Antibiotic residues tests

The U.S. Department of Agriculture Meat and Poultry Antibiotic Residue Testing Program... 

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. 

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]. 

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. 

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. 

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. 

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... 

In some instances additional specialized studies may be required to assess drug-specific toxicological concerns. For example, hypersensitivity tests may be required for the -lactam antibiotics FDA has recently been concerned with how this standard human food safety assessment process accurately determines the safe concentration of antibiotic residues based on the traditional toxicological end-points. Of particular concern was the impact of low levels of antibiotics on the intestinal microflora. 

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. 

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). 

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. 

In the context of residue control, antibiotic residues in milk are the easiest and most practical to accommodate. In the past, inhibitor tests sensitive primarily to -lactam antibiotics were used to control drug residues in milk. More recently, otlier drugs have become the focus of concern, and new concepts for the detection of antibiotics in milk arc repeatedly being reported (31). 

In assays in which bromocresol purple indicator is incorporated into the medium (disc assay for penicillin, disc assay with indicator), a clear bluish zone around the disk appears if samples contain inhibitory substances. When addition of penicillinase to positive milk samples eliminates the zone, the inhibitors can be roughly identified as -lactams. Among these tests, the 3 h Bacillus stearothermophilus disc assay was selected in 1981 as the regulatory test for detecting antibiotic residues in milk. Although particularly sensitive to -lactams, this test is relatively insensitive to a number of commonly used antibiotics (20). 

For very rapid on-farm screening of antibiotic residues in animal tissues, the swab test on premises (STOP) has been widely employed (86). This test involves inserting a cotton swab directly into meat tissues, allowing it to absorb tissue fluids. The swab is the removed and placed on a test plate to be incubated with Bacillus subtilis ATCC 6633 spores at 29 C overnight for evidence of inhibition around the swab. 

In addition, a number of other assays have been developed for screening antibiotic residues in body fluids of animals at slaughter. They include an agar plate assay for detecting tilmicosin in bovine serum (88), modified CFT and BR-test assays for screening penicillin G residues in plasma of healthy steers (89), a modified CFT for screening plasma and urine samples from healthy market... 

Besides physicochemical methods, the use of microbiological growth-inhibition assays to test meat and milk for the presence of antibiotics residues is popular over a long period of time. These tests use antibiotic-sensitive bacterial reporter strains, such as Bacillus subtilis and Bacillus stearothermophilus var. calidolactis. These bacteria are inoculated under optimal conditions with and without sample. After culturing, results are read from visible inhibition zones or from the color change of the bacterial suspension in agar gels [6]. 

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