Nationwide microbiological baseline data collected by sponge sampling during 1997 and 1998 for cattle, swine, turkeys, and geese.

During 1997 and 1998, the U.S. Food Safety and Inspection Service completed nationwide microbiological baseline studies on four separate categories of livestock and poultry. Data were collected by sponge-sampling techniques. These studies were designed to provide nationwide estimates of the prevalence of Salmonella and prevalence and concentrations of Escherichia coli in cattle (n = 1,881), swine (n = 2,127), turkeys (n = 1,396), and geese (n = 102) in establishments under federal inspection. Salmonella prevalence ranged from 1.2% in cattle to 6.9% in swine, 13.7% in geese, and 19.6% in turkeys. The prevalence of E. coli was 16.6% in cattle (geometric mean = 0.26 CFU/cm2), 44.1% in swine (mean = 0.78 CFU/cm2), 92.7% in turkey (mean = 2.46 CFU/cm2), and 96.5% in geese (mean = 1.97 CFU/cm2). These values are similar to or somewhat lower than previous baseline values obtained for steers and heifers, cows and bulls, market hogs, and young turkeys. This study is the first in which nationwide microbiological baseline data have been compiled for geese. These data will be useful to individuals working with hazard analysis critical control point plans and risk assessment and to the research and academic communities.

Comparison of monolayer and bilayer plates used in antibiotic assay.

Standard curves of 5 antibiotics were determined in an antibiotic assay using bilayer and monolayer agar plates and AOAC-specified test organisms and agar media. Micrococcus luteus ATCC 9341a and antibiotic medium No. 2 were used to prepare the penicillin G standard curve. The same organism and antibiotic medium No. 11 were used to prepare the erythromycin standard curve. Standard curves for streptomycin, tetracycline, and gentamicin were prepared, respectively, with antibiotic medium No. 5 and Bacillus subtilis ATCC 6633, antibiotic medium No. 8 and B. cereus ATCC 11778, and antibiotic medium No. 11 and Staphylococcus epidermidis ATCC 12228. Assays of inhibition by meat fortified with penicillin, streptomycin, gentamicin, tetracycline, erythromycin also were performed on monolayer and bilayer plates. Differences in standard curves and inhibitory responses obtained with monolayer and bilayer plates were < 10%. Thus, monolayer plates are acceptable for use in analyses of meat and poultry for antibiotics residues, with savings in laboratory resources and time.

Development of a multispecies identification field test by modified agar-gel immunodiffusion.

A multispecies identification field test (MULTI-SIFT) was developed for detection of beef, poultry, pork, sheep, horse, and deer in a wide variety of raw meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for detection of single species. MULTI-SIFT was demonstrated to be specific, relatively sensitive, and accurate in the complete speciation of 100 meat samples.

Development of a rapid equine serological test (REST) by modified agar-gel immunodiffusion.

A rapid equine serological test (REST) has been developed for detection of horse meat in a wide variety of raw meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for beef, poultry, pork, and sheep detection. Results show that the REST test was specific, sensitive, and accurate in the analysis of 101 samples.

Development of serological ovine field test (SOFT) by modified agar-gel immunodiffusion.

A serological ovine field test (SOFT) has been developed for detection of lamb or sheep tissue in a wide variety of raw meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for beef, poultry, and pork detection. The SOFT test was demonstrated to be specific, sensitive, and accurate in the analysis of 104 samples.

Development of porcine rapid identification method (PRIME) by modified agar-gel immunodiffusion.

A porcine rapid identification method (PRIME) has been developed for detection of pork in a wide variety of meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for beef and poultry detection. The PRIME test was demonstrated to be specific, sensitive, and accurate in the analysis of samples in the laboratory and in a commercial meat processing establishment.

Detection of poultry and pork in cooked and canned meat foods by enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISA) are described for the detection of poultry and pork in cooked and canned meat foods. These assays are based on species-specific, polyclonal antibodies raised against heat-resistant antigens. The heat-resistant antigens were isolated from raw skeletal muscle tissue of pork and chicken and were found to be immunoreactive even after heating to 120 degrees C for 15 min. The poultry ELISA could detect chicken or turkey at the 126 ppm level, and the pork ELISA could detect pork at the 250 ppm level. Samples of frankfurters, bolognas, pressed meats, canned baby foods, and canned spreads were prepared by simple aqueous extractions.

Detection of beef and poultry by serological field screening tests (ORBIT and PROFIT): collaborative study.

Ten laboratories each analyzed 30 raw meat and raw meat product samples in a collaborative study of the ORBIT (overnight rapid bovine identification test) and PROFIT (poultry rapid overnight field identification test) serological field screening tests for the detection of beef and poultry. Versatility of the tests was shown in the analysis of whole tissue, ground, or emulsified raw meat products. Both tests were demonstrated to be reliable and were capable of detecting adulterants present at the 10% level. The method has been adopted official first action.

Development of poultry rapid overnight field identification test (PROFIT).

A poultry rapid overnight field identification test (PROFIT) has been developed as a screening test which is practical, economical, and easy to perform and interpret for use in field environments to determine the presence of poultry tissue (chicken and turkey) in raw whole tissue or ground/formulated meat products. The basis of the test is an agar-gel immunodiffusion technique used with a printed template pattern and stabilized reagent paper discs. The test shows adequate sensitivity and specificity for its intended purpose. Key components are stable for at least 1 year if they are stored at refrigerator conditions. The design of the test is such that it can be made commercially available as a complete, stable, test kit suitable for use by any type of inspection program concerned with verification of poultry species in meat and/or poultry products that are subject to regulatory or quality controls.

Development of an overnight rapid bovine identification test (ORBIT) for field use.

An Overnight Rapid Bovine Identification Test (ORBIT) has been developed as a serological screen test for species verification of raw, whole tissue, bovine meat products. The test, an agar-gel immunodiffusion technique, uses stabilized reagent paper discs and prepared agar plates that have a printed template for correct placement of test components. This test is reliable, practical, economical, and easily performed in the field, such as at a meat import inspection station. The only nonbovine species found to react in the test are the bovine-related species of American bison (buffalo) and water buffalo (from Australia); however, these rare-occurring species do not present a problem for the intended application of the test. Stability of all test components, when stored in a refrigerator, is excellent for at least 1 year. The nature and stability of the test make it suitable for commercial development into test kits which should be highly practical and economical for wide availability and application of this procedure to meat inspection programs concerned with species verification.

Detection and quantitation of chloramphenicol by competitive enzyme-linked immunoassay.

A competitive enzyme-linked immunoassay for the detection and quantitation of chloramphenicol has been developed. The binding of specific rabbit antibody to solid-phase-bound chloramphenicol was competitively inhibited by free chloramphenicol in the sample to be assayed. Antibody not displaced was indicated by using a commercially available, enzyme-linked, anti-rabbit immunoglobulin preparation and reacted with added substrate. Enzyme activity, measured spectrophotometrically, was inversely proportional to the concentration of chloramphenicol in the sample. Quantitation of the antibiotic was linear to 100 ng/ml, with a lower limit of detection of 1 ng/ml (P less than 0.05). Specificity was demonstrated by the lack of inhibition by any of 31 selected antimicrobial agents or chemicals tested in the assay. Chloramphenicol sodium succinate and thiamphenicol, an experimental antibiotic similar in structure to chloramphenicol, were the only drugs found to produce cross-reactions. In addition to excellent sensitivity and specificity, the assay was shown to have good precision and economy and could be completed in approximately 24h.

Association of toxic capsule and cell wall mucopeptide with virulence in Gaffkya tetragena.

Nine strains of organisms morphologically and physiologically identified as Gaffkya tetragena were obtained from various sources to study their pathogenicity. Initial virulence analysis of all strains by mouse intraperitoneal injection of viable cells revealed that only three strains, recently isolated from and associated with respiratory infections in hospitalized patients, caused death of mice within 48 hr. The ld(50) for these virulent, encapsulated strains was 1 x 10(7) to 6 x 10(7) viable organisms. To associate virulence with a toxic component, the following fractions were purified from all strains: capsular material, cell walls, mucopeptide preparations from cell walls and whole cells, grouplike material, cytoplasmic material, and culture filtrate with and without added reducing agent. Rabbit and mouse dermal toxicity testing of these fractions revealed that the capsular material, cell walls, and mucopeptide preparations of the virulent strains were toxic. None of the nonvirulent strains contained toxic components, with the exception of one strain which yielded capsular material equal in toxicity to that of the virulent strains. The capsular material induced a soft pustular lesion persisting for approximately 22 days. Cell walls and mucopeptide preparations produced a hard nodular lesion, identical to that produced by autoclaved whole cells, that persisted for 25 to 30 days. One strain may represent a virulence intermediate between the virulent and nonvirulent strains, since it contains toxic capsular material but nontoxic cell wall mucopeptide. The results indicate that the virulence of this organism is associated with toxic capsular material and cell wall mucopeptide.