by bioautography are reported. 

Although some European countries still accept the results of the four plate test as confirming the presence of antibiotic residues in samples ( ), other work indicates that FPT test is not necessarily reliable. The occurrence of natural microbial inhibitors in tissues has frequently been noted (4,9,49,82), It has also been frequently observed that the results obtained by microbial and physicochemical procedures sometimes differ considerably (9,10,45,82,86), Results obtained in our laboratory suggest that even inactivation by penicillinase may not be totally specific for B-lactam antibiotics (W), The specificity of immunoassay procedures depends on the specificity of the antibody used in the test (95), Specific antisera are not widely available at present. Physicochemical procedures are therefore essential for identification and confirmation of suspect residues detected by microbiological tests. 

Validation of the purification process and of the analytical tests used for characterization of the final protein product is especially important. The purification process must be validated to ensure that it is adequate to remove extraneous substances such as chemicals used in purification, column contaminants, endotoxin, antibiotics, residual cellular proteins, inactive protein, and viruses. Analytical tests are validated by using other analytical... 

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