Filter sterilized carcass rinsate for recovery of Salmonella species with various concentrations of cetylpyridinium chloride.

Controlling Salmonella in poultry processing continues to be important to processors and consumers. Cetylpyridinium chloride (CPC) has proven to be effective in vitro in controlling Salmonella. This study evaluated the recovery of Salmonella after overnight storage in 4°C filter-sterilized carcass rinsate containing CPC from 0.44 to 909 ppm (µg/mL). Ten Salmonella serotypes (18 strains), of which 6 serotypes are commonly isolated from poultry products, were grown in Bacto-Tryptic Soy Broth overnight at 37°C. Serial dilutions of a CPC/propylene glycol solution were prepared in 24-well tissue culture plates containing filter-sterilized carcass rinsate. Approximately 107 cfu/mL of each Salmonella serotype was added to the appropriate wells. Inoculated plates were stored overnight at 4°C. After storage, triplicate plates of brilliant green agar with sulfapyridine (BGS) were surface inoculated with 10 µL of the contents for each well, streaked for isolation, and incubated at 37°C for 24 h. Three replications were conducted. The presence of typical colonies on BGS plates was recorded as growth and verified through biochemical and serological testing. Of the serotypes chosen, Salmonella Kentucky, Dublin, and Enteritidis were the least resistant to CPC with a median minimum inhibitory concentration (MIC) of 14.22 µg/mL (range from 3.55 to 56.88 µg/mL); S. Typhimurium demonstrated a median MIC of 114.00 µg/mL (range from 28.44 to 114.00 µg/mL). Residual CPC potentially remaining attached to a carcass or in the weep after processing could potentially alter which Salmonella serotype is recovered from a carcass rinse due to different growth patterns during regulatory testing, with a potential for more virulent strains not to be recovered.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Characterizing the immune response of chickens to Campylobacter jejuni (Strain A74C).

Campylobacter is one of the major foodborne pathogens causing bacterial gastroenteritis worldwide. The immune response of broiler chickens to C. jejuni is under-researched. This study aimed to characterize the immune response of chickens to Campylobacter jejuni colonization. Birds were challenged orally with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or with 0.5 mL of 0.85% saline. Campylobacter jejuni persisted in the ceca of challenged birds with cecal colonization reaching 4.9 log10 CFU/g on 21 dpi. Campylobacter was disseminated to the spleen and liver on 7 dpi and was cleared on 21 dpi from both internal organs. Challenged birds had a significant increase in anti-Campylobacter serum IgY (14&21 dpi) and bile IgA (14 dpi). At 3 dpi, there was a significant suppression in T-lymphocytes derived from the cecal tonsils of birds in the challenge treatment when compared to the control treatment after 72 h of ex vivo stimulation with Con A or C. jejuni. The T-cell suppression on 3 dpi was accompanied by a significant decrease in LITAF, K60, CLAU-2, IL-1ß, iNOS, and IL-6 mRNA levels in the ceca and an increase in nitric oxide production from adherent splenocytes of challenged birds. In addition, on 3 dpi, there was a significant increase in CD4+ and CD8+ T lymphocytes in the challenge treatment. On 14 dpi, both pro and anti-inflammatory cytokines were upregulated in the spleen, and a significant increase in CD8+ T lymphocytes in Campylobacter-challenged birds' ceca was observed. The persistence of C. jejuni in the ceca of challenged birds on 21 dpi was accompanied by an increase in IL-10 and LITAF mRNA levels, an increase in MNC proliferation when stimulated ex-vivo with the diluted C. jejuni, an increase in serum specific IgY antibodies, an increase in both CD4+ and CD8+ cells, and a decrease in CD4+:CD8+ cell ratio. The balanced Th1 and Th2 immune responses against C. jejuni might explain the ceca's bacterial colonization and the absence of pathology in Campylobacter-challenged birds. Future studies on T lymphocyte subpopulations should elucidate a pivotal role in the persistence of Campylobacter in the ceca.

Research Note: Effect of organic acid mixture on growth performance and Salmonella Typhimurium colonization in broiler chickens.

Feed additives can be alternatives to antibiotics for routinely encountered pathogens in the poultry production. The objective of this study was to understand effects of organic acid mixture on growth parameters and Salmonella Typhimurium (ST) colonization in broilers. Organic acid mixture is a feed-grade buffered formic acid and sodium formate mixture (Amasil NA). A total of 800 1-day-old Cobb500 males were fed one of the five dietary treatments: a negative control diet without ST challenge (NC), positive control diet with ST challenge (PC), 0.3% organic acid mixture with ST, 0.6% organic acid mixture with ST, and 0.9% organic acid mixture with ST. Treatments were assigned to 20 pens with 40 chicks/pen and 4 replicates of each treatment. Chickens were challenged with 107 CFU/mL of nalidixic acid-resistant ST (STNAR) 4-D posthatch. In the grower phase, feed conversion rate was significantly reduced in the 9% organic acid mixture compared with the PC. The body weight and body weight gain (BWG) were not affected either in the starter or grower phases. However, in the finisher phase, the nonchallenged NC had higher BWG than the PC (P < 0.05), whereas there were no differences in BWG among the NC and organic acid mixture fed groups. In addition, there was a significant effect of organic acid mixture on the colonization of cecal STNAR. At 9 dpi, cecal STNAR was 3.28 log10 in the PC that was reduced to 2.65 log10 at 0.3%, 1.40 log10 at 0.6%, and 0.84 log10 in 0.9% organic acid mixture. At 24 dpi, cecal STNAR recovery was 0.81, 0.99, 0.53, and 0.33 log10 in the PC and 0.3, 0.6, and 0.9% organic acid mixture, respectively. Similarly, at 38 dpi, cecal STNAR was 0.26, 0.11, 0.33, and 0 log10 in the PC, 0.3, 0.6, and 0.9%, respectively. These results show that organic acid mixture can be one dietary strategy to control ST infection and maintain efficient growth performance.

Effect of dietary fructooligosaccharide supplementation on internal organs Salmonella colonization, immune response, ileal morphology, and ileal immunohistochemistry in laying hens challenged with Salmonella enteritidis.

A study was conducted to evaluate the efficacy of fructooligosaccharides (FOS) in controlling the infection of Salmonella Enteritidis (SE) in White Leghorns. A total of 30 laying hens (white leghorns W-36) were challenged both orally and cloacally with approximately 108 colony-forming units of nalidxic acid resistant SE (SENAR) and divided into 3 treatments: 1) SENAR challenged + 0.0% FOS, 2) SENAR challenged + 0.5% FOS (Nutraflora), and 3) SENAR challenged + 1.0% FOS. SENAR recovery via fecal shedding was measured at 3- and 6-d post-infection (dpi), whereas in the ceca and internal organs, SENAR recovery was measured at 7-d post-infection. In the first experiment, there was a 1.0 log10 and a 1.3 log10 reduction in cecal SENAR by supplementation of FOS at 0.5 and 1.0%, respectively. In the second experiment, there was a 0.6 log10 and a 0.8 log10 reduction in cecal SENAR by supplementation of FOS at 0.5 and 1.0%, respectively. Fecal shedding was significantly lower in 1.0% FOS supplemented groups compared to SENAR challenge 0.0% FOS. There was no significant difference among the 3 treatments on SENAR recovery in liver with gall bladder and ovaries. However, the frequency of positive SENAR in the ovaries (10 to 40%) in SENAR challenge 0.0% FOS was significantly lower than liver with gall bladder (60 to 80%) in both experiments. There was a significant upregulation of toll-like receptor-4 in 1.0% FOS and interferon gamma in both 0.5 and 1.0% FOS. Histologic measurements of ileal villi height and crypt depth were similar across all treatments. Immunohistochemistry analyses of ileal samples showed that immunoglobulin A positive cells increased as FOS concentration increased reaching significance at 1.0% as well as altered cytokine gene expression in the ileum. Further, FOS supplementation also reduced cecal SENAR and feces SENAR levels. Collectively, the results suggest that dietary supplementation with FOS may impair SE pathogenesis while modulating humoral immunity within the gut-associated lymphoid tissue.

Impact of desiccation and heat exposure stress on Salmonella tolerance to acidic conditions.

In a recent study, the pH of commonly used Salmonella pre-enrichment media became acidic (pH 4.0 to 5.0) when feed or feed ingredients were incubated for 24 h. Acidic conditions have been reported to injure or kill Salmonella. In this study, cultures of four known feed isolates (S. montevideo, S. senftenberg, S. tennessee, and S. schwarzengrund) and four important processing plant isolates (S. typhimurium, S. enteritidis, S. infantis, and S. heidelberg) were grown on meat and bone meal and later subjected to desiccation and heat exposure to stress the microorganism. The impact of stress on the isolates ability to survive in acidic conditions ranging from pH 4.0 to 7.0 was compared to the non-stressed isolate. Cell injury was determined on xylose lysine tergitol 4 (XLT4) and cell death determined on nutrient agar (NA). When measured by cell death in non-stressed Salmonella, S. typhimurium was the most acid tolerant and S. heidelberg was the most acid sensitive whereas in stressed Salmonella, S. senftenberg was the most acid tolerant and S. tennessee was the most acid sensitive. The pH required to cause cell injury varied among isolates. With some isolates, the pH required for 50% cell death and 50% cell injury was similar. In other isolates, cell injury occurred at a more neutral pH. These findings suggest that the pH of pre-enrichment media may influence the recovery and bias the serotype of Salmonella recovered from feed during pre-enrichment.

Bacterial Isolates from the Chicken Gizzard and Ceca with In Vitro Inhibitory Activity against Salmonella typhimurium.

Bacterial isolates (197) obtained from the gizzard and ceca of 20 broiler and 40 specific-pathogen-free chickens, 21 days to 8 months of age, were evaluated for inhibitory activity against Salmonella typhimurium . One-hundred forty strains were characterized as gram negative and oxidase negative, typical of the Enterobacteriaceae . Five of the gram-negative and oxidase-negative isolates demonstrated inhibitory activity against six strains of S. typhimurium after 10- and 20-fold concentration and ammonium sulfate precipitation of the cell-free supernatant fluid from a culture grown in M9 minimal medium. Three isolates were identified as lactobacilli, 40 other strains exhibited Gram stain, oxidase, and catalase reactions typical of the Lactobacillus spp., and three known lactobacilli were included in the evaluation. Limited inhibitory activity was exhibited by these 46 isolates when tested against six S. typhimurium strains. Fourteen other strains not characterized as presumptive enterobacteria or lactic acid bacteria demonstrated little or no inhibitory activity against the six test strains.