Effect of thiram on avian growth plate chondrocytes in culture.

Thiram is a dithiocarbamate pesticide that causes tibial dyschondroplasia (TD), a growth plate defect, in poultry. Deaths of transitional zone chondrocytes appear to interrupt endochondral bone development leading to the broadening of growth plate. The mechanism of action of thiram on chondrocytes is not well understood. Since proteins play major roles in different aspects of cell's metabolism, growth, and survival, the objective of this study was to find whether thiram produces proteomic changes that could impair the development of chondrocytes. The chondrocytes, isolated from proximal tibial growth plates, were cultured with or without a sub-lethal concentration of thiram for 48 hr, and the cell proteins were extracted, and subjected to 2-D gel electrophoresis. The gel images were compared and statistically evaluated using Melanie software to identify differentially expressed protein spots. Of a total of 72 identifiable spots 3 were down-regulated and 2 up-regulated in thiram treated chondrocytes. In-gel trypsin digestion of the protein spots followed by their characterization by matrix-assisted laser desorption ionization-time-of- flight (MALDI-TOF) mass spectrometry identified 25 spots comprising of 23 proteins. Two of 3 down-regulated proteins were identified as a heat shock protein 70 (HSP 70) and a GALE (UDP-galactose-4 epimerase) protein isoform I. The up-regulated proteins were Serpin H1, a protein involved in collagen metabolism and a redox sensor NmrA-like (NMRAL) family domain protein-1. Both GALE and NMRAL proteins are implicated in energy metabolism and redox regulation whereas the HSP 70 protects cells against stress, and implicated in chondrocyte hypertrophy, an important event in endochondral bone formation. The failure of chondrocyte protective mechanisms such as associated with protection against cellular stress and energy metabolism appear to be the likely cause for chondrocyte death induced by thiram.

Efficient separation and sensitive detection of Listeria monocytogenes using an impedance immunosensor based on magnetic nanoparticles, a microfluidic chip, and an interdigitated microelectrode.

Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning, and sometimes death, among immunosuppressed people and abortion among pregnant women. In this study, magnetic nanoparticles with a diameter of 30 nm were functionalized with anti-L. monocytogenes antibodies via biotin-streptavidin bonds to become immunomagnetic nanoparticles (IMNPs) to capture L. monocytogenes in a sample during a 2-h immunoreaction. A magnetic separator was used to collect and hold the IMNPs-L. monocytogenes complex while the supernatants were removed. After the washing step, the nanoparticle-L. monocytogenes complex was separated from the sample and injected into a microfluidic chip. The impedance change caused by L. monocytogenes was measured by an impedance analyzer through the interdigitated microelectrode in the microfluidic chip. For L. monocytogenes in phosphate-buffered saline solution, up to 75% of the cells in the sample could be separated, and as few as three to five cells in the microfluidic chip could be detected, which is equivalent to 10(3) CFU/ml of cells in the original sample. The detection of L. monocytogenes was not interfered with by other major foodborne bacteria, including E. coli O157:H7, E. coli K-12, L. innocua, Salmonella Typhimurium, and Staphylococcus aureus. A linear correlation (R(2) = 0.86) was found between the impedance change and the number of L. monocytogenes in a range of 10(3) to 10(7) CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when the IMNPs-L. monocytogenes complexes existed in mannitol solution. Finally, the immunosensor was evaluated with food sample tests; the results showed that, without preenrichment and labeling, 10(4) and 10(5) CFU/ml L. monocytogenes in lettuce, milk, and ground beef samples could be detected in 3 h.

Caprylic Acid reduces enteric campylobacter colonization in market-aged broiler chickens but does not appear to alter cecal microbial populations.

Campylobacter is a leading cause of foodborne illness in the United States, and epidemiological evidence indicates poultry products to be a significant source of human Campylobacter infections. Caprylic acid, an eight-carbon medium-chain fatty acid, reduces Campylobacter colonization in chickens. How caprylic acid reduces Campylobacter carriage may be related to changes in intestinal microflora. To evaluate this possibility, cecal microbial populations were evaluated with denaturing gradient gel electrophoresis from market-age broiler chickens fed caprylic acid. In the first trial, chicks (n = 40 per trial) were assigned to four treatment groups (n = 10 birds per treatment group): positive controls (Campylobacter, no caprylic acid), with or without a 12-h feed withdrawal before slaughter; and 0.7% caprylic acid supplemented in feed for the last 3 days of the trial, with or without a 12-h feed withdrawal before slaughter. Treatments were similar for trial 2, except caprylic acid was supplemented for the last 7 days of the trial. At age 14 days, chicks were orally challenged with Campylobacter jejuni, and on day 42, ceca were collected for denaturing gradient gel electrophoresis and Campylobacter analysis. Caprylic acid supplemented for 3 or 7 days at 0.7% reduced Campylobacter compared with the positive controls, except for the 7-day treatment with a 12-h feed withdrawal period. Denaturing gradient gel electrophoresis profiles of the cecal content showed very limited differences in microbial populations. The results of this study indicate that caprylic acid's ability to reduce Campylobacter does not appear to be due to changes in cecal microflora.

Trisodium Phosphate and Cetylpyridinium Chloride Spraying on Chicken Skin to Reduce Attached Salmonella typhimurium.

Spraying treatments with trisodium phosphate (TSP) and cetylpyridinium chloride (CPC) were evaluated for their effectiveness in reducing Salmonella typhimurium attached to chicken skins. Chicken skins with an area of 38.5 cm2 were cut from the breast areas of pre-chill chicken carcasses, mounted in a plastic holder, and inoculated with S. typhimurium . The inoculated skins were sprayed with tap water, 10% (wt/vol) TSP, or 0.1 % CPC solutions at 10, 35, or 60°C and 206.8, 413.7, 620.5, 827.4, or 1034.2 kPa for 30 s. After spraying, each skin was rinsed with tap water, transferred to a plastic bag containing 50 ml buffered peptone water, and stomached for 1 min. The stomaching water was collected, diluted serially, plated on xylose lysine tergitol 4 (XLT4) agar and Petrifilm aerobic count plates, and incubated for 18 to 24 h at 37°C. The results showed that tap water spraying reduced S. typhimurium by 0.7 to 1.6 log, while the reduction ranges for TSP and CPC spraying treatments were 1.6 to 2.3 and 1.5 to 2.5 log, respectively. Greater reductions in the numbers of S. typhimurium were obtained in TSP spraying treatments in the high pressure range (620.5 to 1034.2 kPa) and in CPC spraying treatments at 10°C.

Changes in Eggshell Surface Microstructure after Washing with Cetylpyridinium Chloride or Trisodium Phosphate.

The effects of egg-washing chemicals on microstructural changes of the eggshell were examined. Fresh-laid eggs were washed with solutions of cetylpyridinium chloride (CPC) at 10, 50, or 100 ppm, or trisodium phosphate (TSP) at 0.5, 1.0, or 5.0% and changes in the shell surface were examined using scanning electron microscopy (SEM). As the concentration of CPC increased, the eggshell surface became pitted and the cuticle layer became thinner. TSP caused the cuticle layer to fissure and flake and even cuticle-free areas were observed at 5.0%. When the porosity of eggs was measured by using the blue lake staining method, there were significant differences between control and TSP- or CPC-washed eggs as observed by SEM. These results suggest that depending on the types of chemicals used in the wash water, different microstructural changes occur in eggshell surfaces, and the more damaged eggshell surfaces allow more bacterial penetration.

Morphological Changes of Salmonella typhimurium Caused by Electrical Stimulation in Various Salt Solutions.

To better understand what physical changes occur in bacteria subjected to low-voltage, low-current electrical stimulation (ES), morphological changes in Salmonella typhimurium killed by ES were examined using electron microscopy. Cells (107 CFU/ml) were suspended in 0.015 M NaCl, 0.015 M Na2CO3, or 0.005 M Na3PO4·12H2O solutions and pulsed electric signals at 10 mA/cm2 current, 1 kHz frequency and 50% duty cycle were applied until more than 90% of the cells were dead. In NaCl solutions, cells were swollen before collapsing into amorphous bodies. In Na2CO3 solutions, cells showed irregular surfaces and the cytoplasm became less dense at the centers of the cells. In Na3PO4 solutions, the cytoplasm was scattered into small aggregates within the cell and many fibrils were formed outside. The results of this research suggest that the mechanisms of destruction of S. typhimurium by ES depend on the salt used an electrolyte.

Reduction of Salmonella and Campylobacter on Chicken Carcasses by Changing Scalding Temperature.

Chickens were processed at three scalding temperatures, 52, 56, or 60°C, and the numbers of Salmonella and Campylobacter attached to the fully processed carcasses in each group were compared. For Salmonella , carcasses scalded at 52 or 56°C showed ~ 0.3 to 0.5 lower log numbers than carcasses at 60°C (P < 0.05). There were no significant differences between the carcasses at 52 and 56°C. For Campylobacter , carcasses scalded at 56°C showed ~ 0.7 lower log counts than the carcasses at 60°C (P < 0.05) in the first two trials; however, no difference was observed in a third trial. Although the reduction of bacteria attached to the chicken carcasses was not as great as shown in previous attachment studies using skin samples (1.0 to 1.4 log cycles), these results show that reductions in bacterial numbers on chicken carcasses can be achieved by simply changing the scalding temperature.

Electrical Treatment of Poultry Chiller Water to Destroy Campylobacter jejuni.

To control bacterial contamination in poultry processing, pulsed electricity in combination with a salt was evaluated as an electrical pasteurization method to kill Campylobacter jejuni in poultry chiller water. Chiller water from a poultry processing plant was mixed with either sodium chloride (NaCl) or trisodium phosphate (Na3PO4 12H2O or TSP) at 0.1 %, 0.2% or 0.3% concentration, and inoculated with C. jejuni at 1 × 106 CFU/ml. The inoculated chiller water was treated at 4°C for 20 min using pulsed electrical signals at 10mA/cm2 current, 1 kHz frequency and 50% duty cycle. Samples taken at different intervals were serially diluted, pre-enriched in Brucella-FBP broth, plated on Campy-Cefex agar and incubated, and colony-forming units (CFU) were counted. The results showed that C. jejuni was reduced and that the bacterial death rate was dependent upon pH of the salt solution, salt concentration, and treatment time. In the electrical treatments, C. jejuni was reduced linearly on log scale in the chiller water mixed with TSP, but nonlinearly with NaCl. Bacterial destruction rate was accelerated by higher concentrations or higher pH of NaCl or TSP added to chiller water.

Rapid Fluorescence Concentration Immunoassay for Detection of Salmonellae in Chicken Skin Rinse Water.

Salmonellae in chicken carcass rinse water or enrichment broth were detected with a fluorescence concentration immunoassay (FCIA) using intact salmonellae cells as the reacting particles. Sample Solutions and reagents were added to a 96-well fluoricon assay plate and incubated 30 min in the chamber of a fluorescence concentration analyzer at room temperature. The bound and free components were separated by automated vacuum filtration and washings through the membrane filters equipped at the bases of the wells. The fluorescent signals were concentrated through vacuum filtration and were read at a wavelength of 485/535 nm. A cut-off value of the arbitrary fluorescence units was calculated by adding two standard deviations to the mean value of the negative control. When the arbitrary fluorescence units of a sample were above the cut-off value, it was scored positive indicating the presence of salmonellae in the test suspensions. It was found the FCIA method could detect salmonellae at 1.25 × 104 cells per mi in pure culture and was specific enough to screen salmonellae from four types of common chicken carcass contaminating bacteria. By coupling with 18-h direct enrichment in selenite cystine broth of the whole carcass rinse, the detection of salmonellae in chicken carcass rinse water was accomplished in less than 24 h.

Effect of Trisodium Phosphate on Campylobacter Attached to Post-Chill Chicken Carcasses.

Trisodium phosphate (TSP) was evaluated as a means to reduce Campylobacter on chicken carcasses. Post-chill chicken carcasses were dipped into a 10% TSP solution at 50°C for 15 s. After storing the TSP-treated carcasses for 0, 1 or 6 days at 4°C, the carcasses were subjected to the recovery of Campylobacter . The incidence and reduction of Campylobacter attached to the carcasses were measured using a nitrocellulose (NC) membrane lift, conventional culture method, and a most probable number (MPN) technique. In trials 1 and 2, the incidence of Campylobacter was measured. For 1 day-stored groups, Campylobacter was present on 96 and 100% of control carcasses and present on 24 and 28% of TSP-treated carcasses as measured by NC membrane lift method. The reduction was less (4 to 36%) when measured by culture method. For carcasses immediately subjected for the recovery of cells after treatment, there was no difference between TSP-treated and control carcasses by either NC membrane or culture method. In trial 3, the reduction levels of Campylobacter were quantified by using a MPN method. The levels of Campylobacter on carcasses were decreased by 1.5 and 1.2 logs in 1- and 6-day stored, TSP-treated carcasses, respectively (p < 0.05). However, TSP treatment at 10°C reduced the level of Campylobacter only by 0.16 log (p > 0.10).

A Membrane Blotting Procedure for Detection of Salmonella on Chicken Carcasses.

Salmonellae attached to artificially contaminated chicken carcasses were identified in less than 24 h using a nitrocellulose membrane lift method. Six pieces of nitrocellulose membrane were pressed onto each chicken carcass at the breast, the thigh, and the back in order to lift the salmonellae from the carcasses. After direct incubation of the membranes on xylose lysine tergitol 4 agar overnight at 37°C, the appearance of black colonies on the white membrane was considered a positive presumptive test for salmonellae. Immunoassay of black colonies on the membrane with Salmonella -specific antiserum confirmed that the black colonies were salmonellae . The nitrocellulose membrane lift technique was shown to be comparable in sensitivity by direct enrichment of whole carcass rinse in selenite cystine broth.