Kinome Analysis of Cattle Peripheral Lymph Nodes to Elucidate Differential Response to Salmonella spp.

Salmonella spp., contained within the peripheral lymph nodes (PLNs) of cattle, represents a significant source of contamination of ground beef. Herein is the first report where species-specific kinome peptide arrays designed for bovine biology were used to further the understanding of Salmonella spp. within these PLNs. For the purpose of this research, multiple comparisons of sub-iliac lymph nodes were made to include nodes from feedlot cattle that were infected with Salmonella spp. to those that were non-infected; seasonal differences in feedlot cattle harvested in either August or January; cull dairy cows compared to feedlot cattle; and PLNs from cattle experimentally inoculated with Salmonella spp. versus naturally infected animals. The first comparison of Salmonella-positive and -negative PLNs found that considering the kinotypes for these animals, the major distinguishing difference was not the presence or absence of Salmonella spp. in the PLNs but the concentration. Further, the majority of pathways activated were directly related to immune responses including innate immunity, thus Salmonella spp. within the PLNs activates the immune system in that node. Results from the comparison of feedlot cattle and cull dairy cows suggests that a Salmonella spp.-negative animal, regardless of type, has a more consistent kinome profile than that of a Salmonella spp.-positive animal and that the differences between feedlot and cull dairy cattle are only pronounced when the PLNs are Salmonella spp. positive. PLNs collected in the winter showed a much more consistent kinome profile, regardless of Salmonella status, suggesting that in the winter these cattle are similar, and this is not affected by the presence of Salmonella spp., whereas significant variability among kinotypes was observed for PLNs collected in the summer. The most distinct clustering of kinotypes observed in this study was related to how the animal was infected with Salmonella spp. There were significant differences in the phosphorylation state of the immune response peptides between experimentally and naturally infected animals, suggesting that the immune system is activated in a significantly different manner when comparing these routes of infection. Increasing our understanding of Salmonella spp. within cattle, and specifically within the PLNs, will ultimately help design effective pre-harvest intervention strategies as well as appropriate experimentation to validate those technologies.

Innovative Treatments Enhancing the Functionality of Gut Microbiota to Improve Quality and Microbiological Safety of Foods of Animal Origin.

The gastrointestinal tract, or gut, microbiota is a microbial community containing a variety of microorganisms colonizing throughout the gut that plays a crucial role in animal health, growth performance, and welfare. The gut microbiota is closely associated with the quality and microbiological safety of foods and food products originating from animals. The gut microbiota of the host can be modulated and enhanced in ways that improve the quality and safety of foods of animal origin. Probiotics-also known as direct-fed microbials-competitive exclusion cultures, prebiotics, and synbiotics have been utilized to achieve this goal. Reducing foodborne pathogen colonization in the gut prior to slaughter and enhancing the chemical, nutritional, or sensory characteristics of foods (e.g., meat, milk, and eggs) are two of many positive outcomes derived from the use of these competitive enhancement-based treatments in food-producing animals.

Antagonistic Effects of Lipids Against the Anti-Escherichia coli and Anti-Salmonella Activity of Thymol and Thymol-ß-d-Glucopyranoside in Porcine Gut and Fecal Cultures In Vitro.

Strategies are sought to reduce the carriage and dissemination of zoonotic pathogens and antimicrobial-resistant microbes within food-producing animals and their production environment. Thymol (an essential oil) is a potent bactericide in vitro but in vivo efficacy has been inconsistent, largely due to its lipophilicity and absorption, which limits its passage and subsequent availability in the distal gastrointestinal tract. Conjugation of thymol to glucose to form thymol-ß-d-glucopyranoside can decrease its absorption, but in vivo passage of effective concentrations to the lower gut remains suboptimal. Considering that contemporary swine diets often contain 5% or more added fat (to increase caloric density and reduce dustiness), we hypothesized that there may be sufficient residual fat in the distal intestinal tract to sequester free or conjugated thymol, thereby limiting the availability and subsequent effectiveness of this biocide. In support of this hypothesis, the anti-Salmonella Typhimurium effects of 6 mM free or conjugated thymol, expressed as log10-fold reductions of colony-forming units (CFU) ml-1, were diminished 90 and 58%, respectively, following 24-h in vitro anaerobic fecal incubation (at 39°C) with 3% added vegetable oil compared to reductions achieved during culture without added oil (6.1 log10 CFU ml-1). The antagonistic effect of vegetable oil and the bactericidal effect of free and conjugated thymol against Escherichia coli K88 tested similarly were diminished 86 and 84%, respectively, compared to reductions achieved in cultures incubated without added vegetable oil (5.7 log10 CFU ml-1). Inclusion of taurine (8 mg/ml), bile acids (0.6 mg/ml), or emulsifiers such as polyoxyethylene-40 stearate (0.2%), Tween 20, or Tween 80 (each at 1%) in the in vitro incubations had little effect on vegetable oil-caused inhibition of free or conjugated thymol. Based on these results, it seems reasonable to suspect that undigested lipid in the distal gut may limit the effectiveness of free or conjugated thymol. Accordingly, additional research is warranted to learn how to overcome obstacles diminishing bactericidal activity of free and conjugated thymol in the lower gastrointestinal tract of food-producing animals.

Controlling the Colonization of Clostridium perfringens in Broiler Chickens by an Electron-Beam-Killed Vaccine.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Evaluation of Thymol-ß-d-Glucopyranoside as a Potential Prebiotic Intervention to Reduce Carriage of Zoonotic Pathogens in Weaned and Feeder Pigs.

The gut of food-producing animals is a reservoir for foodborne pathogens. Thymol is bactericidal against foodborne pathogens but rapid absorption of thymol from the proximal gut precludes the delivery of effective concentrations to the lower gut where pathogens mainly colonize. Thymol-ß-d-glucopyranoside is reported to be more resistant to absorption than thymol in everted jejunal segments and could potentially function as a prebiotic by resisting degradation and absorption in the proximal gut but being hydrolysable by microbial ß-glycosidase in the distal gut. Previous in vitro studies showed bactericidal effects of thymol-ß-d-glucopyranoside against Campylobacter, Escherichia coli, and Salmonella enterica serovar Typhimurium in the presence but not absence of intestinal microbes expressing ß-glycosidase activity, indicating that hydrolysis was required to obtain antimicrobial activity. Presently, the oral administration of thymol-ß-d-glucopyranoside was studied to examine the effects on intestinal carriage of Campylobacter, E. coli, and S. Typhimurium in swine. The effects of thymol-ß-d-glucopyranoside or thymol on antimicrobial sensitivity of representative E. coli isolates and characterized Salmonella strains were also explored. Results from two in vivo studies revealed little antimicrobial effects of thymol-ß-d-glucopyranoside on Campylobacter, E. coli, or S. Typhimurium in swine gut. These findings add credence to current thinking that hydrolysis and absorption of thymol-ß-d-glucopyranoside and thymol may be sufficiently rapid within the proximal gut to preclude delivery to the distal gut. Antibiotic susceptibilities of selected bacterial isolates and strains were mainly unaffected by thymol. Further research is warranted to overcome obstacles, preventing the delivery of efficacious amounts of thymol-ß-d-glucopyranoside to the lower gut.

Disinfectant and antimicrobial susceptibility studies of the foodborne pathogen Campylobacter jejuni isolated from the litter of broiler chicken houses.

Foodborne illness is an ongoing problem worldwide and is caused by bacteria that invade the food chain from the farm, slaughter house, restaurant or grocery, or in the home and can be controlled by strategies using biocides (antiseptics and disinfectants). Susceptibility profiles were determined for 96 Campylobacter jejuni strains obtained in 2011-2012 from broiler chicken house environments to antimicrobials and disinfectants as per the methods of the Clinical and Laboratory Standards Institute and TREK Diagnostics using CAMPY AST Campylobacter plates. Low prevalence of antimicrobial resistance was observed in C. jejuni strains to tetracycline (TET; 21.9%), ciprofloxacin (CIP; 13.5%), and nalidixic acid (NAL; 12.5%). The resistance profiles had a maximum of 3 antimicrobials, CIP-NAL-TET, with TET being the main profile observed. No cross-resistance was observed between antimicrobials and disinfectants. The C. jejuni strains (99%) were resistant to triclosan, 32% were resistant to chlorhexidine, and they all were susceptible to benzalkonium chloride. The strains had low-level minimum inhibitory concentrations (MICs) to the disinfectants P-128, Food Service Sanitizer, F-25 Sanitizer, Final Step 512 Sanitizer, OdoBan, dioctyldimethylammmonium chloride, didecyldimethylammonium chloride (C10AC), benzyldimethyldodecylammonium chloride (C12BAC), and benzyldimethyltetradecylammonium chloride (C14BAC). Intermediate MICs against DC&R, cetylpyridinium bromide hydrate, hexadecylpyridinium chloride, ethylhexadecyldimethylammonium bromide, and hexadecyltrimethylammonium bromide with elevated intermediate MICs against Tek-Trol, benzyldimethylhexadecylammonium chloride, tris(hydroxylmethyl)nitromethane (THN), and formaldehyde. The highest MIC were obtained for povidone-iodine. The components THN and the benzylammonium chlorides C12BAC and C14BAC were responsible for the inhibition by DC&R. The components C10AC and C12BAC may act synergistically causing inhibition of C. jejuni by the disinfectant P-128. The formaldehyde component in DC&R was not effective against C. jejuni compared with the ammonium chloride components. Its use in disinfectants may result in additional unnecessary chemicals in the environment. Didecyldimethylammonium chloride is the most effective ammonium chloride component against C. jejuni.

Methylsulfonylmethane Exhibits Bacteriostatic Inhibition of Vancomycin-Resistant Enterococcus faecium, In Vitro.

The aim of this study was to evaluate the antibacterial properties of methylsulfonylmethane (MSM) on vancomycin-resistant Enterococcus faecium (VRE). Bacterial proliferation was measured spectrophotometrically during growth in brain heart infusion broth with 0%, 3%, 5%, 7%, 10%, 12%, and 16% MSM. To assess the mechanism of inhibition, VRE was grown overnight with 0-16% MSM and enumerated on unmedicated and medicated (3-16% MSM) brain heart infusion agar (BHIA). Viability studies were performed to evaluate the impact of 10-16% MSM on VRE over 7 days. Absorbance data indicated a dose-dependent inhibition from 0% to 7% MSM and no increase in optical density in 10-16% MSM. VRE enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM partially recovered. No growth was observed when BHIA contained 10-16% MSM. There was little effect on VRE growth in 10% MSM over 7 days. VRE displayed a population rebound on day 6 when exposed to 12% MSM, and elimination by day 6 in 16% MSM. Regrowth after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. Cell elimination in 16% MSM suggests inhibition of an essential metabolic function from which the bacterium could not recover.

A Preliminary Study on the Presence of Salmonella in Lymph Nodes of Sows at Processing Plants in the United States.

Salmonella-contaminated lymph nodes (LN), when included into edible meat products, are a potential source of Salmonella foodborne disease. In this survey, ventral superficial cervical and mandibular LN were tested for the presence of Salmonella from two sow processing plants in the midwestern United States. Results indicate that both LN can be contaminated with Salmonella; mandibular LN have higher prevalence (p < 0.05) of Salmonella than cervical LN (16% vs. 0.91%), and the majority (>90%) of Salmonella isolates are pan-susceptible or resistant to one antimicrobial, while 9.78% of isolates were multi-drug-resistant (MDR-resistant to three or more classes of antimicrobials). Intervention methods to prevent foodborne disease could include elimination of these LN from pork products or inclusion of LN only into products that are destined for cooking. Integrated multi-faceted intervention methods need to be developed to reduce Salmonella in the food chain.

Evaluation of two commercially-available Salmonella vaccines on Salmonella in the peripheral lymph nodes of experimentally-infected cattle.

BACKGROUND: Salmonella is a common inhabitant of the ruminant gastrointestinal tract, where it often resides asymptomatically and may be shed into the feces. More recently it was discovered that Salmonella may be contained within the peripheral, non-mesenteric lymph nodes, where it is impervious to in-plant pathogen control interventions and may serve as a source of Salmonella-contamination of ground beef. Over the past 10 years considerable research effort has been expended at understanding how this pathogen gets to these lymph nodes, the duration of infection, and, most importantly, screening and developing potential intervention strategies that may be employed on farm prior to the animal being presented for slaughter. METHODS: Utilizing an experimental model of Salmonella inoculation of bovine peripheral lymph nodes (PLNs), two pilot vaccine experiments were conducted to evaluate two Salmonella vaccines: Salmonella Newport Bacterial Extract (Experiment I) and Endovac-Bovi® (Experiment II) on preventing Salmonella acquisition by these nodes. In Experiment I, 4 months following the booster vaccination, 30 steers were inoculated with three Salmonella serotypes intradermally: Newport, Montevideo, and Anatum administered to the right legs, left legs, and to the caudal thorax and abdomen, respectively. Cattle were inoculated every other day over the course of five days (three total inoculation events) and 6 and 12 days following the final Salmonella inoculation, 16 and 14 head in each treatment were euthanized, respectively. In Experiment II, 12 head of Holstein steers were utilized. Seven days following the booster and weekly thereafter for 3 weeks (four total inoculation events), cattle were inoculated as above and euthanized 7 days following final inoculation. Right and left sub-iliac, popliteal and pre-scapular lymph nodes were collected in each experiment, weighed and cultured for Salmonella. RESULTS: In Experiment I, no treatment differences were observed in Salmonella prevalence 6 days post-inoculation (necropsy 1). However, in vaccinated cattle at the second necropsy, a reduction (p = 0.05) in Salmonella prevalence was observed in the sub-iliac and pre-scapular lymph nodes as well as when all nodes were evaluated collectively (p = 0.04). In Experiment II, the vaccine reduced (p = 0.03) Salmonella prevalence in the right popliteal and tended (p = 0.09) to decrease prevalence in both popliteal lymph nodes. CONCLUSION: Under these experimental conditions, the data generated provide evidence of a partial vaccine effect on Salmonella within PLNs and indicate that further research may be warranted.

Whole-Genome Sequence of Aeromonas hydrophila CVM861 Isolated from Diarrhetic Neonatal Swine.

Aeromonas hydrophila are ubiquitous in the environment and are highly distributed in aquatic habitats. They have long been known as fish pathogens but are opportunistic human pathogens. Aeromonas spp. have persisted through food-processing safeguards and have been isolated from fresh grocery vegetables, dairy, beef, pork, poultry products and packaged ready-to-eat meats, thus providing an avenue to foodborne illness. A beta-hemolytic, putative Escherichia coli strain collected from diarrheic neonatal pigs in Oklahoma was subsequently identified as A. hydrophila, and designated CVM861. Here we report the whole-genome sequence of A. hydrophila CVM861, SRA accession number, SRR12574563; BioSample number, SAMN1590692; Genbank accession number SRX9061579. The sequence data for CVM861 revealed four Aeromonas-specific virulence genes: lipase (lip), hemolysin (hlyA), cytonic enterotoxin (ast) and phospholipid-cholesterolacyltransferase (GCAT). There were no alignments to any virulence genes in VirulenceFinder. CVM861 contained an E. coli resistance plasmid identified as IncQ1_1__M28829. There were five aminoglycoside, three beta-lactam, and one each of macrolide, phenicol, sulfonamide, tetracycline and trimethoprim resistance genes, all with over 95% identity to genes in the ResFinder database. Additionally, there were 36 alignments to mobile genetic elements using MobileElementFinder. This shows that an aquatic pathogen, rarely considered in human disease, contributes to the resistome reservoir and may be capable of transferring resistance and virulence genes to other more prevalent foodborne strains such as E. coli or Salmonella in swine or other food production systems.

Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic.

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

Nitro-treatment of composted poultry litter; effects on Salmonella, E. coli and nitrogen metabolism.

Poultry litter is a potentially valuable crude protein feedstuff for ruminants but must be treated to kill pathogens before being fed. Composting kills pathogens but risks losses of nitrogen due to volatilization or leaching as ammonia. Treatment of poultry litter with ethyl nitroacetate, 3-nitro-1-propionate, ethyl 2-nitropropionate (at 27 µmol/g), decreased numbers of experimentally-inoculated Salmonella Typhimurium (>1.0 log10 compared to controls, 4.2 ± 0.2 log10 CFU/g) but not endogenous Escherichia coli early during simulated composting. By day 9 of simulated composting, Salmonella and E. coli were decreased to non-detectable levels regardless of treatment. Some nitro-treatments preserved uric acid and prevented ammonia accumulation, with 18% more uric acid remaining and 17-24% less ammonia accumulating in some nitro-treated litter than in untreated litter (18.1 ± 3.8 µmol/g and 3.4 ± 1.4 µmol/g, respectively). Results indicate that nitro-treatment may help preserve uric acid in composted litter while aiding Salmonella control.

Bacillus subtilis PB6 Supplementation in Weaned Holstein Steers During an Experimental Salmonella Challenge.

To evaluate the effects of a patented Bacillus subtilis probiotic, weaned Holstein steers, not shedding Salmonella (n = 40; ∼90 kg), were supplemented (CLO) or not (CON) with CLOSTAT® (13 g/hd per day; Kemin Industries, Des Moines, IA) in a starter ration for 35 d. The calves were assigned to one of four treatments in a 2 × 2 factorial design with CLO and CON calves that were orally administered Salmonella (STM) or not (NoSTM). Calves were challenged with 1.6 × 106 colony-forming unit (CFU) Salmonella Typhimurium (resistant to 50 µg/mL nalidixic acid) in 1 L of milk replacer on day 0. Blood samples were collected through jugular catheters every 6 h for 96 h, and body temperature was measured every 5 min through indwelling rectal temperature recording devices. Five calves from each treatment were harvested 48 h postchallenge, and the remaining calves were harvested 96 h postchallenge. During necropsy, tissues were collected for the isolation and quantification of the inoculated STM from various tissues. The CLOSTM group had reduced STM concentrations in the jejunum, ileum, and transverse colon 48 h after the challenge (p ≤ 0.03), but were not different 96 h postchallenge (p > 0.05). Decreased (p < 0.01) pyrexia was observed after the challenge in CLOSTM calves when compared with CONSTM calves. White blood cells and lymphocyte counts were increased (p ≤ 0.05) in CLOSTM calves after the challenge in comparison with other treatments. In calves given STM, the CLO group had greater feed intake before and after the challenge (p < 0.01) compared with the CON group. Increased serum IL-6 and IFN-γ concentrations were observed in the CONSTM group compared with other treatments. Overall, CLO reduced Salmonella presence and concentrations in gastrointestinal tissues while simultaneously reducing the severity of the challenge as indicated by blood parameters and the reduced febrile response.

Inhibition and Interactions of Campylobacter jejuni from Broiler Chicken Houses with Organic Acids.

Campylobacter jejuni is a bacterium that causes major diarrheal disease worldwide and is also one of the top five foodborne pathogens encountered in the United States. Poultry is a major source of C. jejuni, and a high-risk factor for contracting campylobacteriosis. Organic acids are used in the United States during food animal processing for removal of bacterial contamination from animal carcasses. Six organic acids were evaluated in inhibition studies of 96 C. jejuni strains obtained from shoe covers used in broiler chicken houses at different poultry farms in several states by determining the susceptibilities of the C. jejuni strains, along with the pH values at the molar minimum inhibitory concentrations (MICMs). The undissociated and dissociated organic acid concentrations were calculated at the MICMs with the Henderson-Hasselbalch equation. The results for the 96 C. jejuni strains were treated similarly for each different organic acid. Campylobacter jejuni inhibition did correlate with the dissociated organic acids, but did not correlate with pH or with the undissociated organic acids. When the concentrations of dissociated organic acids decreased, the C. jejuni strains were not disinfected. A carcass wash using organic acids should have the concentration of dissociated acid species carefully controlled. It is suggested to maintain a dissociated acid concentration for propionic, l-lactic, formic, citric, butyric, and acetic acids at 24, 40, 36, 21, 23, and 25 mM, respectively, and at these dissociated organic acid levels an acid wash would be expected to remove or inhibit 97% or more of the C. jejuni bacteria studied here. However, studies must be undertaken to confirm that the suggested concentrations of dissociated organic acids are adequate to remove C. jejuni bacteria in the field vs. the laboratory. Due to propionate, l-lactate, formate, butyrate, and acetate being utilized by C. jejuni, these organic acids may not be appropriate for use as a carcass wash to remove C. jejuni surface contamination. Of all tested organic acids, dissociated citric acid was the most efficient at inhibiting C. jejuni.

Establishment and characterization of a competitive exclusion bacterial culture derived from Nile tilapia (Oreochromis niloticus) gut microbiomes showing antibacterial activity against pathogenic Streptococcus agalactiae.

This study reports the characterization of the microbial community composition, and the establishment and dynamics of a continuous-flow competitive exclusion culture (CFCEC) derived from gut microbiomes of Nile tilapia (Oreochromis niloticus) specimens reared on aquaculture farms in Colombia. 16S rRNA gene amplicon Illumina sequencing was used to identify taxonomical changes in the CFCEC microbial community over time. The CFCEC was developed from adult tilapia from two farms in Colombia, and CFCEC samples were collected over two months. The pH varied from 6.25 to 6.35 throughout culturing, while anaerobic and aerobic cell counts stabilized at day 9, at 109 CFU mL-1 and were maintained to day 68. A variation in the CFCEC bacterial composition was observed over time. Cetobacterium was the most abundant in the first two days and coincided with a higher CFCEC supernatant antimicrobial effect against the fish pathogen Streptococcus agalactiae. Antimicrobial activity against S. agalactiae disappeared by day 3. Changes in bacterial composition continued to day 33 with Lactococcus spp. becoming the most abundant member of the community. In conclusion, the study of the CFCEC from intestinal tract of Nile tilapia (Oreochromis niloticus) by 16S rRNA gene sequencing allowed identification of predominant bacterial genera in the continuous-flow competitive exclusion culture exhibiting antibacterial activity against the fish pathogen Streptococcus agalactiae.

Disinfectant and Antimicrobial Susceptibility Profiles of Campylobacter coli Isolated in 1998 to 1999 and 2015 from Swine and Commercial Pork Chops.

Susceptibility profiles were determined for 111 Campylobacter coli strains obtained in 1998 to 1999 and 2015 from market age pigs and pork chops against 22 disinfectants and 9 antimicrobials. Resistance to tetracycline (TET) was observed in 44.4% of 1998 to 1999 strains, and the antibiotic resistance profile was TET. But strains obtained in 2015 from swine and retail pork chops had 75% TET resistance and the antibiotic resistance profile was TET, followed by azithromycin-erythromycin-TET-telithromycin-clindamycin. Antimicrobial resistance increased in 2015 strains. All strains were resistant to triclosan, and 84.1% and 95.8% of strains in 1998 to 1999 and 2015, respectively, were chlorhexidine resistant. All strains were susceptible to benzalkonium chloride. There was a shift toward higher susceptibility to chlorhexidine, triclosan, P-128, OdoBan, CPB, and CPC in 2015 swine and pork chop strains compared with 1998 to 1999 strains. The disinfectants Tek-Trol and providone-iodine, tris(hydroxylmethyl)nitromethane (THN) and formaldehyde demonstrated the highest susceptibilities. Didecyldimethylammonium chloride (C10AC) appeared to be about equally effective as benzyldimethyltetradecylammonium chloride (C14BAC) for inhibiting C. coli, and both were more effective than C8AC and C12BAC, but C16BAC was not efficient at inhibiting C. coli. The BACs, C12BAC and C14BAC, were the most effective ingredients in DC&R. Also, C12BAC and C14BAC, or these two in synergy with C10AC were responsible for inhibition of C. coli at high P-128 MICs. No cross-resistance was observed between antibiotics and disinfectants. The continued use of THN and formaldehyde in DC&R should be evaluated since these components are not effective, and their inclusion adds unwanted chemicals in the environment. PRACTICAL APPLICATION: Campylobacter species cause diarrheal disease throughout the world. Disinfectants are often used on the farm, in veterinary medicine, by the food processing industry, in restaurants, and in consumer's homes. Limited information is available in the literature showing how disinfectants or disinfectant components may affect the many different foodborne pathogens, and, specifically, Campylobacter coli studied here. The knowledge generated in this study concerning the interactions of a broad array of disinfectants against C. coli may well affect the types of disinfectants and disinfectant formulations allowable for use by medical personnel, producers, food processors, restaurants, and consumers.

Inhibition of calmodulin increases intracellular survival of Salmonella in chicken macrophage cells.

Calcium (Ca2+) is a pivotal intracellular second messenger and calmodulin (CaM) acts as a multifunctional Ca2+-binding protein that regulates downstream Ca2+ dependent signaling. Together they play an important role in regulating various cellular functions, including gene expression, maturation of phagolysosome, apoptosis, and immune response. Intracellular Ca2+ has been shown to play a critical role in Toll-like receptor-mediated immune response to microbial agonists in the HD11 chicken macrophage cell line. The role of that the Ca2+/CaM pathway plays in the intracellular survival of Salmonella in chicken macrophages has not been reported. In this study, kinome peptide array analysis indicated that the Ca2+/CaM pathway was significantly activated when chicken macrophage HD11 cells were infected with S. Enteritidis or S. Heidelberg. Further study demonstrated that treating cells with a pharmaceutical CaM inhibitor W-7, which disrupts the formation of Ca2+/CaM, significantly inhibited macrophages to produce nitric oxide and weaken the control of intracellular Salmonella replication. These results strongly indicate that CaM plays an important role in the innate immune response of chicken macrophages and that the Ca2+/CaM mediated signaling pathway is critically involved in the host cell response to Salmonella infection.

Paenibacillus 79R4, a potential rumen probiotic to enhance nitrite detoxification and methane mitigation in nitrate-treated ruminants.

The effects of supplemental nitrate administered alone or with a denitrifying ruminal bacterium, designated Paenibacillus 79R4 (79R4) intentionally selected for enhanced nitrate- and nitrite-metabolizing ability, on select rumen fermentation characteristics was examined in vivo. Rumen and blood samples were collected from cannulated Holstein steers one day prior to and one day after initiation of treatments applied as three consecutive intra-ruminal administrations of nitrate, to achieve the equivalent of 83 mg sodium nitrate/kg body weight day, given alone or with the nitrite-selected 79R4 (provided to achieve 106 cells/mL rumen fluid). Results revealed a day effect on methane-producing activity, with rates of methane production by ruminal microbes being more rapid when collected one day before than one day after initiation of treatments. Nitrate-metabolizing activity of the rumen microbes was unaffected by day, treatment or their interaction. A day by treatment interaction was observed on nitrite-metabolizing activity, with rates of nitrite metabolism by rumen microbes being most rapid in populations collected one day after initiation of treatment from steers treated with nitrate plus 79R4. A day by treatment interaction was also observed on plasma methemoglobin concentrations, with concentrations being lower from steers one day after initiation of treatments than from collected one day prior to treatment initiation and concentrations being lowest in steers treated with nitrate plus 79R4. A major effect of treatment was observed on accumulations of most prominent and branched chain volatile fatty acids produced and amounts of hexose fermented in the rumen of animals administered nitrate, with concentrations being decreased in steers administered nitrate alone when compared to steers treated with nitrate plus the 79R4. These results demonstrate that the nitrite-selected Paenibacillus 79R4 may help prevent nitrite toxicity in nitrate-treated ruminants while maintaining benefits of reduced methane emissions and preventing inhibition of fermentation efficiency by the microbial ecosystem.

Interactions of organic acids with Campylobacter coli from swine.

Campylobacter coli is a bacterial species that is a major cause of diarrheal disease worldwide, and Campylobacter spp. are among the top 5 foodborne pathogens in the United States. During food production organic acids (OAs) are often used to remove bacteria from animal carcasses. The interactions of six OAs with 111 C. coli strains obtained from swine and retail pork chops were studied by determining the molar minimum inhibitory concentrations (MICMs) of the C. coli strains, and the pH at the MICMs. The Henderson-Hasselbalch equation was used to calculate the concentrations of the undissociated and dissociated OAs at the MICMs of the C. coli strains. The results for the 111 different C. coli strains obtained from different locations were treated as a single group for each OA since many of the C. coli strains behaved similarly to each different OA. Inhibition of C. coli was not dependent on pH or on the undissociated OA species, but C. coli inhibition correlated with the dissociated OA species. Therefore, if the concentration of the dissociated OAs decreases from optimum, one may then expect that C. coli bacteria would escape disinfection. The concentration of the dissociated OA should be carefully controlled in a carcass wash. We suggest maintaining a concentration of the dissociated acetic, butyric, citric, formic, lactic and propionic acids at 29, 23, 11, 35, 22 and 25 mM, respectively, when using a carcass wash with these OAs to remove C. coli bacteria. However, due to C. coli utilization of acetate, formate, lactate and propionate, these four OAs may not be the best choice to use for a carcass wash to remove C. coli contamination. Of the six OAs, citric acid was the most efficient at inhibiting C. coli.

Effect of waste milk pasteurization on fecal shedding of Salmonella in preweaned calves.

The objective of the current research was to determine if pasteurization of nonsaleable waste milk influences fecal Salmonella concentrations and prevalence, or antimicrobial susceptibility and serotype of the cultured isolates. Holstein dairy calves (n = 211) were housed on a single commercial dairy in the southwestern United States and randomly allotted to be fed either pasteurized (PWM; n = 128 calves) or nonpasteurized waste milk (NPWM; n = 83 calves). Fecal samples were collected via rectal palpation or from freshly voided, undisturbed fecal pats, weekly during the first 4 wk of the animal's life and then again at weaning. Eight total collections were made and 1,117 fecal samples cultured for Salmonella. One isolate from each culture-positive fecal sample was preserved for antimicrobial susceptibility screening and serotyping. Sixty-nine percent of the fecal samples were culture positive for Salmonella with no difference due to treatment (67.7 and 69% Salmonella positive for PWM and NPWM treatments, respectively). Few fecal samples (178/1,117; 15.9%) contained Salmonella concentrations above the limit of detection (∼1 cfu/g of feces) with concentrations ranging from 1.0 to 6.46 cfu (log10)/g of feces. Concentration was not affected by treatment. Seventeen different serotypes were identified, the majority of which were Montevideo and Anatum. A greater percentage of Typhimurium (87 vs. 13%), Muenchen (88 vs. 12%), and Derby (91 vs. 9%) were recovered from calves fed PWM compared with NPWM-fed calves. Conversely, Newport (12.5 vs. 86%), Bredeney (22.2 vs. 77.8%), and Muenster (12.5 vs. 87.5%) were lower in PWM compared with NPWM treatments. The majority (66.7%) of isolates were susceptible to all of the antibiotics examined. Results from this one commercial dairy suggest that milkborne Salmonella is not an important vector of transmission in dairy neonates, nor does pasteurization of waste milk influence fecal shedding of this pathogen. Caution should be used, however, when extrapolating results to other farms as Salmonella contamination of milk on farm is well documented. The potential benefits of pasteurization in disease prevention outweigh the potential risks of feeding a nonpasteurized product and warrants incorporation into any calf-rearing program using nonsaleable waste milk for feeding young dairy neonates.