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ABSTRACT: Lactic acid bacteria (LAB) play a key role in regulating the balance of gut microbiota and serve as a suitable alternative to antibiotics. This study aims to evaluate the characteristics of 2 LAB isolates Lactiplantibacillus plantarum Lp71 (L. plantarum Lp71) and Enterococcus faecium Ef72 (E. faecium Ef72), and their roles in alleviating Salmonella Enteritidis infection. Sixty 1-day-old chicks were randomly divided into 4 groups which treated with or without L. plantarum Lp71 and E. faecium Ef72 mixture for 21 d, and then intestinal samples were collected for gut microbiota analysis, pathological and immunohistochemical analysis at 24 h post infection with or without Salmonella Enteritidis on the 22nd d. The results showed that L. plantarum Lp71 and E. faecium Ef72 had the ability to anti-acid and anti-bile salt. Salmonella Enteritidis infection damaged the intestinal epithelial barrier and reduced the expression level of tight junction proteins (ZO-1, Claudin-1, Occludin). Oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could alleviated the damages to intestinal epithelial barrier by Salmonella Enteritidis infection. Salmonella Enteritidis could cause abnormal Akkermansia muciniphila proliferation and decrease the diversity of cecal microbiota in chicks. These conditions could have further led to reduce gut microbiota health index (GMHI), and improve microbial dysbiosis index (MDI). Moreover, oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could effectively prevent the aforementioned infection outcomes and increase the abundance proportions of the several key functions in metabolic pathways metabolic pathways such as transcription and signal transduction mechanisms. In summary, L. plantarum Lp71 and E. faecium Ef72 could be the probiotics candidates that used to prevent the damage from enteric pathogens such as Salmonella Enteritidis in broiler chicks.

The antipathogenic efficacy of starch/nanoclay biocomposites containing Thymus fedtschenkoi Ronniger essential oil (TFEO) and thymoquinone (TQ) against Listeria monocytogenes and Salmonella enteritidis inoculated in chilled chicken fillets during 12-days of storage was investigated. A dose-response antibacterial effect was observed for all active ingredients (TFEO (0.5 and 1 %) and TQ (0.03 and 0.06 %) individually and in a combination incorporated into starch nanocomposite coating based on well diffusion assay. The inhibitory effect against inoculated L. monocytogenes was better than S. enteritidis for all treatments in chicken fillets. It was more influential at combined concentrations versus both bacteria at an undetected level for L. monocytogenes and a reduction of S. enteritidis by 2.4 log CFU/mL at the end of cold storage. Starch bio-nanocomposites enriched with TFEO and TQ can be proposed as an innovative bioactive packaging material to control the growth of pathogens and increase the microbial safety of meat and meat products.

ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACT: In the face of the increasingly severe problem of antibiotic resistance, phage therapy is regarded as a highly potential alternative. Compared with traditional antimicrobial agents, a key research area of phage therapy is the study of phage-resistant mutant bacteria. To effectively monitor and prevent this resistance, it is crucial to conduct in-depth exploration of the mechanism behind phage resistance. In this study, a strain of Salmonella enteritidis (sm140) and the corresponding phage (Psm140) were isolated from chicken liver and sewage, respectively. Using the double-layer plate method, successfully screened out phage-resistant mutant strains. Whole-genome resequencing of 3 resistant strains found that the wbaP gene of all 3 strains had mutations at a specific position (1,118), with the base changing from G to A. This mutation causes the gene-encoded glycine to be replaced by aspartic acid. Subsequent studies found that the frequency of this gene mutation is extremely high, reaching 84%, and all mutations occur at the same position. To further explore the relationship between the wbaP gene and phage resistance, knockout strains and complement strains of the wbaP gene were constructed. The experimental results confirmed the association between the wbaP gene and phage resistance. At the same time, biological characteristics and virulence were evaluated for wild strains, resistant strains, knockout strains, and complement strains. It was found that mutations or deletions of the wbaP gene lead to a decrease in bacterial environmental adaptability and virulence. Through systematic research on the mechanism and biological characteristics of phage resistance, this study provides important references and guidance for the development of new phage therapies, promoting progress in the field of antimicrobial treatment. At the same time, the emergence of phage resistance due to wbaP gene mutations is reported for the first time in salmonella, providing a new perspective and ideas for further studying phage resistance mechanisms.

ABSTRACT: In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Salmonella Enteritidis (SE) is a significant global cause of foodborne illness, often linked to egg contamination. This study evaluated the inhibitory effects of eight bacteriophages (phages) against three SE strains isolated from poultry environments. The most effective phages were selected to formulate different phage cocktails, to enhance the efficacy and prolong inhibition. Four phage cocktails were tested at a multiplicity of infection (MOI) of 100 in tryptic soy broth (TSB), and at MOIs of 100 and 1000 in liquid egg white (EW) and egg yolk (EY) with storage at 8 °C for up to 30 days (d). The effectiveness of the phage cocktails varied significantly among bacterial strains, yet all demonstrated significant reductions compared to the positive control in liquid culture (P

ABSTRACT: Salmonella and Campylobacter are common foodborne pathogens in chickens, but their persistence mechanisms within flocks are not fully understood. In this study, 4 groups of SPF Leghorn chickens (n = 50) were orally inoculated with 108 Salmonella Enteritidis and 108 Campylobacter jejuni, housed in BSL-2 rooms inside containers with autoclaved bedding and beetles (n = 200). Phase I (wk 1–3): the infected chickens remained in the containers and were then euthanized while beetles and litter remained in the container (group A), beetles were removed and litter remained in the container (group B), beetles remained and litter was removed (group C), and beetles and litter were removed (group D). Phase II (wk 5–7): autoclaved bedding was added to containers in groups C and D, and new SPF chickens (n = 50) were introduced and kept. Phase III (wk 8–20): all chickens were euthanized, and the litter and/or beetles remained in the containers for 17 wk. The prevalence of Salmonella Enteritidis and Campylobacter was significantly higher when detected by PCR compared to culture. In phase II, when infected chickens were removed and new chickens were introduced, 1 fecal sample in group B and 3 litter samples in groups B and C were found positive for Salmonella Enteritidis, and Campylobacter was still detected in groups A, B, and C litter samples, but not in beetles. In phase III, when all chickens were removed, Salmonella Enteritidis was identified in beetle samples from group A and the litter samples of all tested groups A, B, and C, and C. jejuni was positive in litter samples from groups A and B but not in the beetle. Sixty-nine days after removing infected chickens, culturable Salmonella was still found in beetles. Salmonella and Campylobacter were detectable in litter up to 127 d after removing infected chickens. This study highlights the transmission of Salmonella and Campylobacter via beetles and litter to new flocks in successive rearing cycles. Intensive control programs should target insect exclusion and implement strict poultry litter management or litter changes between flocks.

ABSTRACT: The poultry industry has not been spared from the prevalent incidence of diseases caused by invasive pathogens, especially Salmonella. Due to the pressing need to identify a suitable antibiotic alternative for use in poultry production, this study investigated the efficacy of red osier dogwood (ROD) extract on the growth, blood parameters, gut morphology, and Salmonella excretion in broiler chickens orally challenged with Salmonella Enteritidis (SE). A 4 × 2 factorial experiment was conducted based on 2 main factors, namely dietary treatments, and SE challenge. A total of 404, one-day-old male Ross broiler chicks were randomly assigned to 4 dietary treatments; 1) Negative control (NC), 2) NC + 0.075 ppm of Trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, 3) NC + 0.3% ROD extract, and 4) NC + 0.5% ROD extract. The absence of SE in the fecal samples obtained from chick delivery boxes was confirmed on d 0. On d 1, half of the birds were orally gavaged with 0.5 mL of phosphate-buffered saline each (noninfected group) and the remaining with 0.5 mL of 3.1 × 105 CFU/mL SE (infected group) in all treatment groups. Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On 1-, 5-, 12-, and 18-day postinfection (DPI), cloacal fecal samples were collected on the 6 birds/cage to assess SE excretion. Average weight gain (AWG), average feed intake (AFI), feed conversion ratio (FCR), and mortality were determined weekly. On d 21, 10 chickens/treatment were euthanized to perform hematology, gut histomorphometry, serum immunoglobulins G and M (IgG and IgM), and superoxide dismutase measurements. Both ROD extract levels did not affect (P > 0.05) growth performance; however, the SE-infected birds showed increased (P < 0.05) AFI and FCR throughout the experimental period. Regardless of the SE-infection, both ROD extract levels improved (P < 0.05) duodenal villus height: crypt depth compared to other treatments. 0.5% ROD extract improved (P < 0.05) ileal villus width (VW) of noninfected birds and ileal crypt depth of infected birds, but it decreased (P < 0.05) the ileal VW of infected birds, compared to other treatments. The SE-infected birds showed lower (P < 0.05) lymphocytes (L) but increased (P < 0.05) heterophils (H), H:L, and monocytes (MON). Both ROD extract levels did not affect (P > 0.05) white blood cell differential, while dietary 0.3% ROD extract increased (P < 0.05) MON of the birds, regardless of infection model. Regardless of infection model, both TMP/SDZ and 0.5% ROD extract reduced the concentration of IgM in the serum, compared to the control and 0.3% ROD (P = 0.006). Conclusively, both ROD extract levels improved duodenal histomorphology and body defense against SE infection in broiler chickens; however, the 0.3% ROD extract was better.

ABSTRACT: Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.

SUMMARY: A study was conducted to assess the effectiveness of yeast-derived products in the recovery of Salmonella Enteritidis (SE) in White Leghorns. A total of 32 hens at 38 wk of age were challenged both orally (OR) and intracloacally (IC) with nalidixic acid resistant SE (SENAR). Hens were individually housed in conventional wire laying cages and randomly allocated to 4 treatments (T) with 8 hens per replicate: 1) No SENAR challenge (T1; basal diet), 2) SENAR challenge (T2; basal diet), 3) SENAR challenge: basal diet with a blend of Saccharomyces-Torula yeasts and yeast cell walls (T3; intact yeast), and 4) SENAR challenge: basal diet with enzymatically-modified yeast products blend (T4; enzyme-modified yeast). The excreta samples were 100% positive for SENAR at 3 d postinfection (dpi) in T2, T3, and T4. By 6 dpi, excreta samples were 87, 62 and 37% positive for T2, T3, and T4, respectively. Hens were euthanized at 7 dpi to harvest liver with gall bladder (L/GB), spleen, ovaries, and ceca. The recovery of SENAR in L/GB was 75, 25, and 37% in T2, T3, and T4 (P < 0.05), whereas that of the spleen was 62, 25 and 37% in T2, T3 and T4 (P < 0.05), respectively. Treatments 3 and 4 reduced SENAR counts (cfu/g) in the ceca from 4.3 log10 to 2.7 and 1.95 log10, respectively, compared to T2 (P < 0.05). The cecal count of SENAR was 1.6 log10 lower in T3 and 2.3 log10 lower in T4 compared to T2. Both yeast derived products reduced Salmonella in internal organs and the ceca of laying hens. Enzymatically-modified yeast (T4) was better than its intact counterpart (T3) in reducing Salmonella counts in the ceca.

Salmonella Enteritidis (SE) is an important foodborne pathogen primarily causing human disease through contaminated food and water. In the current study, to assess the effect of Salmonella Enteritidis infection on the immune system and the microbial diversity of cecum and oviduct in chickens, twelve 24-week-old SE-negative White Leghorn layers were randomly selected and divided into 2 groups. Chickens in the challenge group were orally inoculated with SE, and chickens in the control group received an equal amount of sterilized Phosphate Buffered Saline solution. Serum and tissue samples (cecum, oviduct, ovary, liver, spleen, and pancreas) were collected at 7 days and 14 days post-infection (dpi). Quantitative PCR was used to detect the expression of Toll-like receptors (TLRs) in the cecum, oviduct and ovary. To understand the influence of SE infection on the microbial profile of the cecum and oviduct, microbial community composition of the cecal contents and oviducal contents were analyzed through 16S rRNA sequencing. Results showed that SE infection caused damage to the digestive organs, reproductive organs, and immune organs in laying hens. The expression of TLR1a, TLR1b, TLR2, TLR4, TLR5, TLR7 and TLR15 in the cecum were induced, and the content of IFN-γ, TNF-α, IL-2 and IL-18 in serum increased after SE infection. The composition of the microbial community significantly changed in cecal content, the dominant phylum of Firmicutes increased, and Bacteroidetes decreased significantly. In the oviduct, the microbial diversity became complicated, the dominant bacteria Faecalibacterium was significantly increased, and Bacteroides was significantly decreased. This study investigated the effects of SE infection in laying hens, including host innate immunity, the expression of TLRs, and changes in the composition of microbes in the cecum and reproductive tract. Our results may provide a scientific basis for the Salmonella Enteritidis control in chicken, the maintenance of oviduct function, and the guarantee of clean egg production.

Objectives: To enhance monitoring of high-burden foodborne pathogens, there is opportunity to combine pangenome data with network analysis. Methods: Salmonella enterica subspecies Enterica serovar Enteritidis isolates were referred to the New South Wales (NSW) Enteric Reference Laboratory between August 2015 and December 2019 (1033 isolates in total), inclusive of a confirmed outbreak. All isolates underwent whole genome sequencing. Distances between genomes were quantified by in silico multiple-locus variable-number tandem repeat analysis (MLVA) as well as core single nucleotide polymorphisms (SNPs), which informed the construction of undirected networks. Centrality-prevalence spaces were generated from the undirected networks. Components on the undirected SNP network were considered alongside a phylogenetic tree representation. Results: Outbreak isolates were identified as distinct components on the MLVA and SNP networks. The MLVA network-based centrality-prevalence space did not delineate the outbreak, whereas the outbreak was delineated in the SNP network-based centrality-prevalence space. Components on the undirected SNP network showed a high concordance to the SNP clusters based on phylogenetic analysis. Conclusions: Bacterial whole-genome data in network-based analysis can improve the resolution of population analysis. High concordance of network components and SNP clusters is promising for rapid population analyses of foodborne Salmonella spp. owing to the low overhead of network analysis.

ABSTRACT: Salmonella Enteritidis is a major foodborne pathogen that causes enteric illnesses in humans, primarily through the consumption of contaminated poultry meat and eggs. Despite implementation of traditional disinfection approaches to reduce S. Enteritidis contamination, egg-borne outbreaks continue to occur, raising public health concerns and adversely affecting the popularity and profitability for the poultry industry. Generally Recognized as Safe (GRAS) status phytochemicals such as Trans-cinnamaldehyde (TC) have previously shown to exhibit anti-Salmonella efficacy, however, the low solubility of TC is a major hurdle in its adoption as an egg wash treatment. Therefore, the present study investigated the efficacy of Trans-cinnamaldehyde nanoemulsions (TCNE) prepared with emulsifiers Tween 80 (Tw.80) or Gum Arabic and lecithin (GAL) as dip treatments, at 34°C, for reducing S. Enteritidis on shelled eggs in presence or absence of 5% chicken litter. In addition, the efficacy of TCNE dip treatments in reducing trans-shell migration of S. Enteritidis across shell barrier was investigated. The effect of wash treatments on shell color were evaluated on d 0, 1, 7, and 14 of refrigerated storage. TCNE-Tw.80 or GAL treatments (0.06, 0.12, 0.24, 0.48%) were effective in inactivating S. Enteritidis by at least 2 to 2.5 log cfu/egg as early as 1 min of washing time (P < 0.05). In presence of organic matter, nanoemulsions (0.48%) reduced S. Enteritidis counts by ∼ 2 to 2.5 log cfu/egg as early as 1 min, (P < 0.05). Nanoemulsion wash also inhibited trans-shell migration of S. Enteritidis, as compared to control (P < 0.05). The nanoemulsion wash treatments did not affect shell color (P > 0.05). Results suggest that TCNE could potentially be used as an antimicrobial wash to reduce S. Enteritidis on shelled eggs, although further studies investigating the effect of TCNE wash treatments on organoleptic properties of eggs are necessary.

ABSTRACT: With the subsisting restrictions on the use of antibiotics in poultry production, the use of plant extracts has shown some promising antimicrobial capacity similar to antibiotics; however, such capacity is largely dependent on their total polyphenol concentration and profile. Given the emerging antimicrobial potential of red osier dogwood (ROD) extract, the study aimed to investigate the pharmacodynamic effect of ROD extract on the ileal and cecal microbiota of broiler chickens challenged orally with Salmonella Enteritidis (SE). A 21 d 4 × 2 factorial experiment was conducted based on 2 main factors, including diets and SE challenge. A total of 384 one-day-old mixed-sex Cobb-500 broiler chicks were randomly allotted to 4 dietary treatments; Negative control (NC), NC + 0.075 mg trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, and NC containing either 0.3 or 0.5% ROD extract. On d 1, half of the birds were orally challenged with 0.5 mL of phosphate-buffered saline (Noninfected group) and the remaining half with 0.5 mL of 3.1 × 105 CFU/mL SE (Infected group). Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On d 21, 10 birds/treatment were euthanized and eviscerated to collect ileal and cecal digesta for gut microbiota analysis. The ileal and cecal microbiota was dominated by phyla Firmicutes, Proteobacteria, and Actinobacteriota. The SE infection decreased (P < 0.05) the relative abundance of Proteobacteria and Actinobacteriota in the ileum and ceca, respectively, however, it increased (P < 0.05) Proteobacteria in the ceca. Both 0.3 and 0.5% ROD extracts (P < 0.05) depressed the relative abundance of Actinobacteriota in the ileum but marginally improved (P < 0.05) it in the ceca compared to the TMP/SDZ treatment. Dietary TMP/SDZ increased (P < 0.05) genus Bifidobacterium at the ileal and cecal segments compared to other treatments. Dietary 0.3 and 0.5% marginally improved (P < 0.05) Bifidobacterium in the ceca and depressed (P < 0.05) Weissella and was comparably similar to TMP/SDZ in the ileum. Regardless of the dietary treatments and SE infection, alpha diversity differed (P < 0.05) between ileal and cecal microbiota. Beta diversity was distinct (P < 0.05) in both ileal and cecal digesta along the SE infection model. Conclusively, both ROD extract levels yielded a pharmacodynamic effect similar to antibiotics on ileal and cecal microbiota.

Background: Salmonella Enteritidis is one of the main pathogens of foodborne diseases and an important pathogen causing diarrhea in yaks. Antibiotics are the mainstay of treatment for salmonellosis, but the widespread use of antibiotics has increased Salmonella resistance. Probiotics have been shown to antagonize Salmonella and reduce Salmonella infection. Bacillus pumilus is one of the microbial feed additives approved by the Chinese Ministry of Agriculture for use in animal breeding, which has the effect of improving animal growth performance and immunity, among others. Therefore, this paper explored the anti-infective effect of Bacillus pumilus against Salmonella., Results: Bacillus pumilus SMU5927 significantly enhances the intestinal mechanical barrier and reduces the number of Salmonella transferred to the organs. Bacillus pumilus SMU5927 ameliorated intestinal tissue damage and attenuated intestinal inflammatory responses in mice. In addition, Bacillus pumilus increased the ratio of the Firmicutes/Bacteroidetes in the intestinal flora, increased the abundance of beneficial bacteria such as Lactobacillus, and decreased the abundance of harmful bacteria., Conclusion: This study confirmed the role of Bacillus pumilus SMU5927 in preventing and attenuating Salmonella damage and provided ideas for the development of novel antimicrobial drugs., Competing Interests: Declaration of competing interest None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C-20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log 10  CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R 2  = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of -0.11 log 10  CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R 2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products., Competing Interests: Declaration of competing interest The authors declare that they have no financial or non-financial interests that could be perceived as potential conflicts of interest regarding this research. None of the authors have received any form of compensation, including honoraria or consultancy fees, from any organization that may gain or lose financially from the publication of this manuscript. The authors have no financial or personal relationships that could bias this work or create a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Adaptation to sublethal amounts of ethanol enables Salmonella Enteritidis to survive under normally lethal ethanol conditions, which is referred to as the ethanol tolerance response (ETR). To uncover mechanisms underlying this adaptative response, RNA-seq and RT-qPCR techniques were employed to reveal global gene expression patterns in S. Enteritidis after sublethal ethanol treatment. It was observed that 811 genes were significantly differentially expressed in ethanol-treated cells compared with control cells, among which 328 were up-regulated and 483 were down-regulated. Functional analysis revealed that these genes were enriched in different pathways, including signal transduction, membrane transport, metabolism, transcription, translation, and cell motility. Specifically, a couple of genes encoding histidine kinases and response regulators in two-component systems were up-regulated to activate sensing and signaling pathways. Membrane function was also influenced by ethanol treatment since ABC transporter genes for transport of glutamate, phosphate, 2-aminoethylphosphonate, and osmoprotectant were up-regulated, while those for transport of iron complex, manganese, and ribose were down-regulated. Accompanied with this, diverse gene expression alterations related to the metabolism of amino acids, carbohydrates, vitamins, and nucleotides were observed, which suggested nutritional requirements for S. Enteritidis to mount the ETR. Furthermore, genes associated with ribosomal units, bacterial chemotaxis, and flagellar assembly were generally repressed as a possible energy conservation strategy. Taken together, this transcriptomic study indicates that S. Enteritidis employs multiple genes and adaptation pathways to develop the ETR.

ABSTRACT: Salmonella Pullorum is one of the most important avian pathogenic bacteria due to widespread outbreaks accompanied by high mortality. It has been demonstrated that the Salmonella Enteritidis live vaccine strain Sm24/Rif12/Ssq is able to induce cross-immunity protection against Salmonella Gallinarum and Salmonella Infantis, however, it is unknown whether this vaccine is effective against Salmonella Pullorum infection. In the present study, the Hubbard parent chickens were orally administrated this vaccine at 1-day-old, 40-day-old, and 131-day-old respectively, and challenged by Salmonella Pullorum at 157-day-old to evaluate the protective effect of the Salmonella Enteritidis live vaccine strain Sm24/Rif12/Ssq. After each vaccination, the vaccine strain could be recovered from cloacal swabs within a week, whereas no vaccine strain was re-isolated from environmental samples throughout the experiment. Vaccination for the breeder chickens with Salmonella Enteritidis Sm24/Rif12/Ssq could relieve swollen liver (P = 0.0066) caused by Salmonella Pullorum infection and decrease Salmonella Pullorum colonization level in spleen (P = 0.0035), whereas no significant difference was found in the bacterial counts of liver, ovary and oviduct of vaccinated chickens. These results suggested that the Salmonella Enteritidis live vaccine strain Sm24/Rif12/Ssq was high safety and effective against Salmonella Pullorum infection to a certain extent.

Foodborne infections caused by Salmonella spp. are among the most common foodborne diseases in the world. We isolated a lytic phage against extended-spectrum beta-lactam producing S. Enteritidis strain PT1 derived from chicken carcass. Results from electronmicrography indicated that phiPT1 belonged to the family, Siphoviridae, in the order, Caudovirales. Phage phiPT1 was stable at temperatures from 4 °C to 60 °C and inactivated at 90 °C. phiPT1 retained a high titer from pH 4 to pH 10 for at least 1 h. Nevertheless, it displayed a significant decrease (p 0.05). The phage-antibiotic combination analysis (PAS) indicated that synergism was not detected at higher phiPT1 titer (1012 PFU/mL) with all tested antibiotics at all subinhibitory concentrations. However, synergistic activities were recorded at 0.25 × MIC of four tested antibiotics: cefixime, gentamicin, ciprofloxacin, and aztreonam in combination with phage at 104, 106 and 108 PFU/mL (ΣFIC ≤0.5). Synergism was detected for all antibiotics (0.1 × MIC) except meropenem and colistin in combination with phiPT1 at 104, 106 and 108 PFU/mL (ΣFIC ≤0.5). Synergism also displayed at the lowest concentrations of all antibiotics (0.01 MIC) in combination with phiPT1 at all titers except 1012 PFU/mL. Such characteristic features make phiPT1 to be a potential candidate for therapeutic uses.

Salmonella infections are responsible for a large burden of disease worldwide. Non-typhoidal Salmonella (NTS) species cause a myriad of disease manifestations, particularly amongst severely immunocompromised individuals. We present a rare case of endocarditis caused by the NTS species Salmonella Enteritidis in an individual living with HIV and hepatitis C. In this case, endocarditis was complicated by embolization and acute arterial occlusion of the left arm, as well as mitral valve perforation resulting in cardiac failure. A review of the available literature shows few cases of NTS causing endocarditis in people living with HIV, with the earliest reported case in 1983. Our case demonstrates the potential complications of NTS endocarditis and highlights the importance of evaluating patients with NTS-associated blood stream infection for cardiovascular involvement. Prompt surgical intervention in addition to appropriate antimicrobial therapy is essential to reduce the high morbidity and mortality associated with NTS endocarditis.

In food industries, microbial contaminations are difficult to control due to the recurrent formation of biofilms that hinders antimicrobials penetration and efficiency. An understanding of Salmonella Enteritidis biofilms behavior under flow conditions is a key to develop efficient preventive and control strategies. S. Enteritidis biofilms displayed 5.96, 6.28 and 6.80 log CFU cm−2 under 0.006 cm s−1, 0.045 cm s−1, and 0.087 cm s−1 flow velocities, respectively. Biofilms exposed to higher nutrient conditions under greater flow rates, induced significantly more biofilm biomass. To control biofilms, the disinfection efficiency of thymol (THY) was assessed under dynamic conditions by encapsulation it into two types of nanocapsules: monolayer (ML) nanocapsules prepared with a single carrier material (maltodextrin), and layer-by-layer (LBL) nanocapsules prepared by combining two carrier materials (maltodextrin and pectin). A combined mixture of ML and LBL nanocapsules at ½ their minimal inhibitory concentrations induced 99.99% eradication of biofilms developed under the highest flow conditions, after 5 h. ML nanocapsules decreased significantly bacterial counts during the first 0.5 h, while LBL nanocapsules eliminated the remaining bacterial cells and ensured a protection from bacterial contamination for up to 5 h by releasing THY in a sustained manner over time due to the thicker shell wall structure.

ABSTRACT: Salmonellosis causes massive economic losses globally every year. Especially in poultry, numerous drug-resistant bacteria have emerged; thus, it is imperative to find alternatives to antibiotics. As a probiotic, Clostridium butyricum (C. butyricum) provides the latest strategy for inhibiting the proliferation of Salmonella. This study aimed to evaluate the effects of C. butyricum on intestinal environment and gut microbiome under Salmonella infection. In this study, we modeled the infection of Salmonella using specific pathogen-free (SPF) chicks and found that the use of C. butyricum directly reduced the number of Salmonella colonizations in the spleen and liver. It also alleviated the histopathological changes of the liver, spleen, and cecum caused by Salmonella Enteritidis (S. Enteritidis). In addition, S. Enteritidis increased the expression of pro-inflammatory IL-6 in the cecum on day 6 postinfection. Interestingly, we found that C. butyricum changed PPAR-γ transcript levels in the cecum on day 6 postinfection. Analysis of the chick gastrointestinal microbiome showed that Salmonella infection increased the relative abundance of Subdoligranulum variabile. Further analysis found that Salmonella challenge significantly reduced the relative abundance of Faecalibacterium prausnitzii and C. butyricum increased the relative abundance of anaerobic bacteria in the gut on day 6 postinfection. Moreover, early supplementation of C. butyricum restored the epithelial hypoxia in S. Enteritidis infection in chicks. The results suggest that C. butyricum restores epithelial hypoxia caused by S. Enteritidis, improves the stability of intestinal flora, and inhibits the proliferation of Salmonella.

ABSTRACT: In this study, we investigate the effect of the probiotic E. faecium 669 strain on the gut integrity of broilers and the effect on intestinal colonization with Salmonella Enteritidis. In the in vivo experiment, 120-day-old broilers (Ross 308) were divided into 4 equally sized groups. Group A received the probiotic as a single dose by spray at d 18 of incubation and group B received the probiotic in the drinking water daily throughout the experiment. Group C was untreated control. Group D received the antibiotic Apramycin sulfate in the drinking water. Broilers in all four groups were challenged with S. Enteritidis by oral gavage at d 8 of life. From d 9 to 12, a cloacal swab was collected from all broilers for culturing on Salmonella selective media to determine the shedding. At d 12, birds were euthanized and S. Enteritidis in ceca were enumerated and intestinal samples for histology and host gene expression were collected. The group receiving the probiotic in the drinking water shed significantly less S. Enteritidis compared to the untreated control group at all times. The group receiving a single probiotic application before hatch showed a reduced shedding of Salmonella at d 9 and 10. S. Enteritidis was not detected in the ceca of the antimicrobial treated broilers. Histology of jejuni samples and host gene expression showed that intestinal integrity was enhanced by adding probiotic to the drinking water.Overall, the study shows that pre-hatch and daily application of the probiotic strain E. faecium 669 reduces the colonization of broilers with S. Enteritidis and daily application enhances gut integrity. Application of the probiotic E. faecium strain can be recommended as a method to reduce the colonization of broilers with S. Enteritidis and enhance their gut integrity.

ABSTRACT: Salmonella is one of the most common Gram-negative pathogens and seriously threatens chicken farms and food safety. This study aimed to establish a multiplex polymerase chain reaction (PCR) approach for the identification of different Salmonella enterica subsp. enterica. The citE2 gene and interval sequence of SPS4_00301–SPS4_00311 existed in all S. enterica subsp. enterica serovars by genomic comparison. By contrast, a 76 bp deletion in citE2 was found only in Salmonella Pullorum. Two pairs of special primers designed from citE2 and interval sequence were used to establish the multiplex PCR system. The optimized multiplex PCR system could distinguish Salmonella Pullorum and non-Salmonella Pullorum. The sensitivity of the optimized multiplex PCR system could be as low as 6.25 pg/μL and 104 colony-forming units (CFU)/mL for genomic DNA and Salmonella Pullorum cells, respectively. The developed multiplex PCR assay distinguished Salmonella Pullorum from 33 different Salmonella enterica subsp. enterica serotypes and 13 non-target species. The detection of egg samples artificially contaminated with Salmonella Pullorum, Salmonella Enteritidis, and naturally contaminated 69 anal swab samples showed that results were consistent with the culture method. These features indicated that the developed multiplex PCR system had high sensitivity and specificity and could be used for the accurate detection of Salmonella Pullorum in clinical samples.

Salmonella is a major zoonotic foodborne pathogen that persists on poultry farms worldwide. The present study aimed to survey the prevalence of Salmonella and antimicrobial resistance of Salmonella enterica serovar Enteritidis (S. Enteritidis) recovered from broiler chickens in Shandong Province, China. A total of 280 Salmonella isolates were identified from 923 broiler chicken samples between 2013 and 2018. Among the isolates, S. Enteritidis (n = 128, 45.7%) was the predominant serovar, and high antimicrobial resistance rates to piperacillin (PIP) (n = 123, 96.1%), ampicillin (AM) (n = 122, 95.3%), nitrofurantoin (FT) (n = 106, 96.1%), and tetracycline (TE) (n = 93, 72.7%) were observed in S. Enteritidis. A total of 96 (75.0%) S. Enteritidis isolates presented with multidrug resistance, the most frequent of which were the combination of AM, PIP, TE, and FT. Resistance to fluoroquinolone tended to increase during 2013 to 2018. Our findings provide important and updated information about the baseline antimicrobial-resistant data for food safety and a risk assessment of S. Enteritidis from broiler chickens in Shandong Province and will be helpful for future surveillance activities to ensure the safety of the chicken supply.

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.

The transmission of Salmonella to humans via contaminated eggs is an international public health concern. S. Enteritidis is deposited inside eggs after colonizing reproductive tissues of infected hens. Diverse housing facility characteristics and flock management practices influence Salmonella persistence and transmission in poultry, but the food safety consequences of different housing systems for laying hens remain unresolved. The present study compared the horizontal transmission of infection and invasion of internal organs during the first 2 wk after experimental S. Enteritidis and S. Kentucky infection of laying hens in indoor cage-free housing. Groups of 72 hens were housed in isolation rooms simulating commercial cage-free barns, and 1/3 of the hens in each room were orally inoculated with either S. Enteritidis (2 rooms) or S. Kentucky (2 rooms). At 6 d and 12 d postinoculation, 12 inoculated and 24 contact-exposed hens in each room were euthanized, and samples of liver, spleen, ovary, oviduct, and intestinal tract were removed for bacteriologic culturing. All orally inoculated hens were positive for intestinal colonization by S. Enteritidis at 6 d postinfection, and 70.8% of contact-exposed hens had become colonized by 12 d. S. Enteritidis was isolated from 100% of livers and 50.0% of ovaries from inoculated birds at 6 d and from 41.7% of livers and 10.4% of ovaries from contact-exposed birds at 12 d. The majority of both orally inoculated and contact-exposed hens were positive for intestinal colonization by S. Kentucky at 6 d, but S. Kentucky was found in other internal organs of both inoculated and contact-exposed hens significantly (P

The objective of this study was to evaluate the efficacy of flushing surfaces with untreated feed vs. the use of 2 different dry chemical sanitizers on residual surface and feed Salmonella Enteritidis contamination. First, a Salmonella-negative batch of poultry feed was mixed in 9 laboratory-scale paddle mixers. A feed sample was collected, and targeted locations on surfaces within the mixer were swabbed to confirm Salmonella-negative. Next, a Salmonella-positive batch of poultry feed was mixed, sampled, and mixer surfaces swabbed. Mean Salmonella Enteritidis contamination across all 9 mixers were 3.63 cfu/g for sampled feed and 1.27 cfu/cm2 for surface contamination. Next, the mixers manufactured one of the following treatments (3 mixers/treatment): 1) none (control); 2) a commercially available essential oil blend; or 3) rice hulls treated with a 10% concentration of a propriety blend of medium-chain fatty acids (MCFA). After each treatment, each mixer manufactured another 2 batches of Salmonella-free feed (sequence 1 and sequence 2). Feed samples were collected, and surfaces were swabbed between each batch of feed. Manufacturing sequence (P 0.05) impacted feed or surface contamination of Salmonella Enteritidis. There was Salmonella-positive residue in the batch of feed manufactured immediately after the positive control batch. However, no Salmonella residue was detected in batches of feed treated with either the commercial essential oil blend or MCFA. Low levels of Salmonella residue were observed from either feed (0.7 cfu/g for commercial essential oil blend) or surfaces (0.1 cfu/cm2 for MCFA) manufactured in sequence 1, but no residue was observed in sequence 2. These data suggest that sequencing of feed during manufacturing reduces Salmonella-positive contamination within animal food and on manufacturing surfaces, particularly after the second batch or with the use of chemical treatments.

Salmonella Enteritidis (SE) infection is not only a leading cause of poor production performance and compromised animal welfare in broilers but also a potential threat to public health. Two experiments were conducted to evaluate the effects of dietary supplemental vitamin C (VC) on SE challenged-broilers. In experiment 1, one hundred eighty 1-day-old Arbor Acre broilers were randomly allocated into 3 treatments, with 0, 500, or 1,000 mg/kg VC included in the diet. In experiment 2, dietary VC at 0 or 500 mg/kg, with or without SE challenge was applied in a 2 × 2 factorial arrangement in 6 randomized complete blocks. In experiment 1, addition with 500 mg/kg VC increased BW and infectious bursal disease (IBD) titer of broilers on 35 D (P 0.05) compared with the control group. In experiment 2, SE challenge depressed BW on 11 and 21 D (P

ABSTRACT: Bacillus coagulans (B. coagulans) have proven to be effective in improving the development of gut immunity and microbiome, and offering protection against pathogens, especially in young animals. The newborn chicks are highly vulnerable to the foodborne pathogenic Salmonella infections, leading to high mortality and economic loss. However, whether B. coagulans can protect young chickens from Salmonella-induced intestinal mucosal damage by modulating the development of intestinal epithelium remains unclear. In this study, B. coagulans with excellent anti-Salmonella property was selected and used. The results showed that B. coagulans alleviated the morphological damage, intestinal inflammation and body weight loss caused by Salmonella enteritidis (S. enteritidis) infections. B. coagulans significantly increased the crypt depth. Furthermore, the goblet cell loss and downregulating of mucin 2 induced by S. enteritidis were all relieved by B. coagulans treatment. Consistently, the expression of the related genes of Notch signaling pathway was also upregulated in the S. enteritidis group but inhibited by B. coagulans. In addition, B. coagulans improved the levels of immunoglobulin A, superoxide dismutase, total antioxidant capacity, and avian beta-defensin 2 in the intestinal mucosa. This study demonstrated that B. coagulans could regulate the development of intestinal epithelium, protect the intestinal barrier, thus relieve infections with S. enteritidis in chicks, which can be used as alternatives to antibiotics in poultry feed.

ABSTRACT: Salmonella enterica serovar Enteritidis is a bacterial pathogen that contributes to poultry production losses and human foodborne illness. The bacterium elicits a broad immune response involving both the innate and adaptive components of the immune system. Coordination of the immune response is largely directed by cytokines. The objective of the current study was to characterize the expression of a select set of cytokines and regulatory immune genes in three genetically diverse chicken lines after infection with S. Enteritidis. Leghorn, Fayoumi and broiler day-old chicks were orally infected with pathogenic S. Enteritidis or culture medium. At 2 and 18 h postinfection, spleens and ceca were collected and mRNA expression levels for 7 genes (GM-CSF, IL2, IL15, TGF-β1, SOCS3, P20K, and MHC class IIβ) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-β1, SOCS3 and P20K in the spleen and on P20K and MHC class IIβ in the cecum. Comparing challenged vs. unchallenged birds, the expression of SOCS3 and P20K mRNA were significantly higher in the spleen and cecum, while MHC class IIβ mRNA was significantly lower in spleen. Combining the current RNA expression results with those of previously reported studies on the same samples reveals distinct RNA expression profiles among the three genetic chicken lines and the 2 tissues. This study illustrates that these diverse genetic lines have distinctively different immune response to S. Enteritidis challenge within the spleen and the cecum.

SUMMARY: Producers and consumers associated with small scale backyard egg production tend to reuse egg cartons. Egg cartons are also reused for arts and craft projects, gardening, and organization units for small items. The reuse of egg cartons is primarily driven by economic or ecological reasons. The ability of zoonotic bacteria, such as Salmonella Enteritidis (SE), to survive on the eggshell surface and a variety of food packaging materials makes the reuse of egg carton risky. This study was aimed at determining the scope of cross-contamination of SE between eggshell and different egg carton types using 2 experiments. Unwashed eggs from end-of-lay white Leghorn hens were used in the experiments. Two different SE strains were used with 3 independent tubes of inocula from each strain as replicates. In Experiment 1, 216 eggs from each SE strain (72/replicate) were inoculated with 10 μL of SE inoculum (~ 9.95 log cfu/mL), allowed to dry in room temperature, and placed in nonadjacent wells of noninoculated plastic, polystyrene foam, and pulp egg cartons. Egg cartons of each type were then stored either at refrigeration (4°C) or room temperature (25°C). After 24 h eggs were discarded and the carton-wells were swabbed for SE recovery. In Experiment 2, wells of egg cartons were inoculated with SE and uninoculated eggs were placed in them and stored similar to Experiment 1. A total of 216 wells within the egg cartons were inoculated for each SE strain (72/replicate). Eggshell samples were collected for SE recovery. Only 3 samples were detected positive for SE in Experiment 1 and no effect of carton type, SE strain, or incubation temperature was observed. In Experiment 2, 8 eggshell samples were SE positive – 6 from polystyrene foam and 2 from plastic carton. Statistical difference was observed for pulp versus polystyrene foam only (P < 0.05). These results indicate that transfer of SE between egg carton and eggshell surface is possible and that the risk of cross-contamination is associated with type of carton material.

ABSTRACT: Contaminated eggs are a leading source of human Salmonella infections and this problem continues to challenge public health authorities and egg industries around the world. Salmonella invasion of the ovaries and oviducts of infected laying hens can result in bacterial deposition inside the edible portions of developing eggs. The introduction, persistence, and transmission of salmonellae in commercial egg-laying flocks are influenced by flock management practices, but the food safety ramifications of different types of laying hen housing remain unresolved. The present study assessed the frequency of internal contamination of eggs after experimental Salmonella Enteritidis and S. Typhimurium infection of laying hens in indoor cage-free housing. Groups of 72 hens were housed on wood shavings in isolation rooms simulating commercial cage-free barns with community kick-out nest boxes and perches and 1/3 of the hens in each room were orally inoculated with 8.0 × 107 cfu of 2-strain mixtures of either S. Enteritidis (2 rooms) or S. Typhimurium (2 rooms), and the entire internal contents of all eggs laid 5 to 30 d postinoculation in nest boxes or on the flooring substrate were cultured to detect Salmonella. Contaminated eggs were laid between 8 and 28 d postinoculation. The overall incidence of S. Enteritidis isolation from eggs (3.41%) was significantly (P = 0.0005) greater than S. Typhimurium (1.19%). The contamination frequencies associated with the 2 egg collection locations were not significantly different (P > 0.05). These results demonstrate that oral infection of a relatively small proportion of laying hens in indoor cage-free housing with invasive Salmonella serovars can result in the production of internally contaminated eggs at low frequencies over a period of nearly a month postinoculation.

ABSTRACT: Salmonella enterica serovar Enteritidis (SE) has consistently been the most common serotype associated with the foodborne Salmonellosis worldwide. In this study, the effect of a dietary direct-fed microbial (DFM) and yeast cell walls (YCW) under a challenge of nalidixic acid resistant SE strain using layer chicks has been investigated. A total of 160 newly hatched Dekalb White female chicks were randomly assigned into 2 experimental groups (80 birds/treatment), control group (CON) and treatment group (DY). Chicks were fed ad libitum a non–medicated-corn-soy based diet and DY was supplemented with the combination of DFM and YCW. At 8 days of age, 2.1 × 109 CFU/bird of the SE was given to all chicks by oral administration. On 3 days postinoculation (dpi), 20 chicks/group were euthanized and all cecal contents were collected for analysis. On 6, 10, and 14 dpi, the cecal contents were sampled from 16 chicks per group. The number of SE in the cecal contents was counted using culture-based methods. A 16S rRNA-based microbiota analysis was performed for additional microbial profiling. The CON and DY showed difference (P ≤ 0.05) in β diversity throughout the trial. Prevalence of SE in cecal contents was lower (P ≤ 0.05) in DY across all time-points. Lower abundance of Salmonella spp. was also shown in DY by liner discriminant analysis effect size (LEfSe). DY increased (P ≤ 0.05) diversity of bacterial species in the cecal contents in DY at 10 and 14 dpi. For the SE challenged birds, SE reduction in DY was observed at 3 dpi and until the end of the trial at 14 dpi confirming a numerically larger difference between groups as well as an increase in bacterial species diversity in DY. It could be hypothesized that the SE reduction shown immediately after the challenge and the greater SE reduction shown after 10 dpi may be the synergistic effect of the combined feed additives.

ABSTRACT: The presence of Salmonella serotypes is a major safety concern of the food industry and poultry farmers. This study aimed to isolate and identify Salmonella spp. from a chicken processing facility by PCR and pulsed-field gel electrophoresis (PFGE). In addition, the biofilm-forming abilities of the isolated bacteria on stainless steel, silicone rubber, plastic, and chicken skin were also investigated. PCR was used for the confirmation of Salmonella serotypes, and then gene similarity within the same serotype was analyzed by PFGE. As a result, 26 S. Enteritidis isolates were detected at a high rate from both food contact surfaces and chicken products during processing. All of them were 100% genetically identical to the same bacteria. The results indicated that the virulence factors and effective biofilm-forming ability of S. Enteritidis isolates could affect human health and economic revenue. It was also suggested that the visual observation of food and food contact surfaces could be a great concern in the future. The continuous monitoring of S. Enteritidis molecular and biofilm characteristics is needed to increase food safety.

ABSTRACT: The present study describes the generation of Salmonella enteritidis (SE) ghosts with a surface decorated Salmonella Typhimurium (ST) flagellin (FliC) antigen for immune enhancement and strain-specific protection. The ghosts were generated by biological means using pJHL184::fliC temperature inducible plasmid where the lysis occurs by phage PhiX174 lysis gene E expression. Being an inactivated strain, no environmental contamination was observed by fecal shedding upon inoculation into the chicken. To test the protective immune responses, ghost vaccination was conducted via the intramuscular route using chicken as the model organism. The development of antigen-specific humoral, cell-mediated, and protective immune responses was assessed. Compared to vector alone and phosphate-buffered saline (PBS) control groups, pJHL184::fliC ghost could generate significantly high antigen-specific IgY and cell-mediated immune (CMI) responses measured by a peripheral blood mononuclear cell proliferation, flow cytometer, and cytokine responses elicited by stimulated splenic T-cells (P < 0.05). The adjuvant effect induced by FliC was demonstrated by elicitation of Toll-like receptor 5 (TLR5). To test the protection efficacy, chickens were challenged with both SE and ST wild type (WT) strains, and the protection efficacy was assessed by determining the presence of challenging strains in the spleen and liver, and by assessing the histopathological alterations. Complete clearance of the challenged strain and least inflammatory signs were evident in the SE ghosts vaccinated group compared to the vector and PBS control. The elimination of both SE and ST in chicken organs ensures the intramuscular immunization of the present SE ghost vaccine can reduce SE and ST contamination levels in chicken that can be beneficial to prevent enteric infections in humans.

Salmonella Enteritidis (SE) are important zoonotic pathogens, and can be easily transferred to humans by contaminated animal products. Epidemic surveys of SE are necessary in current modern large-scale chicken farms. In this study, Salmonella strains were isolated from possibly infected samples collected at 3 independent farms, and their serotype, drug resistances, virulence genes, and genetic similarity were analyzed by molecular genetic analysis technologies including multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 346 Salmonella strains were isolated from 3,598 samples (9.61%); 329 isolates were identified as SE (95.09%) and 308 isolates were multidrug resistant (93.62%). Virulotyping based on 6 virulence genes showed high similarity in SE isolates of each farm, with the exception of 2 isolates. All SE isolates were found to be the same ST11 type by MLST, and 22 strains of 150 SE isolates selected at random were found to belong to 1 cluster by PFGE and the same SET1 type by CRISPR. WGS results further revealed that these isolates belonged to the same clonal cluster, with high genetic similarity of 99.80 to 100.00%. All these results indicated that these SE isolates were overwhelmingly dominant and demonstrated high genetic similarity, which revealed that the same SE clone might be transmitted in these farms.

An animal infection model was evaluated on sheep and goats to confirm which species infected with Salmonella enterica serovar Enteritidis C StR (SE13) would provide a consistent and high frequency of Salmonella colonization in lymph nodes (LNs) without causing undue animal morbidity. Sheep and goats (n = 5) were intradermally inoculated with Salmonella, postincubated for 7 days, and euthanized. Superficial cervical, medial iliac, subiliac, mammary, and popliteal LNs were excised from each carcass. Goat LNs had approximately 53% greater Salmonella level compared to sheep. Also, Salmonella was inconsistently recovered from the sheep LNs. Thus, goats were selected to determine the ability of carcass vascular rinsing (with and without bacteriophages) to reduce Salmonella in infected LNs. Goats with similar characteristics were grouped together before being randomly assigned to 3 postharvest treatments; control (CN, not vascularly rinsed; n = 10), vascularly rinsed with a standard Rinse & Chill® solution (RC; 98.5% water and a blend of saccharides and phosphates; n = 10), or vascularly rinsed with a standard Rinse & Chill® solution plus the addition of bacteriophages (BP; n = 10). Rinse & Chill® system was able to successfully deliver a mean 7.0 log PFU/g to the S. Enteritidis-infected LNs (mean 3.5 log CFU/g). However, neither Rinse & Chill® without bacteriophages nor with bacteriophages caused Salmonella reduction (P > 0.05) compared to the nonrinsed goat carcasses., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Egg washing guidelines vary across countries; however, since 2020, Korea has required that all eggs produced from farms with more than 10,000 laying hens must be washed through egg grading and packing (GP) plant. This study investigated the prevalence and characterization of non-typhoidal Salmonella in eggs after washing at GP plants. In total, 16,800 eggs were collected from 60 egg GP plants located inside commercial layer farms, and 840 pooled eggshell and egg contents were tested for Salmonella, respectively. Of the 60 GP plants tested, 11 (18.3%) and 12 (20.0%) plants were positive for Salmonella spp. In the eggshells and egg contents, respectively. In particular, High Salmonella prevalence in the eggshells and egg contents occurred most often in farms with laying hens older than 80 weeks (33.3% and 40.0%, respectively). However, among 840 pooled eggshells and egg content samples, only 19 (2.3%) of each sample type were positive only for non-typhoidal Salmonella spp. The most common Salmonella serovar in both eggshells and egg contents was S. Infantis, which was found in five (8.3%) of 60 GP plants for both samples types. The other Salmonella serovars detected in eggshells were S. Bareilly (5.0%), S. Agona (3.3%), S. Enteritidis (1.7%), and S. Montevideo (1.7%), whereas those detected in egg contents were S. Enteritidis (5.0%), S. Agona (3.3%), S. Newport (3.3%), S. Senftenberg (3.3%), and S. Derby (1.7%). Of the 19 virulence genes tested, 14 genes were detected in all Salmonella. Interestingly, the spvB gene was detected only in S. Enteritidis, and the sefC gene was detected only in S. Enteritidis and S. Senftenberg. Moreover, all S. Infantis isolates showed multidrug resistance (MDR) against five or more classes, and the other serovars only showed MDR against three to four classes or no MDR. These results suggest that comprehensive surveillance and advanced management approaches for egg GP plants are required to minimize egg contamination with non-typhoidal Salmonella., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest., (Copyright © 2024. Published by Elsevier Ltd.)

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAP TM ). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods., Competing Interests: Disclosures The study was conceived, developed, and analyzed by J.G.L, C.G, P.C-G, and S.S-N from CECAV which declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gas-forming pyogenic liver abscess (GFPLA) is considered to be a very severe form of PLA and carries a high mortality. Klebsiella pneumoniae is the most common pathogen responsible for the disease, whereas cases where Salmonella is cited as the cause are very uncommon. We report the first case of a 53-year-old lady suffering from GFPLA due to Salmonella, who was successfully treated with surgical drainage. To the best of our knowledge, this is the first case of GFPLA caused by Salmonella enteritidis to be reported in the English literature.

In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human) by Enterobacterial Repetitive Intergenic Consensus (ERIC) -PCR, as well as to assess their plasmid profiling and antimicrobial resistance. A total of 30 Salmonella Enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats) and 15 from clinical samples were collected in Tehran. Identification of isolates as Salmonella was confirmed by using conventional standard biochemical and serological tests. Multiplex-PCR was used for serotyping of isolates to identify Salmonella Enteritidis. Antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among Salmonella Enteritidis isolates. No resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. The highest resistance (96.7%) was observed to nitrofurantoin. Seven plasmid profiles (P1–P7) were detected, and a 68-kb plasmid was found in all isolates. Two different primers; ERIC and (GTG)5 were used for genotyping, which each produced four profiles. The majority of clinical and food isolates fell into two separate common types (CTs) with a similar percentage of 95% by ERIC-PCR. Using primer (GTG)5, 29 isolates incorporated in three CTs with 70% of isolates showing a single banding pattern. Limited genetic diversity among human and food isolates of Salmonella Enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. These results confirmed that ERIC-PCR genotyping has limited discriminatory power for Salmonella Enteritidis of different origin.

Salmonella have been implicated in foodborne disease outbreaks globally and is a pressing concern in the South African small-scale sector due to inadequate hygiene standards and limited regulatory oversight, leading to a higher risk of foodborne diseases. By investigating irrigation water and leafy green vegetables produced by small-scale growers and sold through unregulated supply chains, this study was able to determine the presence, serotype distribution, virulence gene profiles, antibiotic resistance, and genetic diversity of Salmonella isolated from these sources. From 426 samples, 21 Salmonella-positive samples were identified, providing 53 Salmonella isolates. Of these, six different Salmonella serotypes and sequence types (STs) were identified, including Salmonella II 42:r: ST1208 (33.96%; n = 18), Salmonella Enteritidis: ST11 (22.64%; n = 12), Salmonella II 42:z29: ST4395 (16.98%; n = 9), Salmonella Havana: ST1524 (15.09%; n = 8), Salmonella Typhimurium: ST19 (9.43%; n = 5), and Salmonella IIIb 47:i:z: ST7890 (1.89%; n = 1). A total of 92.45% of the isolates were found to be multidrug-resistant, showing high rates of resistance to aztreonam (88.68%; n = 47), ceftazidime (86.79%; n = 46), nalidixic acid (77.36%; n = 41), cefotaxime (75.47%; n = 40), cefepime (71.70%; n = 38), and streptomycin (69.81%; n = 37). All isolates possessed the aac(6')-Iaa antimicrobial resistance gene, with a range of between 9 and 256 virulence genes. Eleven cluster patterns were observed from Enterobacterial Repetitive Intergenic Consensus sequence analyses, demonstrating high diversity among the Salmonella spp., with water and fresh produce isolates clustering, suggesting water as a potential contamination source. Plasmid replicon types were identified in 41.51% (n = 22) of the isolates, including Col(pHAD28) in Salmonella Havana (5.66%; n = 3), Col156 in Salmonella II 42:z29:- (1.89%; n = 1) and both IncFIB(S) and IncFII(S) in Salmonella Enteritidis (22.64; n = 12), Salmonella Typhimurium (9.43%; n = 5), and Salmonella Havana (1.89%; n = 1). This study highlights the presence of multidrug-resistant and multivirulent Salmonella spp. in the small-scale leafy green vegetable supply chains, underscoring the need for the development of a "fit-for-purpose" food safety management system within this system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Globally, nontyphoidal Salmonella (NTS) causes approximately 150 million foodborne illnesses annually; many of which are linked to poultry products. Thus, improving food safety interventions in the poultry sector can reduce foodborne illness associated with prevalent NTS serotypes. Bacteriophages (phages) have shown promise as food-safe alternatives to current antimicrobial practices. However, challenges such as limited host range, bactericidal effectiveness in practical production settings, and the risk of developing host resistance remain as barriers for the widespread use of phages in commercial poultry operations. A broad-spectrum three-phage cocktail was evaluated against S. enterica subsp. enterica serotypes Enteritidis, Typhimurium, and Kentucky. The impact of multiplicity of infection (MOI) on NTS growth was assessed in broth at 22°C for 18 hours (h). Then, phage cocktail efficacy was evaluated on raw chicken breast samples inoculated with the NTS cocktail and stored at 10°C or 22°C for 0, 1, and 5 days or 0, 4, 8, and 16 h, respectively. Most probable number (MPN) calculations were performed for NTS counts on chicken after phage treatment and storage at 10°C to account for samples with NTS counts below the detection limit. In general, a higher MOI corresponded to reduced NTS growth; however, residual nutrition in growth media and initial NTS contamination level affected samples treated with the phage cocktail at identical MOIs. On chicken, phage cocktail treatment significantly reduced NTS counts at 10°C and 22°C. After storage at 10°C for 5 days, NTS counts were reduced by >3.2 log compared to the control. After storage at 22°C for 16 h, NTS counts were reduced by >1.7 log compared to the control. Overall, the phage cocktail was effective at reducing a diverse set of prominent NTS strains in broth and on raw chicken breast, highlighting its potential for commercialization and use alongside other hurdles in poultry production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Eggs represent a significant vehicle for Salmonella Enteritidis with the pathogen being transferred to chicks in the hatchery, or to consumers via table eggs. In the following, the efficacy of a gas-phase hydroxyl-radical process for decontaminating hatchery and table eggs was evaluated. Recovery of Salmonella was maximized through holding eggs in tryptic soy broth containing 20% w/v glycerol for 1 h prior to plating. By using this technique, it was possible to recover 63% of the theoretical Salmonella inoculated onto eggs. The continuous hydroxyl-radical reactor consisted of a bank of UV-C lamps (254 nm) that generated hydroxyl-radicals from the degradation of hydrogen peroxide (H 2 O 2 ) mist and ozone gas. The optimal treatment was defined as that which supports a 5 log CFU/egg reduction of Salmonella without negatively affecting egg quality or leaving H 2 O 2 residues. A process of 2% v/v H 2 O 2 delivered at 30 mL/min with a UV-C dose of 19 mJ/cm 2 and ozone (20 ppm) with a total treatment time of 10s was selected. The egg quality metrics (Haugh value, yolk index, albumin pH, yolk pH) did not negatively differ over a 35-day shelf-life at 4 or 25℃ compared to washed eggs or nontreated controls. The cuticle layer of eggs remained intact following hydroxyl-radical treatment. Fertilized eggs (n = 61) treated with the hydroxyl-radicals exhibited the same hatchery rate (75%) as nontreated controls (71-79%) with no defects (unhealed navels or red hocks) being observed. The same hydroxyl-radical treatment could be applied to table eggs to support >5 log CFU/egg reduction of Salmonella and was compatible with egg washing regimes practiced in industry. In comparison, the egg washing process based on sodium hydroxide and chlorine supported a 2.76 ± 0.38 log CFU/egg reduction of Salmonella. The hydroxyl-radical treatment represents a preventative control step to reduce the carriage of Salmonella on hatchery and table eggs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In Sri Lanka, foodborne diseases caused by nontyphoidal Salmonella are of increasing concern. We therefore aimed to characterize the dominant Salmonella serovars in humans, poultry, and aquaculture through a One Health approach., Methods: We collected isolates from different sectors, confirmed their identities using PCR, screened their antibiotic resistance profiles, and determined their antibiotic resistance genes based on whole-genome sequencing., Results: Of the 75 Salmonella isolates identified, the majority of serotypes were unidentified. Both Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) could be isolated from human sources and were also found prevalent in the poultry sector. ST36, ST11 and ST1541 were the dominant serotypes of S. Typhimurium and S. Enteritidis, respectively. Alarmingly, 4% (1/25) of poultry Salmonella isolates were resistant to ciprofloxacin, suggesting an emergence of this phenotype. Moreover, virulence genes were very diverse among S. Enteritidis and S. Typhimurium isolates., Conclusions: With the diversity of unidentified serotypes found and the detection of emerging resistances, our study highlights the importance of a One Health approach to monitoring antibiotic resistance. For public health initiatives in Sri Lanka to be successful in mitigating salmonellosis, all three sectors - humans, aquaculture, and poultry - must be tackled concomitantly in a coordinated manner under the One Health approach because antibiotic resistance genes, and even specific sequence types, may be able to spread across the aforementioned sectors. We anticipate that our results will inform public health policies in Sri Lanka to tackle foodborne illnesses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)