Effect of sampling method on the representative recovery of microorganisms from the surfaces of aquacultured finfish.

The objective of this study was to determine if a gentle rinse procedure was equivalent to the combination of excision and homogenization with a stomacher for the relative removal of various microorganisms from finfish fillets. Fillets of hybrid striped bass and rainbow trout were obtained from local markets and sampled using three methods: rinse (R), excision followed by homogenization in a stomacher (S), and homogenization of fillets following a rinse (RS). Microorganisms were enumerated on selective and nonselective media, and randomly selected colonies from aerobic plate counts were identified using MIDI Sherlock and BIOLOG microbial identification systems. Enrichments and selective media were used for the isolation of Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica. This study confirms previous reports that stomaching is superior to rinsing for enumerating total microbial populations from fish fillets. Rinsing was more effective for rainbow trout than for striped bass. Sampling method did not affect the relative magnitude of plate counts on media selective for aeromonads, pseudomonads, Shewanella, lactic acid bacteria, enterics, and gram-positive cocci. In the compositional analysis of random isolates, R recovered significantly lower fractions of aeromonads than did S or RS, but sampling method did not affect the percent recovery of lactic acid bacteria, pseudomonads, Shewanella, Moraxellaceae, or Cytophaga/Flavobacterium. However, observations suggest that with increased replication, differences among Moraxellaceae, Pseudomonas, and gram positives might be significant. Only one L. monocytogenes colony was isolated, and no Salmonella or Y. enterocolitica, so the effect of sampling method could not be determined for these organisms. Differences in predominant bacterial populations were seen between fish species.

Immunization with Staphylococcus aureus lysate incorporated into microspheres.

Antibiotics are of limited value against Staphylococcus aureus due to development of resistant strains, scar tissue formation, and blockage of ducts due to inflammation. Though macrophages are the predominant cell type in the mammary gland, they are primarily scavenger cells and are not effective against bacteria entering the gland. Neutrophil phagocytosis is the bovine's primary defense against S. aureus mastitis. Attempts to develop vaccines that enhance neutrophil phagocytosis by stimulating production of opsonizing antibodies to S. aureus have met with limited success because of the low immunogenicity of the exopolysaccharide capsule surrounding S. aureus. Staphylococcus aureus can also adhere to and penetrate epithelial tissue. This study was conducted to determine whether lysates of S. aureus encapsulated in biodegradable microspheres would increase the production of opsonizing antibodies to capsule and block adherence. Four groups of four cows each were injected with 1 ml of the respective treatment in the area of the supramammary lymph node and 1 ml in the hip muscle. The treatments were: lysate in NaCl, lysate in Freund's incomplete adjuvant (FICA), lysate in microspheres in NaCl, and lysate in microspheres in FICA. Antigen in microspheres produced a similar antibody response to antigen emulsified in FICA, but to a lesser magnitude. Antigen in microspheres produced antibodies that were more opsonic for neutrophils at 20 and 52 wk postimmunization and inhibited S. aureus adherence to mammary epithelium. Ability to control antigen release and presentation, and the benefit of a single injection for long-term immunity using microspheres warrants additional studies.

Production of antibodies to Staphylococcus aureus serotypes 5, 8, and 336 using poly(DL-lactide-co-glycolide) microspheres.

Staphylococcus aureus is responsible for a major portion of the economic losses due to mastitis. Attempts to produce a vaccine to prevent S. aureus mastitis have been hampered by the low immunogenicity of the polysaccharide, which forms on the surface of the organism when it enters the mammary gland. The polysaccharide inhibits phagocytosis and destruction of the organism by neutrophils. This study was conducted to determine if S. aureus polysaccharide serotypes 5, 8, and 336 conjugated to a protein and incorporated in poly(DL-lactide-co-glycolide) microspheres would enhance the production of opsonizing antibodies to the polysaccharide. Cows were immunized with either polysaccharide conjugates emulsified in Freund's incomplete adjuvant or polysaccharide conjugates encapsulated in poly (DL-lactide-co-glycolide) microspheres emulsified in Freund's incomplete adjuvant. All cows produced sustained antibody titers to the three polysaccharide serotypes. Cows immunized with microspheres had higher antibody titers. Cows in both groups produced increased concentrations of IgG1 and IgG2 antibodies; neither group produced an increase in IgM. Immune sera from cows immunized with conjugates alone increased phagocytosis, which decreased at the end of the study. Sera from cows immunized with conjugates in microspheres increased phagocytosis, which was sustained at the end of the study. Immune sera from both groups decreased bacterial adherence to bovine mammary epithelial cells. These data showed that a single injection of antigen in microspheres produced higher titers and more sustained enhancement of phagocytosis, which could aid in the defense of the cow against S. aureus infections.

Prevention of nonspecific binding of immunoglobulin to Staphylococcus aureus protein A in ELISA assays.

The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.

Prevalence and Distribution of Campylobacter jejuni and Campylobacter coli in Retail Meats.

Nine cooperating laboratories, distributed throughout the United States, determined the interlaboratory reproducibility of a sensitive, selective method for isolation of Campylobacter jejuni and Campylobacter coli from foods, and determined the prevalence and distribution of the organism in retail meats. A double-blind inoculated/recovery experiment demonstrated the ability to detect two cells of C. jejuni and C. coli per g of meat at a rate of 96% among the cooperating laboratories. However, a 7.5% false-positive rate for the presumptive detection of the organism was also reported. Samples of ground beef, beef flank steak, lamb stew meat, broiler chicken, pork sausage (without antimicrobials), and pork chops were selected to assess the presence of campylobacters. Each cooperator purchased five of each of the above samples from the refrigerated case of two retail outlets at quarterly intervals throughout the year. A total of 2,160 retail samples were analyzed for the presence of C. jejuni and C. coli . Results indicated that about 30% of the 360 chickens sampled yielded the organism. Analysis of 1,800 red meat products yielded campylobacters at a rate of about 5.1%. Pork samples yielded C. coli and other meats yielded C. jejuni . Higher numbers of isolations from the red meats were made during June and September (8.6%) as compared with December and March (4.2%). These results provide a baseline, for the prevalence of campylobacters in these selected foods, and also support epidemiologic data associating mishandled foods of animal origin as a potential vehicle in human gastroenteritis.

Selected enrichment broths for recovery of Campylobacter jejuni from foods.

We attempted to shorten the required time for enrichment broth culture for the isolation of Campylobacter jejuni. Enrichment broths described by Doyle and Roman and Park and Stankiewicz and one developed during this study were compared for ability to isolate C. jejuni from raw chicken carcasses. Our medium was a modification of that of Doyle and Roman with the addition of filter-sterilized FBP (0.2% ferrous sulfate, 0.025% sodium metabisulfite, 0.05% sodium pyruvate), 0.1% sodium lauryl sulfate, and 0.075% agar. Initially, laboratory strains were employed in the development of this medium. Subsequently, an indigenous load of C. jejuni obtained from chickens was used to compare media. Isolation rate comparisons were as follows: direct plating, 40%; Doyle and Roman broth, 45% at 7 h and 61% at 16 h; Park and Stankiewicz broth, 53% at 7 h and 60% at 16 h; our broth, 48% at 7 h and 50% at 16 h. In addition to having the highest isolation rate, the enrichment broth of Doyle and Roman showed greatest selectivity. Our inoculation method of indigenous bacteria provided a controlled means for comparison of isolation procedures.

Isolation and identification of ropy bacteria in raw milk.

Approximately 4.2% of 4,000 Maryland-Virginia raw milk tanker samples developed ropiness when incubated at 10 degrees C. Of the 56 bacterial isolates 30 were identified by species. Klebsiella oxytoca and Pseudomonas aeruginosa were isolated most frequently. Other ropy isolates were identified as Pseudomonas spp., Chromobacterium, Flavobacterium multivorum, presumptive Yersinia pestis, Enterobacter agglomerans, Klebsiella pneumoniae, and Pasteurella-Actinobacter spp. Six of the Klebsiella oxytoca isolates were mesophilic (optimum temperatures of 32.0 to 37.8 degrees C) with two isolates having psychrotrophic tendencies (optimum temperature of 26.8 degrees C). All Pseudomonas aeruginosa isolates appeared to be psychrotropic in their temperature requirements (optimum temperature of 23.0 to 31.0 degrees C). Klebsiella oxytoca was significant in preliminary development of the ropy condition. All Klebsiella oxytoca isolates developed ropiness within 24 h. The Pseudomonas spp. and Klebsiella pneumoniae isolates required at long as 7 days to develop detectable ropiness at 10 degrees C. A recommended Klebsiella oxytoca differentiation agar is presented as a rapid screening method during outbreaks where Klebsiella oxytoca is the organism of significance.

Selective method for the isolation of Sporolactobacillus from food and environment sources.

A detection procedure was developed in which a modified Lactobacilli MRS medium was used to enrich for Sporolactobacillus from a variety of foods, feed soil and environmental samples. Each sample was rinsed with 50 ml of a modified Lactobacilli MRS broth containing 1.0% (w/v) alpha-methyl glucoside 0.1% (w/v) potassium sorbate, 0.00224% (w/v) bromocresol green indicator, adjusted to pH 5.5 with acetic acid and incubated at 37 degrees C for 7 d under 5% CO2. Volumes of 2 ml from each sample were heat shocked at 80 degrees C for 5 min and 0.1 ml spread onto plates of Lactobacilli MRS agar (Difco), pH 5.5 and APT agar (BBL), pH 5.5. Plates were incubated for 5 d and suspect colonies were tested for catalase production, benzidine reaction, nitrate reduction, motility and Gram stain reaction. This method was demonstrated to be selective for Sporolactobacillus.

Microbiology of ultrahigh temperature milk.

Ultrahigh temperature thermal processing can sterilize milk. Potential energy savings of a commercially sterile, aseptically packaged, nonrefrigerated milk provide the incentive for eventual introduction of the product in the United States. Attention should be directed to raw milk quality, processing parameters, quality control tests, and thermal inactivation data for spores in the ultrahigh temperature range.

Alternative procedure to screening a large number of plates for a low number of mutants.

An apparatus is described in which tygon tubing replaces the use of petri dishes in the screening of a large volume of agar for an expected low number of mutants.