Brief Summary of Minutes of Annual Meeting

Price, Toro, McKee and Hoerr (Auburn U., AB) explored whether Salmonella-targeted bacteriophages treatment can be integrated with competitive inhibition and vaccination to further reduce colonization of chickens. Tap water caused major reductions in titers of three of five bacteriophage cocktails after 48 h exposure; addition of powdered milk to tap water prevented reductions. Four of five cocktail bacteriophages survived transit through chicks; however, findings indicate need for more work on treatment timing and experimental endpoint. In an experimental treatment/challenge model, the combination of bacteriophage treatment plus competitive exclusion, and the bacteriophage treatment alone, significantly reduced cecal counts of Salmonella compared to untreated controls. Quantitative PCR methods for enumerating Salmonella in cecal samples showed good correlation with traditional colony-counting enumeration methods. Oyarzabal (Auburn U., AB) directly enumerated Campylobacter species from poultry feces on mCCDA, Campy-Cefex (mCC) agar, Campy-Line (CL) and Campy BAP (CB) agar plates. Both mCCDA and mCC are suitable plates for the isolation of Campylobacter from fecal or cecal samples from broiler. CL is too selective and yields a statistically lower number of Campylobacter spp. The 96-microwell plate dilution method with mCC appears to be similar to the counts determine by serial dilution and spread plating. However, these results require a further validation with a larger number of samples. Isolates from different plate media have similar PFGE patterns, suggesting that any differences found in the PFGE profiles may be due to the colonization of the flock with multiple isolates and is not a consequence of the antimicrobial composition of the media. Johnson (University of Arkansas, AR) addressed starvation survival response of L. monocytogenes by detailing viable counts in absence of added carbon and nitrogen sources. Sofos, Belk, and Scanga (Colorado State University, CO) assessed effectiveness of single and multiple preharvest intervention strategies on prevalence of Escherichia coli O157 on/in cattle before transport to harvest. Treatments included Control (CT; No treatment), Bovamine (Bov; a Lactobacillus acidophilus NPC-747 dietary product), NEOMIX (Neo; feeding of neomycin sulfate), E. coli O157:H7 bacterin vaccine (Vac), and all combinations of single treatments. Preharvest mitigation strategies used singly or in combination may be effective in reducing prevalence of E. coli O157 in market-ready feedlot cattle. In a second study they evaluated the behavior of Escherichia coli O157:H7 on beef during aerobic storage, following storage in vacuum packages. The practice of storing fresh beef in vacuum packages for subsequent storage in aerobic retail packing does not promote growth of E. coli O157:H7. The residual antimicrobial effect of hot water followed by lactic acid on both E. coli O157:H7 and natural flora was confirmed. Combined use of temperature control and decontamination with hot water and lactic acid may increase the shelf-life of fresh beef. Sofos et al. characterized growth rates and heat and acid death rates for 25 Listeria monocytogenes strains of various serotypes and sources, including clinical and food isolates associated with outbreaks. Extensive variation was observed among growth and stress resistance characteristics of L. monocytogenes strains. Serotype appeared to play a significant role only with respect to the heat resistance of the organism, with 4b isolates as a group exhibiting lower thermal resistance than isolates representing all other serotypes combined. Outbreak-related isolates of serotype 4b demonstrated higher acid resistance compared to the rest of the strains belonging to this serotype. Selecting Scott A as the sole strain to be used in low temperature or acid challenge studies, due to its clinical origin or the strong epidemiologic association of serotype 4b with human listeriosis, may not assure the assessment of the worst case scenario from a food safety perspective. The antilisterial effect of post-processing antimicrobial treatments on commercially manufactured frankfurters formulated with and without 1.5% potassium lactate-0.05% sodium diacetate was evaluated by Sofos et al. Results indicated that dipping frankfurters in post-processing antimicrobial treatments including Nisaplin resulted in 2.4 to >3.8 log CFU/cm2 reductions of initial L. monocytogenes populations. Furthermore, by applying post-processing antimicrobial dipping treatments to frankfurters formulated with potassium lactate-sodium diacetate, inhibitory and listericidal effects were obtained during storage at 10°C. This approach may be refined and validated for commercial application. Sofos et al evaluated post-processing chemical dipping solutions for their antilisterial effects on commercial smoked sausage formulated with or without 1.5% potassium lactate plus 0.05% sodium diacetate, and contaminated (approximately 3-4 log cfu/cm2) with 10-strain composite Listeria monocytogenes inocula prepared under various conditions. Dipping of sausage formulated with potassium lactate-sodium diacetate in Nisaplin followed by AA or LA would satisfy Alternative 1 of the interim final rule, due to substantial reductions of initial contamination levels and subsequent inhibition of growth during storage. Processors, however, need to develop and validate their own formulations to fit their product specifications and expectations. Sofos et al modeled the effect of drying temperature (52, 57 and 63°C) and pre-drying treatments on inactivation of Listeria monocytogenes on beef jerky. Results of this study confirmed that the acidified pre-drying treatment increased pathogen inactivation during drying, regardless of drying temperature and the models developed appeared to be a promising means of predicting the responses of this bacterium during dehydration of beef jerky. The use of the mathematical models to predict the drying time or survival of L. monocytogenes can be useful in beef jerky processing. Janes (Louisiana State University, LA) developed a direct colony immunoblot (DCI) assay for enumeration of Vibrio vulnificus. Vibrio alginolyticus (Va), V. cholerae (Vc), V. parahaemolyticus (Vp), V. damsela (Vd), V. mimicus (Vm), V. fluvialis (Vf) and several Vv strains, were plated onto Vv agar (VVA), Thiosulfate Citrate Bile Salts sucrose (TCBS) agar, and modified Cellobiose Polymyxin Colistin (mCPC) agar and incubated. Colonies were transferred to polyvinylidene fluoride membranes and treated with anti-H Vv antibodies. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG and color was developed. Our results showed that the DCI from the inoculated VVA plates had less non-specific color development compared to TCBS and m-CPC agar plates for Va and Vm. Vc, Vp. Vf and Vd were not detected by the DCI. At 9 log CFU/ml all Vv strains were detected by the DCI whereas Vp was not detected. In a Vv and Vp mixed culture containing 7.9 log CFU/ml of each bacteria the DCI only detected Vv. Total time duration for enumeration of Vv by the DCI was 31/2 h. Janes conducted a second study to determine the concentration of Cetylpyridinium Chloride (CPC) that effectively reduces Listeria monocytogenes Lm on the surface of white Gulf shrimp. Treatment of shrimp with 1.0% CPC (no water rinse) reduced L. monocytogenes counts by >2 log CFU/g on day 0. The largest reduction (3 logs) occurred for shell-on cook shrimp,. The shelled raw, shelled cooked and peeled raw shrimps treated with 1.0% CPC and a water rinse on day 0 had reductions of 1.05, 2.19 and 0.8 log CFU/g, respectively. Shell-on raw shrimp treated with 0.05, 0.4 and 1.0 % CPC (no water rinse) had reductions on day 3 of 1.26, 1.89, and 2.06 log CFU/g, respectively. By day 9 the shell-on cooked shrimp at 1.0% CPC with or without a water rinse had reduced Lm counts by 3 log CFU/g. Ryser (Michigan State University, MI) is addressing : risk and communication regarding Listeria monocytogenes contamination of deli meats and slicers. Accomplishments include: (1) acquisition of four model 2612 Hobart slicers (including two types of blades), as well as two Berkel slicers that was redesigned for enhanced cleanability; (2) set-up of the three Hobart slicers and one Berkel slicer at a local delicatessen for one year of use and monthly sampling; (3) identification of the product contact areas on the slicers for sampling; (4) optimization of the blade inoculation method using surface-inoculated ham; and (5) collection of initial data on the potential and extent for sequential transfer of L. monocytogenes from the slicer blade to ham and other areas of the slicer during slicing of the product at two different temperatures. They are now focusing on transfer of Listeria from the artificially contaminated G+B German blade to ham with different moisture and fat contents stored at 4 and 22Ú C; work with the remaining two blades for the Hobart slicer and the Berkel slicer will follow. 56 environmental samples per month from the retail deli will be examined for Listeria spp. They will continue to work on the development of mathematical models and risk communication. For the mathematical model, a compartmental model of L. monocytogenes cross-contamination among deli products, deli contact surfaces (e.g., different parts of a slicer), employees hands covered (gloved and ungloved) , and the environment with which hands may come into contact, defined as the immediate environment, will be developed. This model will be used to consider which parameters have the most profound impact upon the prevalence and level of L. monocytogenes contamination occurring during delicatessen slicing. Ryser is also investigating a risk-based approach to determine best consume by dates to control exposure of Listeria monocytogenes in deli meats. Eight L. monocytogenes strains of different pulsed-field gel electrophoresis (PFGE) types from RTE meat products are being used. Growth of L. monocytogenes is being assessed in five different brands each of uncured turkey, cured turkey, ham, and roast beef. These products were selected based on differences in pH, moisture, aw, salt, fat content, as well as presence/absence of nitrite, Listeria growth inhibitors, lactate and diacetate. Whole chubs of cured turkey, uncured turkey, ham and roast beef are purchased from retail delis in major supermarkets on the day of delivery. One chub is being inoculated on the day of delivery. The remaining chubs are held at 4Ú C for inoculation at the midpoint and at the last allowable date for sale. All experiments are being conducted in triplicate using three different lot numbers of the same product from the same manufacturer. Marshall (Mississippi State University, MS) investigated a rapid chill intervention process to control Vibrio vulnificus in raw Gulf Coast oysters. Oysters were harvested during the warmer months (April-September) that were expected to have high numbers of naturally occurring V. vulnificus were subjected to a rapid chill process (10°C) for two weeks using a recirculating water tank containing 12, 16, and 20 ppt salt seawater. Control oysters were placed in a similar tank at 23°C. After two weeks, aerobic plate counts, V. vulnificus counts, and MIDI GC-FAME bacterial profiles were determined. In addition, oysters were removed from the tanks and stored at 4°C for two weeks to simulate retail shelf life. After this storage period, bacterial profiles were again performed. Results demonstrated that the chill process reduced V. vulnificus counts by 2-3 logs but counts were not below FDA target levels of less than 30 MPN/g. Salinity had little effect on counts. Bacterial profiles predominantly shifted from vibrios to psychrotrophic spoilage bacteria such as Pseudomonas and related genera. The oral hygiene antimicrobial cetylpyridinium chloride was tested for its activity against Salmonella Typhimurium attached to ready-to-eat shrimp. Levels up to 1% CPC reduced Salmonella counts by less than 1 log. LeJeune (Ohio State University, OH) reported the need to validate probiotic preparations individually and identified birds as a potential vector for E. coli O257 dissemination among dairy farms. E. coli O157 prevalence in cattle was benchmarked against cattle in a country where E. coli O157 disease in humans is rare. Prevalence rate in cattle may impact human disease incidence. The sensitivity of IMS methods for isolation of E. coli O157 from bovine fecal samples containing less than 1000 CFU/ml was determined to be poor (<30%). Removal of oxytetracyline from conventional swine production has miminal impact on the prevalence of oxytetracycline resistant fecal E. coli in pigs. Dawson and Jiang (CLEMSON UNIVERSITY, SC) are assessing the prevalence of vancomycin resistant enterococci in rendered animal byproducts and during composting. About 200 suspected enterococci isolates were first confirmed as enterococci through biochemical tests and real-time PCR. Enterococci were examined for resistance to vancomycin and for minimum inhibitory concentration (MIC). 76 % of the isolates belong to enterococci genus based on PYR results and approximately 92% based on the other tests. Sixteen enterococci were resistant to vancomycin (VRE). Seventeen showed additional resistance to at least five other antibiotics. Rendered animal by-products contain multiple drug resistant VRE. The fate of VRE during active composting was assessed and antibiotic resistance of VRE isolates was further characterized. Duplicate dairy compost heaps were constructed on an outdoor, fenced site. Enterococci populations inside the composting heaps decreased rapidly from ca.7.0 to 5.0 log cfu/g within 3 days of active composting, followed by a relative slow reduction of cell numbers during a 120-day composting. Except at bottom of the compost heap, neither enterococci nor VRE could be detected inside composting heaps at 60 days. Vancomycin-resistant enterococci accounted for ca. 2-18% of total enterococci populations in initial dairy compost, and were inactivated inside the compost heaps at similar rates as enterococci. However, on the surface, like enterococci, VRE survived very well with ca. 2 ~ 3.5 logs reduction in cell populations at the end of experiment. Isolates of those VRE were further analyzed for the resistance to vancomycin and other antibiotics. Most VRE are resistant to multiple antibiotics, and over 93% of VRE had minimal inhibitory concentration (MIC) e128 µg vancomycin/ml. Our results revealed that a large number of vancomycin-resistant enterococci cells survived active composting on the surface or inside the heaps when temperature was low, and the existence of these VRE could serve as the potential source for disseminating vancomycin-resistance among microbes on farm. Dawson and Jiang studied antimicrobial activity of lysozyme absorbed on immunonanoparticles against L. monocytogenes Scott A. Polystyrene nanoparticles with active carboxyl groups were conjugated with anti-L. monocytogenes through covalent bounding. Immunonanoparticles were then coated with lysozyme by direct adsorption. The antimicrobial activity of lysozyme adsorbed on immunonanoparticles was compared to that of free lysozyme in phosphate buffered saline. Amount of anti-L. monocytogenes and lysozyme and adsorption times were optimized for most efficient inhibition. Nanoparticles were conjugated with 0.04 µg anti-L. monocytogenes per ml, and at that concentration, immunonanoparticles demonstrated enhanced antimicrobial activities of lysozyme. Lysozyme (35 µg/ml) adsorbed on immunonanoparticles reduced the L. monocytogenes Scott A population from 5.2 log CFU/ml to below the detection limit (<1 log CFU/ml) in 3 h. However, when free lysozyme (500 µg/ml) was used, ca. 2.2 log CFU/ml of the L. monocytogenes Scott A remained culturable after 5 h treatment. Our study revealed that the use of immunonanoparticles coated with lysozyme is a more efficient method than direct addition of lysozyme in inhibiting L. monocytogenes. Dawson and Jiang assessed the effects of thickness on in-package pasteurization against Listeria monocytogenes on ready-to-eat meats. The surface heating rate (³) and final surface temperature (±) during in-package pasteurization were determined for different thickness levels of two types of bologna having different (13% and 18%) fat content. Three different thickness levels (4, 12, and 20 mm) corresponding to 1, 3, and 5 slices of bologna were each vacuum-packaged separately in clear polymer pouches after placing thermocouples on the surface. Refrigerated samples were immersed into a water bath set to one of four pre-determined temperatures (60, 70, 80, and 90° C) and time and temperature data were recorded for 10 min. Surface- heating rate was fastest in the thinnest (4mm) and slowest in the thickest (20mm) samples for all four pasteurization temperatures. Surface- heating rate was slower in bologna with higher fat content compared to lower fat bologna. Final surface temperature attained after 3 min was lower with increased thickness levels for all pasteurization temperatures. Thus meat sample thickness and fat content significantly affect surface heating rate and final surface temperature during in-package pasteurization of bologna. Dawson and Jiang investigated efficacy of in-package pasteurization combined with pre-surface application of nisin and/or lysozyme to reduce and prevent the growth of Listeria monocytogenes on bologna. Sterile bologna slices were treated with 0.1 ml (500 AU) of nisin (2 mg/ml = 5000 AU/ml), 0.1 ml (8 AU) of lysozyme (10 mg/ml = 80 AU/ml), or 0.1 ml of nisin + lysozyme (250 AU of nisin + 4 AU of lysozyme) solutions. These samples were inoculated with 0.1 ml of a 9 log10 cfu/ml culture then packaged and subjected to 65 C for 32 seconds (after a 60 s come-up time). This heat treatment was calculated to attain a 3-log reduction. Bologna was sampled immediately and weekly through 12 weeks of refrigerated storage for L. monocytogenes population. The combination of heat with nisin + lysozyme or nisin alone treatments significantly reduced the time required to reduce L. monocytogenes populations compared to heat combined with no antimicrobial or lysozyme. The rate of log reduction at each of the three temperatures did not fit a linear model thus a Weibull model was used to fit data at 65 and 62.5OC while a log-logistic model was used to best describe the log reduction vs. time relationship. Grants Received: Control of Salmonella in Infected Chickens by Combining Application of Bacteriophages, Competitive Exclusion, and Maternal Immunity. USDA NRICGP. PI: Price; Co-I: Toro, McKee, Hoerr. 9/15/06  9/14/09. $298,271. Novel Approaches for Reducing Shedding of Salmonella from Chickens. Animal Health and Disease Research Program, College of Veterinary Medicine, Auburn University. PI: Price; Co-I: Toro. 10/1/05-9/30/07. $36,319. Monoclonal antibody probes for C. jejuni and differential enumeration of Campy from poultry Nannapaneni, R. et al:. CSREES/USDA/Food Safety Consortium. M. G. Johnson. Persistence, and virulence of starved L. monocytogenes. CSREES/USDA/Food Safety Consortium Nannapaneni, R. et al:. M. G. Johnson. Ciprofloxacin resistance and virulence of Campy from poultry. Nannapaneni, R. et al: CSREES/USDA/Food Safety Consortium. M. G. Johnson. USDA/CSREES/NIFSI. AGREEMENT 2004-51110-02160 9/15/2004-9/14/2007. John N. Sofos, PI. . USDA/CSREES/NIFSI.AGREEMENT 2005-51110-03278; 9/15/2005-9/14/2009. John N. Sofos, P.I. USDA/CSREES/NIFSI. AGREEMENT 2006; 8/15/2006-8/14/2009. John N. Sofos, PI. USDA/CSREES/NIFSI. AGREEMENT 2004-51110-02160 9/15/2004-9/14/2007. John N. Sofos, PI. . USDA/CSREES/NIFSI.AGREEMENT 2005-51110-03278; 9/15/2005-9/14/2009. John N. Sofos, P.I. USDA/CSREES/NIFSI. AGREEMENT 2006; 8/15/2006-8/14/2009. John N. Sofos, PI. Microbial Drug Resistance in Vibrio spp. from Oysters. USDA Aquaculture Special Grant, September 2006/October 2007, Funded for $24,000. Lead PI  Beilei Ge, Co  PIs Marlene Janes, Witoon Prinyawiwatkul. Determining the Total Aerobic counts, coliform and E. coli counts, and Yeast and Mold counts for mash samples flooded by Hurricane Rita Cooperative Research Agreement, McIlhenny, October 2005, Funded for $10,000. Lead PI  Marlene Janes. Control of Listeria monocytogenes in crawfish and shrimp processing facilities using copper, copper-based alloys or coatings containing copper ions,USDA Aquaculture Special Grants, September 2005/September 2006, Funded for $25,460. Lead PI Marlene Janes, Co  PIs Jon Bell. Detection of Vibrio vulnificus by direct colony immunoblot,Louisiana Sea Grant College Program, , June 2005/July 2007, Funded for $94,537. Lead PI  Marlene Janes, Co  PIs Janet Simonson and Jon Bell. Ecology and control of pathogenic strains of Vibrio vulnificus and Vibrio parahaemolyticus in U.S. Gulf Coast oysters USDA CSREES NRI CGP, October 2004/September 2007, Funded for $415,504. Lead PI  Lee-Ann Jaykus, Co  PIs Andy De Paola, Marlene Janes, Jon Bell, and John Supan. A risk-based approach to determine best consume by dates to control exposure to L. monocytogenes in deli meats. CSREES/USDA. Todd, E.C.D., E.T. Ryser, L.A. Jaykus. $599,999. . L. monocytogenes contamination of deli meat slicers  risk and communication. NAFSS/USDA Todd, E.C.D., and E.T. Ryser. $225,000. Screening for potential non-pathogenic surrogates for Salmonella enteritidis PT-30 during moist-air convection heating of almonds. Almond Board of California. Marks, B.P., A. Orta-Ramirez, E.T. Ryser, K.D. Dolan. $32,478. Mechanisms of E. coli O157:H7 attachment to and transfer between meat and equipment surfaces. National Cattlemens Beef Association. Ryser, E.T., A. Orta-Ramirez, B.P. Marks, and A.M. Booren. $105,999. Determining the likelihood that Salmonella develops heat resistance during processing of commercial whole muscle products. American Meat Institute. Marks, B.P., A. Orta-Ramirez, A.M. Booren, and E. T. Ryser. $44,290. Efficacy of two commercial sanitizers against L. monocytogenes E. coli O157:H7, and Salmonella on conveyor belts during normal operation. Ecolab, Inc. Ryser, E.T., and B.P. Marks. $34,500. Efficacy of two commercial sanitizers against L. monocytogenes E. coli O157:H7, and Salmonella on conveyor belts during normal operation. Midwest Advanced Food manufacturing Allience. Ryser, E.T., and B.P. Marks. $50,000. Risk and protection factors for foodborne illnesses in perinatal and infant populations. CSREES #2006-51110-03663. Medeiros, L(PI), LeJeune, Jeffery T; Sofos, J Kendall, P. $1,500,000 Phage encoded transfer of antibiotic resistance genes in Salmonella. Nat Cattlemen's Beef Assn. LeJeune, Jeffery T. $59,000 Biophysical and ecological processes impacting the growth and survival of E. coli O157 on and in vegetables. CSREES. Lejeune, Jeffery T; Lee, K, Miller, S. $537,816 The role of European starlings in the epidemiology of E. coli O157 of dairy cattle. CSREES. Lejeune, Jeffery T. $1,146,635 Genetic profiles of bovine-origin salmonellae. Nat Cattlemen's Beef Assn. Lejeune, Jeffery T. $47,163 Incidence, significance, and control of Listeria monocytogenes in the home environment. USDA/ARS. Medeiros, L (PI) Lejeune, Jeffery T; Scharf, R Sofos, J and Kendall, P. $540,000 Communicating and advancing prudent antibiotic use among dairy farmers. USDA/FSIS. Lejeune, Jeffery T; Doohan, D, Hooker, N and Tucker, M. $28,862 Evaluation of a bacteriophage cocktail to reduce Escherichia coli 0157:H7 shedding in beef cattle. Univ of Wyoming. NCBA Subcontract. Lejeune, Jeffery T. $37,073 Development of Pre-harvest Nutritional Management Strategies to Reduce E. coli O157:H7 and Salmonella spp. Gastrointestinal Survival and Shedding. Nat Cattlemen's Beef Assn. Henery Zerby (PI), LeJeune et al. $84,346.00 Phage encoded dissemination of Shiga toxin genes among bovine E. coli. Nat Cattlemen's Beef Assn. Lejeune, Jeffery T. $89,787.00