Efficacy of cetylpyridinium chloride in immersion treatment for reducing populations of pathogenic bacteria on fresh-cut vegetables.

The efficacy of cetylpyridinium chloride (CPC) immersion to reduce the numbers of three pathogenic bacteria (Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7) on three different fresh-cut vegetables (broccoli, cauliflower, and radishes) was studied. The fresh-cut vegetables were inoculated with one of the three pathogenic bacteria at a concentration of 10(5) CFU/ml for 1 h at room temperature and then treated with 0.1 or 0.5% CPC immersion for 1 min. Both Salmonella Typhimurium and E. coli O157:H7 plates were incubated from 48 to 72 h at 37 degrees C, and L. monocytogenes plates were incubated from 72 to 96 h before being counted. The results of three experiments showed that for the average of the three vegetables treated with 0.1 and 0.5% CPC, L. monocytogenes was reduced by 2.85 and 3.70 log CFU/g, Salmonella Typhimurium by 2.37 and 3.15 log CFU/g, and E. coli O157:H7 by 1.01 and 1.56 log CFU/g, respectively, in comparison with the vegetables treated with water only. The 0.5% CPC treatment was significantly different (P < 0.05) from the 0.1% CPC treatment on reduction of L. monocytogenes, Salmonella Typhimurium, and E. coli O157:H7. The CPC residual on the treated vegetables and their washing solutions were evaluated by using high-performance liquid chromatography.

Multiplex PCR detection of Campylobacter jejuni and Arcobacter butzleri in food products.

Arcobacter is a recently described species, previously considered part of the Campylobacter family. A sensitive assay such as that provided by PCR could help to distinguish the closely related Arcobacter from Campylobacter. A PCR method to specifically detect both Campylobacter jejuni and Arcobacter butzleri in the same reaction tube has been developed. C. jejuni and A. butzleri were inoculated into a range of dairy products, raw and ready-to-eat foods. The presence of these two organisms was detected in these test foods by the multiplex PCR assay. A product of 159 bp was apparent when C. jejuni was present, while a 1223 bp product was seen when A. butzleri was present. When both organisms were present, both bands could be detected on the agarose gel. All organisms were confirmed by standard microbiological methods. There was complete agreement between the PCR and standard methods. This PCR assay will allow detection of both organisms within the same PCR tube and can be performed within an 8 h day. The presence of these two human pathogens, which are difficult to distinguish by standard biochemical methods, can now be identified using this PCR assay.

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.

Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain.

A wild type micro-organism producing antibacterial substances has been isolated from a Chinese fermented soybean seasoning and identified as Bacillus subtilis. A crude antibacterial preparation (CABP) was obtained by ammonium sulphate precipitation. Isoelectric focusing assay revealed at least four antimicrobial components in the CABP. However, in SDS-PAGE analysis, only one peptide band displayed antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes. This inhibitory peptide had a molecular weight of approximately 3.4 kDa and a pI value of approximately 4.7. Results of this study suggest that at least one antimicrobial substance produced by this wild type strain of B. subtilis may be a new bacteriocin. Its sensitivity to gastric peptidases and activity against the food-borne pathogens make this bacteriocin potentially useful as an antimicrobial agent in foods.

Polymerase chain reaction for rapid detection of Campylobacter jejuni in artificially contaminated foods.

Campylobacter jejuni was inoculated into a range of raw and ready-to-eat foods. These food samples were used as a test source for detection of this bacterium by the polymerase chain reaction (PCR). Specific detection of Camp. jejuni, as indicated by a PCR product of 159 bp, was possible with all pre-cooked deli-sliced and raw poultry products tested. All vegetables tested were compatible with the PCR assay. Cantaloupe, kiwi and pineapple tested positive while strawberries, watermelon, grapes and apples tested negative. By using a nested PCR assay that yields a single band at 122 bp, positive results were obtained with watermelon and grapes while the apple and strawberries continued to give a negative reaction. These rapid and specific assays for Camp. jejuni are compatible with most foods in insuring the safety of the food product.

Spraying chicken skin with selected chemicals to reduce attached Salmonella typhimurium.

Aqueous solutions of 5% and 10% trisodium phosphate (TSP), 0.1% and 0.5% cetylpyridinium chloride (CPC), 1% and 2% lactic acid (LA), and 0.1% and 0.5% grapefruit seed extract (DF-100) were evaluated in prechill spraying for reducing Salmonella typhimurium attached on chicken skins. Chicken skins were inoculated with S. typhimurium and then sprayed with the selected chemical solutions for 30 sec at 206 kPa and 20 degrees C. After chemical spraying, the skins were rinsed by spraying tap water for 30 sec. Each skin was stomached in buffered peptone water (BPW) for 1 min. The stomaching water was then diluted serially, inoculated onto both xylose lysine tergitol (XLT4) agar and Aerobic Plate Count (APC) Petrifilm, and incubated for 24 hr at 37 degrees C. The results showed that the numbers of Salmonella on the chicken skins after the chemical spraying were significantly lower than those without spray (P < 0.05). The CPC reduced Salmonella by 1.5 to 1.9 log10. TSP resulted in a 2.1 to 2.2 log10 reduction of Salmonella and DF-100 produced a 1.6 to 1.8 log10 reduction of Salmonella. The LA had a number of Salmonella with a 2.2 log10 reduction. The 0.5% CPC resulted a significantly greater reduction in Salmonella than 0.1% CPC. There were no significant differences in Salmonella reduction between different concentrations of the other three chemicals.

Bacterial penetration into eggs washed with various chemicals and stored at different temperatures and times.

Eggs were washed with one of three commercial egg-washing chemicals, including a quaternary ammonium compound (QAC, pH 7.5), sodium carbonate (Na2CO3, pH 12), and sodium hypochlorite (NaOCl, 100 ppm, pH 7.5). One hundred fifty intact-shell eggs were washed at 43.3 degrees C with each of three chemicals. A control group was washed with tap water (H2O, pH 7.0). The washed eggs then were inoculated by immersion for 3 min into an aqueous suspension of Salmonella enteritidis at 10(4) colony-forming units/ml and dried for 30 min. The washed and inoculated eggs were stored at 4 degrees C or 23 degrees C, and bacterial penetration was checked at 0-, 7-, 14-, and 21-day intervals. The effects of egg-washing chemicals on microstructural changes of eggshell and postwash inoculation were examined using electron microscopy and conventional culture methods. Fifteen eggs were used in each sample. The results of microbial tests showed that both QAC and sodium hypochlorite treatments reduced bacterial penetration (less than 3.4% and 6.7%, respectively, on day 1 and 16.7% on day 21). The sodium carbonate treatment facilitated bacterial penetration during egg storage (less than 30% on day 1 and 76.7% on day 21). The eggs washed with tap water had a bacterial penetration rate of less than 6.7% on day 1 and 20% on day 21. As the storage intervals increased to 21 days, the bacterial penetration rate increased. Different storage temperatures (4 degrees C and 23 degrees C) did not cause a significant difference in bacterial penetration rates within 21-day interval. The results of electron microscopy showed that QAC and sodium hypochlorite at 100 ppm resulted in microbiologically clean eggs and did not destroy eggshell surfaces, which protected the eggs against future bacterial recontamination. The alkaline sodium carbonate produced visually clean eggs but altered the eggshell surface, which allowed bacterial recontamination.

Rapid PCR with nested primers for direct detection of Campylobacter jejuni in chicken washes.

Rapid detection of Campylobacter jejuni by PCR directly from foods, without prior growth steps, would be beneficial for the poultry industry. We have previously reported a PCR assay that allows detection of this bacterium after 48 h growth on Campy cefex agar. We have now developed a more rapid nested PCR assay that specifically detects C. jejuni in chicken washes that have not undergone any lengthy growth steps prior to PCR. For the nested reaction, an external set of primers, C-1 and C-4, are used for 24 cycles. At this time, 1 microl of the PCR product is removed and added to a second reaction. The second PCR assay is run with C-1 and an internal primer, C-2, for 24 cycles. A single band on a 4% NuSieve agarose gel at 122 bp was apparent with C. jejuni cells at a sensitivity of 10(2) cfu ml-1. With this method chicken carcasses can be washed and C. jejuni identified all within 1 day. We detected C. jejuni in approximately 80% of four groups of chickens using this method. The identifications have been confirmed by standard microbiological techniques.

Cetylpyridinium chloride (CPC) treatment on poultry skin to reduce attached Salmonella.

Cetylpyridinium chloride (1-hexadecylpyridinium chloride, CPC) was evaluated for its effectiveness in removing or killing salmonellae attached to poultry skin. Two different treatment methods were used: (i) spraying 0.1% CPC solution at 15 degrees C or 50 degrees C against inoculated skin surface for I min at 138 kPa, and (ii) immersing inoculated skin surface in 0.1% CPC solution at room temperature for either 1 min, 1 min plus 2 min holding without CPC, or 3 min. After rinsing, cells on the skins were enumerated by conventional plating as well as direct counting from scanning electron microscopy (SEM). Compared with controls, CPC spraying reduced the numbers of salmonellae by 0.9 to 1.7 log units (87 to 98%) assayed by the plating method (P < 0.05). SEM gave results similar to plating. Generally 50 degrees C CPC spraying showed greater reduction than 15 degrees C CPC spraying; however, the differences were not always significant. Water spraying at either temperature did not show any reduction compared to nonsprayed skins. In the immersion test, significant differences also were noticed among the control and the three other CPC-immersed groups (P < 0.05) as assayed by plating, ranging from 1.0 to 1.6 log units, which were similar to the CPC spraying results. However, no difference was noticed among the three CPC-immersed groups. Direct counting from SEM was not a suitable method for recovering cells in CPC immersion tests because dead cells were still attached to the skin while retaining their intact morphology. On the basis of the amount of CPC used, immersion appears to be more cost-effective than spraying CPC on poultry skin.

Evaluation of a PCR based assay for specific detection of Campylobacter jejuni in chicken washes.

An assay for Campylobacter jejuni based on the polymerase chain reaction was developed in our laboratory and shown to be a sensitive and specific method to identify this bacterium in pure culture. This assay was evaluated as a method to rapidly detect C. jejuni attached to chicken carcasses. Chicken carcasses were sampled for PCR using three methods including pre-enrichment of the washes, direct plating of the washes and differential centrifugation of the washes prior to testing. It was found that plating the wash solutions on Campy Cefex plates prior to performing PCR was the most specific and reliable of the three treatment methods evaluated.

Development and evaluation of the polymerase chain reaction method for diagnosis of Mycoplasma gallisepticum infection in chickens.

A polymerase chain reaction (PCR) method specific for Mycoplasma gallisepticum (MG) was evaluated. The PCR method was found to detect as few as two colour changing units (CCU) of MG and did not give false positive reactions with other avian mycoplasmas. In chickens inoculated with either MG or Mycoplasma synoviae (MS), the PCR method was found to closely correlate with MG culture reisolation methods in chicken intranasally inoculated with MG. All chickens inoculated with MS tested negative using the MG PCR method.

Development of PCR method specific for Marek's disease virus.

A rapid polymerase chain reaction (PCR) assay specific for Marek's Disease Virus (MDV) was developed. This assay was able to detect MDV in inoculated chick kidney cells at dilutions of 10(-5). Negative PCR results were obtained using uninoculated chick cells, Marek's Disease Vaccine (SB), Herpesvirus of Turkeys (HVT) and Fowl Laryngotracheitis Vaccine (LT). Bursae, feathers and kidneys from MDV infected chickens were positive in the PCR assay. The same tissues from normal chickens were negative. This method required only 0.5 h for sample preparatory, 3 h for PCR application and 1 h for electrophoresis. Internal probe hybridization confirmed that the PCR products are from MDV, but this hybridization will not be necessary for future MDV detection.

Detection of antibody to Bordetella avium using a particle concentration fluorescence immunoassay (PCFIA).

A fluorescent assay for detection of antibody to Bordetella avium was developed and evaluated against current serological techniques. Bordetella antigen was coated on submicron polystyrene beads and used in an antibody sandwich technique with commercially available goat anti-turkey fluorescein-labeled IgG. Positive control sera were taken from turkeys from which B. avium was isolated. Negative control sera were from turkeys from which no B. avium was isolated and which tested negative by agglutination and enzyme immunoassay. A commercial fluorescence concentration analyzer was used to quantitate the epifluorescence in a special 96-well plate designed to minimize background fluorescence. The particle concentration fluorescence immunoassay was found to be as sensitive as the enzyme immunoassay, with good reproducibility. It required less time and smaller quantities of sample and reagents.

Comparison and modification of rRNA sequencing methods.

In this study, modification of two methods of RNA sequencing resulted in more definitive sequencing bands. In one method of sequencing, the bands of lane A and lane G sometimes were not clear. Modifications of this method by changing the concentrations of ddATP and ddGTP resulted in the bands of lane A and lane G becoming more readable. Although a second sequencing method was found to have clearer bands than the first method, and the bases immediately downstream from the primer binding site could be read by using r-32P-labeled primer, the bands on the top of lane A still were not clear. Modifications of this second method by changing the ddATP/dATP ratio resulted in the bands of lane A becoming much clearer.

Determination of infectious bursal disease virus titration and neutralization endpoints using fluorogenic staining.

An automated method for determining infectious bursal disease virus (IBDV) titration and neutralization endpoints is described. The method employs the fluorogenic ester carboxyfluorescein-diacetate (CFDA) to stain cell monolayers in 96-well plates and a fluorescence-concentration analyzer. Titration results are compared with immunofluorescence and plaque assay titers. Virus-neutralization endpoint determination is objective, and the endpoints of replicate tests were equivalent or within one dilution of variability. Tests can be automatically screened as any percentage of a positive control or any standard deviation from a negative control.

Effect of Electrical Stimulation on Bacterial Contamination of Chicken Legs.

Electrical stimulation was investigated as a method to eliminate or reduce the number of Salmonella typhimurium attached to chicken legs. Salmonellae-inoculated legs were attached to either cathode or anode or placed in an electrical field. In addition, the effect of electrical stimulation on various bacteria in an electrolyte solution was studied in order to determine the feasibility of using this method to prevent cross-contamination of poultry carcasses during processing. Stimulation was accomplished using a square wave with an amplitude of 8.5 to 14.5 volts, a frequency of 0.33 Hz or 100 KHz, and a duty cycle of 67%. Results indicate that electrical stimulation is effective in killing bacteria in solution and in reducing the number of salmonellae attached to chicken legs when legs are attached to anodes. Slight meat damage did occur, however, when chicken legs were connected to either anode or cathode1.z.

The evaluation of chicken spermatozoa using fluorescent staining in a 96-well format.

An efficient method for the evaluation of spermatozoa using the fluorescent stains carboxyfluorescein diacetate and propidium iodide and an automated fluorescence concentration analyzer was adapted for chicken semen. Arbitrary fluorescence units representing either live or dead spermatozoa were strongly correlated with percentage of added dead spermatozoa and with the direct fluorescent microscope counts of live, dead, and damaged cells.

Efficacy in turkeys of spray vaccination with a temperature-sensitive mutant of Bordetella avium (Art Vax).

Broad-breasted white turkeys were vaccinated with a temperature-sensitive mutant of Bordetella avium (Art Vax) at 2 and 15 days of age and challenged at 22 days of age by contact with infected birds. Necropsy was performed at 35 days of age. Two vaccination protocols (eyedrop/oral and spray cabinet/spray bottle) and two challenge isolates (Arkansas 105 and North Carolina [NC] isolates) were used. Neither the spray nor the eyedrop/oral methods of vaccination prevented infection of the anterior trachea with either of the virulent challenge strains. The spray and eyedrop/oral methods of vaccination were equally effective in reducing the severity of gross lesions in the trachea. The vaccine reduced the severity of gross lesions in the tracheas of turkeys challenged with the NC isolate to a level approximately equal to that observed in unchallenged vaccinated controls, but the vaccine only moderately reduced the severity of lesions in birds challenged with the 105 isolate.

Efficacy of thiabendazole (Mertect 340-F) in controlling mold in turkey confinement housing.

Mertect 340-F (thiabendazole) was evaluated as a mold inhibitor for litter in turkey confinement housing using green oak wood shavings heavily contaminated with mold spores. One day before the addition of shavings, two environmentally controlled rooms were sprayed with Mertect 340-F at a rate of 51.0 ml in 11.37 liters of water (16 fl. oz./6000 sq. ft.). After the litter was added, it was treated with Mertect 340-F at a rate of 33.0 ml in 75.8 liters of water (32 fl. oz./6000 sq. ft.). The litter was treated again at 6 and 12 weeks. Mertect 340-F-treated litter had significantly lower mold spore counts than water-treated litter. Turkeys reared on Mertect 340-F-treated litter displayed no adverse effects. No obvious differences between groups were noted post mortem. Microscopically, however, lungs from turkeys on water-treated litter had a higher incidence of granulomas and mycelia than lungs from birds on Mertect 340-F-treated litter.

The efficacy of a temperature-sensitive mutant vaccine against Northwest Arkansas isolates of Alcaligenes faecalis.

The efficacy of a commercially produced temperature-sensitive mutant Alcaligenes faecalis vaccine was evaluated in turkeys contact-challenged with one of three strains of A. faecalis. In the vaccinated control group, the vaccine strain of A. faecalis colonized the nasal turbinates but not the trachea, caused no clinical signs of turkey coryza, and induced humoral antibodies. In the vaccinated challenged groups, the vaccine reduced both the severity of lesions and the number of birds exhibiting lesions compared with unvaccinated challenged groups, but it did not prevent colonization of challenge strains of A. faecalis.