Salmonella Enteritidis Colonization Research Articles

Research Note: Curbing Salmonella Enteritidis in broiler chickens with palm-free medium-chain fatty acids

Salmonellosis is still one of the most reported zoonoses worldwide and poultry meat is a major source, as chickens are often persistent carriers of Salmonella. Medium-chain fatty acids (MCFA) are known for their strong antimicrobial activity. MCFAs used today in the animal feed industry, however, mainly originate from the palm oil industry, which is notorious for its negative impact on the climate. We investigated the effect of a specific blend of palm-free MCFAs (ranging from C6 to C9) on Salmonella Enteritidis (SE) colonization in broiler chickens and in vitro SE characteristics. Fifty Ross 308 broiler chickens were randomly divided in 2 treatment groups. Chickens received either un-supplemented feed or feed supplemented with 300 ppm MCFAs from D0 onwards. On D7, all chickens were orally inoculated with 1600 CFU of SE. Cloacal swabs (D11) and samples of liver and caeca (D12) of all animals were collected and SE was enumerated. Percentage of SE-positive caecum samples was significantly (P = 0.044) reduced in birds receiving MCFAs compared to those receiving unsupplemented feed (36% vs. 64%). In vitro work performed with the same SE strain showed that preincubating the Salmonella bacteria with MCFAs at a sub-minimal inhibitory concentration significantly (p < 0.05) reduced bacterial adhesion to and invasion in Caco-2 cells, which may explain the observed reduction in intestinal SE colonization in the in vivo trial. Together, these results show that the tested eco-friendly MCFA blend could be a promising tool in the control of Salmonella in broilers.

Characterization of spray-dried plasma-induced immune response in Salmonella-challenged broiler chicks

Spray-dried plasma (SDP) is a functional feed additive that has been established to improve performance and health of livestock. Understanding the effect of SDP in immune response modulation is essential to optimize its use for controlling Salmonella Enteritidis (SE) infection in chickens. This study was conducted to determine the levels of expression of selected cytokine genes in the ileum and cecal tonsil of SE-challenged broiler chicks. In a floor-pen housing, 320 broilers chicks were randomly assigned to 6 treatment groups: CX (unmedicated corn-soybean meal (SBM) basal without SDP), MX (unmedicated corn-SBM basal with antibiotic bacitracin methylene disalicylate (BMD) added at 0.055g/kg diet), PCX (unmedicated corn-SBM basal with SDP added at 30g/kg diet). Treatments SE, MSE, and PSE consisted of chicks inoculated with 7.46 × 108 CFU SE /mL at 1 d of age and given diets similar to CX, MX, and PCX, respectively. Samples of cecal tonsils and ileum were collected on d 3, 7 and 14 post infection for qRT-PCR analysis to determine the expression levels of interleukin (IL)-1β, interferon-γ (IFN-γ), IL-13, IL-17, IL-6, and transforming growth factor (TGF)-β genes. In the ceca tonsils, expression of IFN-γ was not affected by the interaction of Day and Treatment (P > 0.05). The level of the anti-inflammatory cytokine IL-13 was lower in MX and PCX on d 7 whereas high levels were expressed (P < 0.05) in MSE and PSE. In the ileum, expression of IL-17 and IFN-γ was significantly lower (P < 0.05) in PSE and MSE, but only PSE expressed lower IL-6 comparable to unchallenged treatments. On d 28 postchallenge, concentrations of anti-SE IgY and IL-6 protein were higher (P < 0.05) in the SE-challenged treatments compared to the unchallenged treatments. Overall, these results suggest that dietary SDP showed similar potency to BMD in modulating intestinal cytokine response against intestinal SE colonization in broiler chicks and therefore can be considered suitable alternative replacement for antibiotics in broiler production.

Chitosan-nanoparticle-based oral Salmonella enteritidis subunit vaccine elicits cross-protection against Salmonella typhimurium in broilers

Non-typhoidal Salmonella infection is a significant health and economic burden in poultry industry. Developing an oral vaccine to induce robust mucosal immunity in the intestines of birds, especially cross protection against different Salmonella serotypes is challenging. Therefore, a potent oral vaccine platform that can mitigate different serotypes of Salmonella is warranted for the poultry industry. We reported earlier that the Salmonella enteritidis (SE) immunogenic outer membrane proteins (OMPs) and flagellin (FLA) entrapped in mannose chitosan nanoparticles (OMPs-FLA-mCS NPs) administered prime-boost (d-3 and 3-wk later) by oral inoculation elicits mucosal immunity and reduces challenge SE colonization by over 1 log10 CFU in birds. In this study, we sought to evaluate whether the SE antigens containing OMPs-FLA-mCS NPs vaccine induces cross-protection against Salmonella typhimurium (ST) in broilers. Our data indicated that the OMPs-FLA-mCS NPs vaccine induced higher cross-protective antibody responses compared to commercial Poulvac ST vaccine (contains a modified-live ST bacterium). Particularly, OMPs-FLA-mCS-NP vaccine elicited OMPs and FLA antigens specific increased production of secretory IgA and IgY antibodies in samples collected at both post-vaccination and post-challenge timepoints compared to commercial vaccine group. Notably, the vaccine reduced the challenge ST bacterial load by 0.8 log10 CFU in the cecal content, which was comparable to the outcome of Poulvac ST vaccination. In conclusion, our data suggested that orally administered OMPs-FLA-mCS-NP SE vaccine elicited cross protective mucosal immune responses against ST colonization in broilers. Thus, this candidate vaccine could be a viable option replacing the existing both live and killed Salmonella vaccines for birds.

Salmonella Enteritidis T1SS protein SiiD inhibits NLRP3 inflammasome activation via repressing the mtROS-ASC dependent pathway.

Inflammasome activation is an essential innate immune defense mechanism against Salmonella infections. Salmonella has developed multiple strategies to avoid or delay inflammasome activation, which may be required for long-term bacterial persistence. However, the mechanisms by which Salmonella evades host immune defenses are still not well understood. In this study, Salmonella Enteritidis (SE) random insertion transposon library was screened to identify the key factors that affect the inflammasome activation. The type I secretion system (T1SS) protein SiiD was demonstrated to repress the NLRP3 inflammasome activation during SE infection and was the first to reveal the antagonistic role of T1SS in the inflammasome pathway. SiiD was translocated into host cells and localized in the membrane fraction in a T1SS-dependent and partially T3SS-1-dependent way during SE infection. Subsequently, SiiD was demonstrated to significantly suppress the generation of mitochondrial reactive oxygen species (mtROS), thus repressing ASC oligomerization to form pyroptosomes, and impairing the NLRP3 dependent Caspase-1 activation and IL-1β secretion. Importantly, SiiD-deficient SE induced stronger gut inflammation in mice and displayed NLRP3-dependent attenuation of the virulence. SiiD-mediated inhibition of NLRP3 inflammasome activation significantly contributed to SE colonization in the infected mice. This study links bacterial T1SS regulation of mtROS-ASC signaling to NLRP3 inflammasome activation and reveals the essential role of T1SS in evading host immune responses.

Effect of a Saccharomyces cerevisiae Postbiotic Feed Additive on Salmonella Enteritidis Colonization of Cecal and Ovarian Tissues in Directly Challenged and Horizontally Exposed Layer Pullets.

Determining the efficacy of feed-additive technologies utilized as pre-harvest food-safety interventions against Salmonella enterica may be influenced by factors including, but not limited to, mechanism of action, experimental design variables, Salmonella serovar(s), exposure dose, route, or duration in both controlled research and real-world field observations. The purpose of this study was to evaluate the dietary inclusion of a Saccharomyces cerevisiae fermentation-derived postbiotic (SCFP) additive (Diamond V, Original XPC®) on the colonization of cecal and ovarian tissues of commercial pullets directly and indirectly exposed to Salmonella Enteritidis (SE). Four hundred and eighty commercial, day-of-age W-36 chicks were randomly allotted to 60 cages per treatment in two identical BSL-2 isolation rooms (Iowa State University) with four birds per cage and fed control (CON) or treatment (TRT) diets for the duration of study. At 16 weeks, two birds per cage were directly challenged via oral gavage with 1.1 × 109 CFU of a nalidixic-acid-resistant SE strain. The remaining two birds in each cage were thus horizontally exposed to the SE challenge. At 3, 7, and 14 days post-challenge (DPC), 20 cages per group were harvested and sampled for SE prevalence and load. No significant differences were observed between groups for SE prevalence in the ceca or ovary tissues of directly challenged birds. For the indirectly exposed cohort, SE cecal prevalence at 7 DPC was significantly lower for TRT (50.0%) vs. CON (72.5%) (p = 0.037) and, likewise, demonstrated significantly lower mean SE cecal load (1.69 Log10) vs. CON (2.83 Log10) (p = 0.005). At 14 DPC, no significant differences were detected but ~10% fewer birds remained positive in the TRT group vs. CON (p > 0.05). These findings suggest that diets supplemented with SCFP postbiotic may be a useful tool for mitigating SE colonization in horizontally exposed pullets and may support pre-harvest food-safety strategies.

The functional role of fecal microbiota transplantation on Salmonella Enteritidis infection in chicks

The intestinal microbiota plays important roles in animal health and growth. We investigated the efficacy and mechanisms of fecal microbiota transplantation (FMT) from adult SPF chickens against Salmonella Enteritidis (SE) infection in chicks. We transplanted 160 recipient SPF chicks (1-day-old) that were randomly divided into four groups, Ca (challenge), Cb (non-challenge), Fa (FMT and challenge) and Fb (FMT without challenge). The experiment lasted 40 days. We found that FMT reduced mortality as well as liver inflammatory lesions, promoted weight gain, improved immunity, ameliorated the digestion and absorption ability and inhibited SE colonization in the liver of challenged chicks. 16S rRNA gene high-throughput sequencing indicated that SE challenge caused a significant increase in the relative abundance of Parasutterella in the cecal microbiota of the recipient chicks (P < 0.05). FMT led to the maturation of the intestinal flora of recipients and the relative abundance of the Bacteroides, Rikenellaceae_ RC9_ gut_ group, Prevotellaceae_ UCG_ 001, Prevotellaceae_ Ga6A1_ group and Parabacteroides was significantly increased (P < 0.05). FMT from adult SPF chickens regulated the intestinal microbiota of chicks and increased resistance to SE infection.

Effects of a synbiotic combination of Bacillus subtilis and yeast cell wall-derived glucomannan on cecal colonization of Salmonella Enteritidis in layer chickens

Salmonella Enteritidis ( SE ) has been a major foodborne concern associated with poultry and poultry products. Direct-fed microbials ( DFM ), such as Bacillus subtilis strains , and mannan-oligosaccharides ( MOS ) added into poultry feed have emerged as an alternative approach to antimicrobials to reduce Salmonella colonization in chickens. This study was conducted to evaluate the effect of a synbiotic feed additive product (BacPack Q1+1) combining a selected Bacillus subtilis strain and yeast cell wall-derived MOS on SE colonization in the ceca of Hy-line W36 pullets. Day-old chicks were raised in cage units and orally challenged at 3 wk-of-age with a nalidixic acid resistant SE strain at a dose of 1.6 × 10 9 colony forming units ( CFU ) per bird. SE colonization was assessed by enumeration of SE in cecal pouches at 7, 10 and 13 days postinfection ( dpi ). SE was not detectable in the ceca of 68% of synbiotic supplemented pullets compared to 36% of the controls at 13 dpi ( P = 0.049). The findings of this study suggest that synbiotic combinations of DFM and prebiotics could be a useful tool for reducing the prevalence of Salmonella in layer chickens.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Synergistic effects of the early administration of Lactobacillus kefiranofaciens DN1 and Kluyveromyces marxianus KU140723-05 on the inhibition of Salmonella Enteritidis colonization in young chickens

In this study, we aimed to assess the feasibility of the lactic acid bacterium Lactobacillus kefiranofaciens DN1 (LKF_DN1) and the yeast Kluyveromyces marxianus KU140723-05 (KMA5), recently isolated from kefir, as probiotics. Specifically, we evaluated the effect of early administration of these 2 microbes on the inhibition of Salmonella Enteritidis (SE) colonization in neonatal chicks. We also examined the effects of exposure of chicks to probiotics before SE exposure on the reduction in the number of gut SE. A total of 108 1-day-old specific-pathogen-free male layer chicks were used for 3 independent experiments. The experimental chicks were randomly divided into 6 groups (negative control: basal diet [BD] without probiotics and SE; positive control: BD; probiotic group [PG] 1: BD + LKF_DN1; PG2: BD + KMA5; PG3: BD + LKF_DN1 + KMA5; and PG4: BD+ a commercial product IDF-7), all of which, except negative control, were coadministered with SE strain resistant to rifampicin (SERR). We found that the administration of LKF_DN1 and/or KMA5 reduced the number of viable cells of the SERR strain in chicks by up to 1.90 log10, relative to positive control chicks. Compared with late administration (day [D] 10 and D11), early administration (D1 and D2) of the probiotics was more effective in reducing SERR cell numbers in the gut. Furthermore, we detected no significant difference in the reduction of gut SERR cell numbers in chicks from the same groups exposed to the probiotics at D10 and D11 before and after administration with SERR. Collectively, our findings indicate that, as dietary additives, LKF_DN1 and KMA5 showed potential probiotic activity in chicks. Moreover, the combination of the lactic acid bacteria and/or yeast strain was found to rapidly reduce SE numbers in the chick gut and showed a prolonged inhibitory effect against SE colonization. We, thus, propose that the administration of these 2 probiotics, as early as possible after hatching, would be considerably effective in controlling SE colonization in the guts of chicks.

Effects of a mannan-rich yeast cell wall-derived preparation on cecal concentrations and tissue prevalence of Salmonella Enteritidis in layer chickens.

Salmonella Enteritidis (SE) has been the most common Salmonella serotype associated with foodborne infections in the last several years. Dietary applications of yeast-based preparations in feed have shown to reduce Salmonella colonization in chickens augmenting SE control strategies. This study was conducted to evaluate the effects of a mannan-rich yeast cell wall-derived preparation (Actigen®) administered in feed at a rate of 400 g/ton on SE colonization in the cecum and internal organs of commercial layer chickens. Sixteen week-old layer pullets were orally challenged with a selected nalidixic acid resistant SE strain at a dose of 1.7×10^9 colony forming units (CFU) per bird. SE colonization was assessed by evaluating isolation rates from ovary and pooled liver/spleen samples as well as enumeration of SE in cecal pouches one week post-challenge. Recovery rates of SE from the ovaries of directly challenged birds receiving Actigen® were significantly lower (P <0.02) than those in directly challenged birds fed an unsupplemented control diet. Recovery rates of SE from pooled liver/spleen samples were not significantly different between Actigen®-treated pullets and controls (P = 0.22). Using direct plate count methods, cecal SE concentrations were 1 log10 lower (P <0.001) in challenged pullets in the Actigen®-supplemented group than in the challenged controls. The SE concentration distributions in the ceca were similar in groups testing positive and groups testing negative for SE in the ovaries and liver/spleens tissues. As a result, SE concentrations in the ceca could not be directly related to the occurrence or prevalence of SE in these tissues. In conclusion, Actigen® supplementation appears to decrease the prevalence of SE in ovarian tissue and concentrations of SE in cecal contents and may be useful as a tool for reducing the risk of eggshell contamination and transovarian transmission of SE in eggs.

Integrative analysis of gut microbiome and metabolites revealed novel mechanisms of intestinal Salmonella carriage in chicken

Intestinal carriage of Salmonella Enteritidis (SE) in the chicken host serves as a reservoir for transmission of Salmonella to humans through the consumption of poultry products. The aim of the current study was to examine the three-way interaction that occurred between host metabolites, resident gut microbiota and Salmonella following inoculation of SE in two-week-old layer chicks. Our results revealed an overall alteration in gut microbiome and metabolites in association with SE infection. Enriched colonization by different microbial members throughout the course of experimental infection highlighted significant fluctuation in the intestinal microbial community in response to Salmonella infection. As changes in community membership occurred, there was also subsequent impact on differential regulation of interlinked predicted functional activities within the intestinal environment dictated by Salmonella-commensal interaction. Alteration in the overall microbial community following infection also has a ripple effect on the host regulation of cecum-associated metabolic networks. The findings showed that there was differential regulation in many of the metabolites in association with SE colonization in chickens. Perturbation in metabolic pathways related to arginine and proline metabolism as well as TCA cycle was most prominently detected. Taken together, the present findings provided a starting point in understanding the effect of intestinal Salmonella carriage on the microbiome and metabolome of developing young layer chicks.

Response to a Salmonella Enteritidis challenge in old laying hens with different vaccination histories.

Extending the laying period of laying hens is beneficial for economic and sustainability purposes. Because vaccines were designed with a shorter laying period envisaged, it is unclear whether current Salmonella vaccines can provide sufficient levels of protection against infection at an older age. The purpose of this experiment was to determine the efficacy of early rearing vaccination schemes against Salmonella challenge late in the laying period. There were four treatment groups: birds that had not been vaccinated (Group 1), birds vaccinated with live Salmonella Enteritidis (SE) (Group 2), with live and inactivated SE (Group 3), or with live SE and live Salmonella Typhimurium (Group 4). At the end of the laying period, the birds were transported from the laying farm to the research facility where they were orally challenged with 2.06 × 109 colony-forming units SE at around 82 wk of age. Hens were euthanized and bacteriology was performed on cecum, liver, spleen, and follicular fluid samples to determine SE colonization 7 and 14 d after challenge. Clinical and bacteriological findings of hens vaccinated with different vaccination schemes and the non-vaccinated control group were compared. No significant differences in SE colonization were found for vaccinated groups compared to the non-vaccinated control group. This may be a result of waning immunity due to the long time between vaccination and challenge. Also, as vaccination took place in the rearing period in the field, initial levels of immunity may not have been optimal due to shortcomings in the vaccination technique. Furthermore, the results of this study may have been affected by differences in age, breed, and origin between the groups. Therefore, controlled studies from early age onwards are necessary for more accurate comparisons between vaccines.

Evaluation of a Solid Dispersion of Curcumin With Polyvinylpyrrolidone and Boric Acid Against Salmonella Enteritidis Infection and Intestinal Permeability in Broiler Chickens: A Pilot Study

In the present study, in vitro assays were conducted to evaluate the solubility of curcumin (CUR) alone or with polyvinylpyrrolidone (PVP) at different pH, as well as its permeability in Caco-2 cells. Results confirmed that the solid dispersion of CUR with PVP (CUR/PVP) at a 1:9 ratio, significantly increased (P < 0.05) solubility and permeability compared to CUR alone. Then, the antimicrobial activity of CUR/PVP, boric acid (BA), and a combination of 0.5% CUR/PVP and 0.5% BA (CUR/PVP-BA) against Salmonella Enteritidis (SE) was determined using an in vitro digestion model that simulates crop, proventriculus, and intestine. The results revealed that in the proventriculus and intestinal compartments significant reductions of SE were observed in all the experimental treatments, but 1% BA eliminated SE in the intestinal compartment and CUR/PVP-BA showed a synergistic effect on antimicrobial activity against SE. To complement these findings, two independent in vivo trials were conducted to determine the effect of 0.1% CUR/PVP; 0.1% BA; or the combination of 0.05% CUR/PVP (1:9 ratio) and 0.05% BA (CUR/PVP-BA) on the antimicrobial activity against SE, intestinal permeability and inflammatory responses in broiler chickens. BA at 0.1% had no significant in vivo effects against SE. However, the combination of 0.05% BA and 0.05% CUR/PVP and 0.05% BA was sufficient to reduce crop and intestinal SE colonization in broiler chickens in two independent trials, confirming the synergic effect between them. A similar antimicrobial impact against SE intestinal colonization was observed in chickens treated with 0.1% CUR/PVP at a 1:9 ratio, which could be due to the increase in solubility of CUR by PVP. Furthermore, 0.1% CUR/PVP reduced the intestinal permeability of FITC-d and total intestinal IgA, as well as increase the activity of SOD when compared to control, while, CUR/PVP-BA only decreased SOD activity. Further studies to confirm and expand the in vivo results obtained in this pilot study, adding intestinal microbial commensal groups and more inflammatory biomarkers to get a complete description of the effects of BA and CUR deserves further investigation.

Colonization of mature laying hens with Salmonella Enteritidis by oral or intracloacal inoculation

Evidence of Salmonella Enteritidis (SE) in internal organs of White Leghorns once they are inoculated via the oral (OR) or intracloacal (IC) route has not been consistently demonstrated. The aim of the current study was to evaluate OR or IC inoculation route of a nalidixic acid (Nal) resistant SE (SENAR) on the SE colonization of ceca and the invasion of internal organs in mature White Leghorns. Five experiments were conducted, and hens were inoculated with 108 colony-forming units (cfu) of SENAR. Hens were euthanized at 7 and 14 d post inoculation (dpi), and the ceca, spleen, liver with gall bladder (L/GB), and ovaries were collected for bacteriological analyses. The recovery of SENAR in ceca was 100% at 7 dpi. Recovery from the ovaries was lower than the other organs for both routes of inoculation. The SE recovery of L/GB, spleen, and ovaries at 7 dpi was not different between the two routes. By 14 dpi, all organs approached negative, and the recovery rate was similar between OR and IC. Fecal shedding was 100% positive at 3 dpi and reduced to almost 0% by 14 dpi. Mature hens were colonized by SENAR with either OR or IC inoculation when using a larger volume and a higher cfu/mL (0.1 mL OR in experiment 1 vs. 1.0 mL OR and IC in the rest). SENAR showed some translocation into other organs, to a greater extent with IC. The colonization did not persist either in ceca or the internal organs at 14 dpi.

Reducing Colonization and Eggborne Transmission of Salmonella Enteritidis in Layer Chickens by In-Feed Supplementation of Caprylic Acid.

Salmonella Enteritidis (SE) is a major foodborne pathogen responsible for causing gastrointestinal infections in humans, predominantly due to the consumption of contaminated eggs. In layer hens, SE colonizes the intestine and migrates to various organs, including the oviduct, thereby leading to egg yolk and shell contamination. This study investigated the efficacy of caprylic acid (CA), a medium-chain fatty acid, in reducing SE colonization and egg contamination in layers. Caprylic acid was supplemented in the feed at 0%, 0.7%, or 1% (vol/wt) from day 1 of the experiment. Birds were challenged with 10(10) log colony-forming units (CFU)/mL of SE by crop gavage on day 10, and re-inoculated (10(10) log CFU/mL) on day 35. After 7 days post first inoculation, eggs were collected daily and tested for SE on the shell and in the yolk separately. The birds were sacrificed on day 66 to determine SE colonization in the ceca, liver, and oviduct. The consumer acceptability of eggs was also determined by triangle test. The experiment was replicated twice. In-feed supplementation of CA (0.7% and 1%) to birds consistently decreased SE on eggshell and in the yolk (p<0.05). Supplementation of CA at 1.0% decreased SE population to ≈14% on the shell and ≈10% in yolk, when compared to control birds, which yielded ≈60% positive samples on shell and ≈43% in yolk. Additionally, SE populations in the cecum and liver were reduced in treated birds compared to control (p<0.05). No significant difference in egg production, body weight, or sensory properties of eggs was observed (p>0.05). The results suggest that CA could potentially be used as a feed additive to reduce eggborne transmission of SE.

Effect of Plant Derived Antimicrobials on Salmonella Enteritidis Adhesion to and Invasion of Primary Chicken Oviduct Epithelial Cells in vitro and Virulence Gene Expression

Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States and one of the most frequently reported Salmonella serotypes globally. Eggs are the most common food product associated with SE infections in humans. The pathogen colonizes the intestinal tract in layers, and migrates to reproductive organs systemically. Since adhesion to and invasion of chicken oviduct epithelial cells (COEC) is critical for SE colonization in reproductive tract, reducing these virulence factors could potentially decrease egg yolk contamination. This study investigated the efficacy of sub-inhibitory concentrations of three plant-derived antimicrobials (PDAs), namely carvacrol, thymol and eugenol in reducing SE adhesion to and invasion of COEC, and survival in chicken macrophages. In addition, the effect of PDAs on SE genes critical for oviduct colonization and macrophage survival was determined using real-time quantitative PCR (RT-qPCR). All PDAs significantly reduced SE adhesion to and invasion of COEC (p < 0.001). The PDAs, except thymol consistently decreased SE survival in macrophages (p < 0.001). RT-qPCR results revealed down-regulation in the expression of genes involved in SE colonization and macrophage survival (p < 0.001). The results indicate that PDAs could potentially be used to control SE colonization in chicken reproductive tract; however, in vivo studies validating these results are warranted.

Genetic differ in TLR4 gene polymorphisms and expression involved in Salmonella natural and artificial infection respectively in Chinese native chicken breeds

Salmonella enteritidis (SE) is a foodborne pathogen that negatively affects both animal and human health. Genetic variations in response to pathogenic SE colonization or to SE vaccination were measured in chicken resource populations. Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. In this study, TLR4 was investigated the association of TLR4 gene polymorphisms with Salmonella natural infection situation of birds from two distinct Chinese genetic breeds. One SNP G1894C in the second intron of chicken TLR4 (chTLR4) was scanned in the two hens breed, which showed significant association with Salmonella natural infection situation (P<0.05). Genetic variations in response to pathogenic SE colonization also existed in distinct Chinese chicken resource population. In this study, mRNA expression of TLR4 was detected to investigate the association with the effect of artificial SE challenge in heterophil granulocytes and spleen of chicks from two distinct Chinese genetic breeds at 1, 3 and 10 day post-infection during the acute infection period. It clearly showed that young chicks' response to SE infection was regulated by TLR4 mRNA expression. The results suggest that genetics, time, gender, and interactions among these factors, play important roles in TLR4 mRNA basic values and copies modulation of SE mediated immune response in distinct Chinese chickens.

Temporal changes in the expression of avian β-defensins in the chicken vagina during sexual maturation and Salmonella infection

Avian β-defensins (AvβDs) constitute a family of antimicrobial peptides that are critical to innate immunity in chickens, providing protection against microbial pathogens including Salmonella Enteritidis (SE). As apart from the digestive tract another main route of SE colonization in birds is via infection of the oviduct and specifically of the vagina, the aim of this study was to investigate the expression of the complete family of AvβDs, in the chicken vagina in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the vaginal AvβDs expression. Expression analysis revealed that 11 members of the AvβD family were expressed in the chicken vagina. Quantitative real-time PCR analysis revealed that the mRNA abundance of five AvβDs was up regulated and of one AvβD was down regulated with respect to sexual maturation. In addition SE infection resulted in a significant induction of AvβD5, 7, 10, 11, 12 and 14 in the vagina of sexually mature birds, and in a significant induction of AvβD5 and 11 in the vagina of aged birds. These findings provide strong evidence to suggest that an AvβD-mediated immune response mechanism exists in the chicken vagina providing protection against bacterial pathogens including Salmonella species.

Use of bacteriophage for biological control of Salmonella Enteritidis infection in chicken

Bacteriophage ΦCJ07 with broad host ranges for Salmonella strains isolated from sewage effluent were used to reduce Salmonella Enteritidis (SE) infection in chickens. One-day-old chicks challenged with 5×107 colony-forming units/bird of SE were cohabitated with contact chicks and treated with three concentrations (105, 107 and 109 plaque forming units (PFU)/g) of bacteriophage prepared as a feed additive for 21days after challenge. Salmonella in the intestine was quantified and environmental contamination level was examined at 1, 2 and 3weeks after challenge. All treatments reduced intestinal SE colonization in challenged and contact chickens and reduced the environmental contamination level, but the reductions produced by 107 and 109PFU/g of bacteriophage were significant (P<0.05) as compared with untreated controls. In addition, seven out of 10 (70%) contact chickens treated with 109PFU/g of bacteriophage had no detectable intestinal Salmonella at 3weeks after treatment, suggesting that bacteriophage therapy significantly prevented the horizontal transmission of SE. These results provide important insights into preventive and control strategies against SE infection in poultry and indicate that the use of bacteriophage could reduce the incidence of Salmonella food poisoning.

Aerosol Spray Treatment with Bacteriophages and Competitive Exclusion Reduces Salmonella Enteritidis Infection in Chickens

A combination of three different Salmonella-specific bacteriophages (BPs) and one competitive exclusion (CE) product were used to reduce Salmonella Enteritidis (SE) colonization in experimentally infected chickens. Equal numbers of 7-day-old chickens were used in each of three groups: a CE group (treated with CE), a BP group (treated with BP), and a CE-plus-BP group (treated with both products). The CE product was administered via coarse spray at 1 day of age and the cocktail of three BPs was given via spray at 6 days of age using a multiplicity of infection of 10(3) plaque-forming units. All the experimental groups, except a healthy control group, were challenged orally with 2.95 x 10(5) colony-forming units (CFU)/ml of an SE strain at 7 days of age. Seven days postchallenge, the chickens were euthanatized for individual SE detection, quantitative bacteriology, and phage isolation from ceca and an internal organ pool. The qualitative bacteriology demonstrated that the use of the CE product diminished the incidence of SE to 75.7% and the mixture of BPs reduced it to 80%; when CE plus BP were used, the incidence dropped significantly to 38.7% (P < 0.0001), as compared with the infection control group (100%). A significant difference in the incidence was observed between the CE and the CE-plus-BP groups, and the BP and the CE-plus-BP groups (P = 0.0027 and P = 0.0010, respectively). The mean SE cecal count diminished with the use of CE plus BP (1.6 x 10(2) CFU/g, P = 0.0003) compared with the control group (1.56 x 10(5) CFU/g), the CE group (4.23 x 10(3) CFU/g), and the BP group (9.48 x 10(3) CFU/g). On the basis of the present study, it may be concluded that the use of both types of biocontrollers can be an effective method for reducing SE colonization in commercial chickens, but further basic and applied research is needed.