Novel Synergistic Probiotic Intervention: Transcriptomic and Metabolomic Analysis Reveals Ameliorative Effects on Immunity, Gut Barrier, and Metabolism of Mice during Salmonella typhimurium Infection.

Salmonella typhimurium (S. typhimurium), a prevalent cause of foodborne infection, induces significant changes in the host transcriptome and metabolome. The lack of therapeutics with minimal or no side effects prompts the scientific community to explore alternative therapies. This study investigates the therapeutic potential of a probiotic mixture comprising Lactobacillus acidophilus (L. acidophilus 1.3251) and Lactobacillus plantarum (L. plantarum 9513) against S. typhimurium, utilizing transcriptome and metabolomic analyses, a novel approach that has not been previously documented. Twenty-four SPF-BALB/c mice were divided into four groups: control negative group (CNG); positive control group (CPG); probiotic-supplemented non-challenged group (LAPG); and probiotic-supplemented Salmonella-challenged group (LAPST). An RNA-sequencing analysis of small intestinal (ileum) tissue revealed 2907 upregulated and 394 downregulated DEGs in the LAPST vs. CPG group. A functional analysis of DEGs highlighted their significantly altered gene ontology (GO) terms related to metabolism, gut integrity, cellular development, and immunity (p ≤ 0.05). The KEGG analysis showed that differentially expressed genes (DEGs) in the LAPST group were primarily involved in pathways related to gut integrity, immunity, and metabolism, such as MAPK, PI3K-Akt, AMPK, the tryptophan metabolism, the glycine, serine, and threonine metabolism, ECM-receptor interaction, and others. Additionally, the fecal metabolic analysis identified 1215 upregulated and 305 downregulated metabolites in the LAPST vs. CPG group, implying their involvement in KEGG pathways including bile secretion, propanoate metabolism, arginine and proline metabolism, amino acid biosynthesis, and protein digestion and absorption, which are vital for maintaining barrier integrity, immunity, and metabolism. In conclusion, these findings suggest that the administration of a probiotic mixture improves immunity, maintains gut homeostasis and barrier integrity, and enhances metabolism in Salmonella infection.

Identification of key transcription factors and their functional role involved in Salmonella typhimurium infection in chicken using integrated transcriptome analysis and bioinformatics approach.

Salmonella enterica serovar typhimurium is the cause of significant morbidity and mortality worldwide that causes economic losses to poultry and is able to cause infection in humans. Indigenous chicken breeds are a potential source of animal protein and have the added advantage of being disease resistant. An indigenous chicken, Kashmir favorella and commercial broiler were selected for understanding the mechanism of disease resistance. Following infection in Kashmir favorella, three differentially expressed genes Nuclear Factor Kappa B (NF-κB1), Forkhead Box Protein O3 (FOXO3) and Paired box 5 (Pax5) were identified. FOXO3, a transcriptional activator, is the potential marker of host resistance in Salmonella infection. NF-κB1 is an inducible transcription factor which lays the foundation for studying gene network of the innate immune response of Salmonella infection in chicken. Pax5 is essential for differentiation of pre-B cells into mature B cell. The real time PCR analysis showed that in response to Salmonella Typhimurium infection a remarkable increase of NF-κB1 (P˂0.01), FOXO3 (P˂0.01) gene expression in liver and Pax5 (P˂0.01) gene expression in spleen of Kashmir favorella was observed. The protein-protein interaction (PPI) and protein-TF interaction network by STRINGDB analysis suggests that FOXO3 is a hub gene in the network and is closely related to Salmonella infection along with NF-κB1. All the three differentially expressed genes (NF-κB1, FOXO3 and PaX5) showed their influence on 12 interacting proteins and 16 TFs, where cyclic adenosine monophosphate Response Element Binding protein (CREBBP), erythroblast transformation-specific (ETSI), Tumour-protein 53(TP53I), IKKBK, lymphoid enhancer-binding factor-1 (LEF1), and interferon regulatory factor-4 (IRF4) play role in immune responses. This study shall pave the way for newer strategies for treatment and prevention of Salmonella infection and may help in increasing the innate disease resistance.

Gene Co-Expression Network Analysis Reveals the Hub Genes and Key Pathways Associated with Resistance to Salmonella Enteritidis Colonization in Chicken.

Salmonella negatively impacts the poultry industry and threatens animals' and humans' health. The gastrointestinal microbiota and its metabolites can modulate the host's physiology and immune system. Recent research demonstrated the role of commensal bacteria and short-chain fatty acids (SCFAs) in developing resistance to Salmonella infection and colonization. However, the complex interactions among chicken, Salmonella, host-microbiome, and microbial metabolites remain unelucidated. Therefore, this study aimed to explore these complex interactions by identifying the driver and hub genes highly correlated with factors that confer resistance to Salmonella. Differential gene expression (DEGs) and dynamic developmental genes (DDGs) analyses and weighted gene co-expression network analysis (WGCNA) were performed using transcriptome data from the cecum of Salmonella Enteritidis-infected chicken at 7 and 21 days after infection. Furthermore, we identified the driver and hub genes associated with important traits such as the heterophil/lymphocyte (H/L) ratio, body weight post-infection, bacterial load, propionate and valerate cecal contents, and Firmicutes, Bacteroidetes, and Proteobacteria cecal relative abundance. Among the multiple genes detected in this study, EXFABP, S100A9/12, CEMIP, FKBP5, MAVS, FAM168B, HESX1, EMC6, and others were found as potential candidate gene and transcript (co-) factors for resistance to Salmonella infection. In addition, we found that the PPAR and oxidative phosphorylation (OXPHOS) metabolic pathways were also involved in the host's immune response/defense against Salmonella colonization at the earlier and later stage post-infection, respectively. This study provides a valuable resource of transcriptome profiles from chicken cecum at the earlier and later stage post-infection and mechanistic understanding of the complex interactions among chicken, Salmonella, host-microbiome, and associated metabolites.

Transcriptome analysis of PBMCs isolated from piglets treated with a miR-124 sponge construct identified miR124/IQGAP2/Rho GTPase as a target pathway support Salmonella Typhimurium infection.

miR-124 is a significantly up-regulated miRNA in peripheral blood collected from piglets infected with Salmonella Typhimurium, suggesting that it may play an important role in Salmonella pathogenesis. This study focused on the transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) isolated from miR-124 sponge and Salmonella Typhimurium-treated piglets, and trying to investigate the function of miR-124 in Salmonella infection. The transcriptome profiling analysis revealed that 2778 genes in miR-124 sponge + Salmonella Typhimurium treatment versus control, 2271 genes in Salmonella Typhimurium treatment versus control, and 1301 genes in miR-124 sponge + Salmonella Typhimurium versus Salmonella Typhimurium treatment, were differentially expressed, respectively (FDR < 0.05 and fold change > 2.0). Pathway analysis indicated that the MAPK signaling pathway, Ribosome pathway, and T-cell receptor signaling pathway were the most significantly enriched pathway in differentially expressed genes between miR-124 sponge + Salmonella Typhimurium and Salmonella Typhimurium along treatment (FDR < 0.05). Reporter assays and electrophoretic mobility shift assays showed that miR-124 is a crucial regulatory factor that targets IQ motif containing GTPase-activating protein 2 (IQGAP2). Cell culture experiment indicated that miR-124 attenuated the Salmonella Typhimurium-mediated activation of CDC42 and RAC1 (P < 0.05). Cultured PBMCs treated with miR-124 and IQGAP2-siRNA had higher intracellular Salmonella count than control samples, particularly 12 h post-infection (P < 0.05). Immunofluorescence analysis revealed that miR-124 treatment reduced the percentage of LAMP-1-positive phagosomes. The miR-124 could be an important regulator for IQGAP2/Rho GTPase pathway in Salmonella Typhimurium-infected PBMCs, and this pathway could be a target for Salmonella that support its infection in PBMCs in piglets.

Multiplex PCR assay based on the citE2 gene and intergenic sequence for the rapid detection of Salmonella Pullorum in chickens.

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.

Transcriptomic Analysis of the Spleen of Different Chicken Breeds Revealed the Differential Resistance of Salmonella Typhimurium.

Salmonella Typhimurium (ST) is a foodborne pathogen that adversely affects the health of both animals and humans. Since poultry is a common source and carrier of the disease, controlling ST infection in chickens will have a protective impact on human health. In the current study, Beijing-You (BY) and Cobb chicks (5-day-old specific-pathogen-free) were orally challenged by 2.4 × 1012 CFU ST, spleen transcriptome was conducted 1 day post-infection (DPI) to identify gene markers and pathways related to the immune system. A total of 775 significant differentially expressed genes (DEGs) in comparisons between BY and Cobb were identified, including 498 upregulated and 277 downregulated genes (fold change ≥2.0, p < 0.05). Several immune response pathways against Salmonella were enriched, including natural killer-cell-mediated-cytotoxicity, cytokine−cytokine receptor interaction, antigen processing and presentation, phagosomes, and intestinal immune network for IgA production, for both BY and Cobb chickens. The BY chicks showed a robust response for clearance of bacterial load, immune response, and robust activation of phagosomes, resulting in ST resistance. These results confirmed that BY breed more resistance to ST challenge and will provide a better understanding of BY and Cobb chickens' susceptibility and resistance to ST infection at the early stages of host immune response, which could expand the known intricacies of molecular mechanisms in chicken immunological responses against ST. Pathways induced by Salmonella infection may provide a novel approach to developing preventive and curative strategies for ST, and increase inherent resistance in animals through genetic selection.

Differences in caecal microbiota composition and Salmonella carriage between experimentally infected inbred lines of chickens.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication resulting from consumption of contaminated poultry products. Genetic selection of animals that are more resistant to Salmonella carriage and modulation of the gut microbiota are two promising ways to decrease individual Salmonella carriage. The aims of this study were to identify the main genetic and microbial factors that control the level of Salmonella carriage in chickens (Gallus gallus) under controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines N and 61 were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition, using a 16S rRNA gene high-throughput sequencing approach. RESULTS: Our results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Among the 617 operational taxonomic units (OTU) observed, more than 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between the high and low Salmonella carriers, with 39 differentially abundant OTU. Using metagenome functional prediction based on 16S data, several metabolic pathways that are potentially associated to microbiota taxonomic differences (e.g. short chain fatty acids pathways) were identified between high and low carriers. CONCLUSIONS: Overall, our findings demonstrate that the caecal microbiota composition differs between genetic lines of chickens. This could be one of the reasons why the investigated lines differed in Salmonella carriage levels under experimental infection conditions.

Differential immunological response detected in mRNA expression profiles among diverse chicken lines in response to Salmonella challenge.

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-ß1, SOCS3, P20K, and MHC class IIß) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-ß1, SOCS3 and P20K in the spleen and on P20K and MHC class IIß 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 IIß 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.

Intra- and inter-breed variation in immune response to acute and sub-chronic Salmonella infection and commercial immune-stimulant in two-layer breeds.

Salmonella is one of the most hazardous diseases in poultry farms. Markedly, the application of active immunostimulants is illustrated as potential protective agents against infection in poultry farms. Thus, this work aimed to explore inter- and intra-breed variation in response to acute and subchronic Salmonella enteritidis infection in two-layer breeds (one commercial [Hy-line strain] and another native [Fayoumi breed]). Besides exploring the possible protective effect of a commercial immune modulator (STIMULAN) on the two breeds during the acute infection. The ELISA antibody titer in sub-chronic infections and the expression analysis of some selected genes (IL-1ß, LITAF, TGF-ß, HSP90 and HSP70) are used as the clinical signs for acute infections to assess the possible protective role of a commercial immunomodulator (STIMULAN). Five groups were used during the acute experiment: G1-control; G2a-susceptible; G2b-resistant birds, G3-which received STIMULAN and G4-which received the infection + STIMULAN. The groups with sub-chronic infections include G1 (control), G2 (high antibody titer) and G3 (low antibody titer). The gene expressions among the susceptible birds during acute infection of both breeds are nearly similar. They only differ in the expression of HSP90 in the Fayoumi breed. However, the resistant birds vary in their gene expression profile. The effect of STIMULAN as a feed additive in non-infected birds was an up-regulation of LITAF, TGF-ß, HSP90 in Fayoumi. Moreover, a powerful stimulatory role was observed when both breeds were infected. Both breeds were asymptomatic during the sub-chronic infection. Although, the increased expression of inflammatory-related genes in the Hy-line was considered as an indication of infection persistence. Fayoumi is capable of immune clearance for this infection. Thus, the Fayoumi breed is more resistant to acute Salmonella infection. HSP90 plays a vital role in its resistance. We recommend the use of STIMULAN as an immunomodulator during Salmonella infection.

Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes.

Salmonella enterica serovar Gallinarum biovar Pullorum (bvP) and biovar Gallinarum (bvG) are the etiological agents of pullorum disease (PD) and fowl typhoid (FT) respectively, which cause huge economic losses to poultry industry especially in developing countries including India. Vaccination and biosecurity measures are currently being employed to control and reduce the S. Gallinarum infections. High endemicity, poor implementation of hygiene and lack of effective vaccines pose challenges in prevention and control of disease in intensively maintained poultry flocks. Comparative genome analysis unravels similarities and dissimilarities thus facilitating identification of genomic features that aids in pathogenesis, niche adaptation and in tracing of evolutionary history. The present investigation was carried out to assess the genotypic differences amongst S.enterica serovar Gallinarum strains including Indian strain S. Gallinarum Sal40 VTCCBAA614. The comparative genome analysis revealed an open pan-genome consisting of 5091 coding sequence (CDS) with 3270 CDS belonging to core-genome, 1254 CDS to dispensable genome and strain specific genes i.e. singletons ranging from 3 to 102 amongst the analyzed strains. Moreover, the investigated strains exhibited diversity in genomic features such as virulence factors, genomic islands, prophage regions, toxin-antitoxin cassettes, and acquired antimicrobial resistance genes. Core genome identified in the study can give important leads in the direction of design of rapid and reliable diagnostics, and vaccine design for effective infection control as well as eradication. Additionally, the identified genetic differences among the S. enterica serovar Gallinarum strains could be used for bacterial typing, structure based inhibitor development by future experimental investigations on the data generated.

Tolerogenic Immunoregulation towards Salmonella Enteritidis Contributes to Colonization Persistence in Young Chicks.

Long-term survival and the persistence of bacteria in the host suggest either host unresponsiveness or induction of an immunological tolerant response to the pathogen. The role of the host immunological response to persistent colonization of Salmonella Enteritidis (SE) in chickens remains poorly understood. In the current study, we performed a cecal tonsil transcriptome analysis in a model of SE persistent infection in 2-week-old chickens to comprehensively examine the dynamics of host immunological responses in the chicken gastrointestinal tract. Our results revealed overall host tolerogenic adaptive immune regulation in a major gut-associated lymphoid tissue, the cecal tonsil, during SE infection. Specifically, we observed consistent downregulation of the metallothionein 4 gene at all four postinfection time points (3, 7, 14, and 21 days postinfection [dpi]), which suggested potential pathogen-associated manipulation of the host zinc regulation as well as a possible immune modulatory effect. Furthermore, delayed activation in the B cell receptor signaling pathway and failure to sustain its active state during the lag phase of infection were further supported by an insignificant production of both intestinal and circulatory antibodies. Tug-of-war for interleukin 2 (IL-2) regulation between effector T cells and regulatory T cells appears to have consequences for upregulation in the transducer of ERBB2 (TOB) pathway, a negative regulator of T cell proliferation. In conclusion, this work highlights the overall host tolerogenic immune response that promotes persistent colonization by SE in young layer chicks.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Pathoadaptation of the passerine-associated Salmonella enterica serovar Typhimurium lineage to the avian host.

Salmonella enterica is a diverse bacterial pathogen and a primary cause of human and animal infections. While many S. enterica serovars present a broad host-specificity, several specialized pathotypes have been adapted to colonize and cause disease in one or limited numbers of host species. The underlying mechanisms defining Salmonella host-specificity are far from understood. Here, we present genetic analysis, phenotypic characterization and virulence profiling of a monophasic S. enterica serovar Typhimurium strain that was isolated from several wild sparrows in Israel. Whole genome sequencing and complete assembly of its genome demonstrate a unique genetic signature that includes the integration of the BTP1 prophage, loss of the virulence plasmid, pSLT and pseudogene accumulation in multiple T3SS-2 effectors (sseJ, steC, gogB, sseK2, and sseK3), catalase (katE), tetrathionate respiration (ttrB) and several adhesion/ colonization factors (lpfD, fimH, bigA, ratB, siiC and siiE) encoded genes. Correspondingly, this strain demonstrates impaired biofilm formation, intolerance to oxidative stress and compromised intracellular replication within non-phagocytic host cells. Moreover, while this strain showed attenuated pathogenicity in the mouse, it was highly virulent and caused an inflammatory disease in an avian host. Overall, our findings demonstrate a unique phenotypic profile and genetic makeup of an overlooked S. Typhimurium sparrow-associated lineage and present distinct genetic signatures that are likely to contribute to its pathoadaptation to passerine birds.

Transcriptional response of blood leukocytes from turkeys challenged with Salmonella enterica serovar Typhimurium UK1.

Non-typhoidal Salmonella is one of the most common causes of bacterial foodborne disease and consumption of contaminated poultry products, including turkey, is one source of exposure. Minimizing Salmonella colonization of commercial turkeys could decrease the incidence of Salmonella-associated human foodborne illness. Understanding host responses to these bacteria is critical in developing strategies to minimize colonization and reduce food safety risk. In this study, we evaluated bacterial load and blood leukocyte transcriptomic responses of 3-week-old turkeys challenged with the Salmonella enterica serovar Typhimurium (S. Typhimurium) UK1 strain. Turkeys (n = 8/dose) were inoculated by oral gavage with 108 or 1010 colony forming units (CFU) of S. Typhimurium UK1, and fecal shedding and tissue colonization were measured across multiple days post-inoculation (dpi). Fecal shedding was 1-2 log10 higher in the 1010 CFU group than the 108 CFU group, but both doses effectively colonized the crop, spleen, ileum, cecum, colon, bursa of Fabricius and cloaca without causing any detectable clinical signs in either group of birds. Blood leukocytes were isolated from a subset of the birds (n = 3-4/dpi) both pre-inoculation (0 dpi) and 2 dpi with 1010 CFU and their transcriptomic responses assayed by RNA-sequencing (RNA-seq). At 2 dpi, 647 genes had significant differential expression (DE), including large increases in expression of immune genes such as CCAH221, IL4I1, LYZ, IL13RA2, IL22RA2, and ACOD1. IL1ß was predicted as a major regulator of DE in the leukocytes, which was predicted to activate cell migration, phagocytosis and proliferation, and to impact the STAT3 and toll-like receptor pathways. These analyses revealed genes and pathways by which turkey blood leukocytes responded to the pathogen and can provide potential targets for developing intervention strategies or diagnostic assays to mitigate S. Typhimurium colonization in turkeys.

Salmonella Schwarzengrund, Akuafo, and O:16 isolated from vacuum-packaged beef produced in the state of Mato Grosso, Brazil.

INTRODUCTION: Salmonella spp. is a pathogen associated with foodborne infections, mainly in foods of animal origin. In this context, the present study investigated the occurrence of Salmonella serotypes, genotypes and the antimicrobial resistance profiles of strains in fresh beef produced in Mato Grosso, Brazil. METHODOLOGY: A total of 107 samples from 13 different slaughterhouses in the Mato Grosso were analyzed. Suggestive Salmonella spp. colonies detected during the biochemical screening were submitted to DNA extraction, and hilA gene amplification was used for the PCR reaction. Antimicrobial resistance analyses were performed using 17 antimicrobial agents from eight different classes by the disk diffusion method. Strains exhibiting multiple drug resistances were submitted to PCR genotyping based on repetitive sequences (rep-PCR), using a commercial semiautomatic DiversiLab® system. RESULTS: A total of 5.6% (6/107) of the samples tested positive by the conventional method and were confirmed by PCR, namely two S. Akuafo, two non-typable Salmonella enterica strains, one Salmonella O:16 serovar, and one S. Schwarzengrund. The antimicrobial resistance profiles indicated resistance to gentamicin (30%), tetracycline, nitrofurantoin, and trimethoprim + sulfamethoxazole (16%). Genotyping indicated a 70% difference between S. Schwarzengrund and the non-typable Salmonella strains. No genetic similarities were observed between the six Salmonella isolates based on rep-PCR, including two S. Akuafo. CONCLUSIONS: The results obtained herein corroborate that Salmonella serovar Schwarzengrund is commonly isolated in animal products in the state of Mato Grosso, Brazil, also highlighting the presence of two unusual Salmonella serovars in beef (Akuafo and O:16).

Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health.

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination.

Chicken cecal DNA methylome alteration in the response to Salmonella enterica serovar Enteritidis inoculation.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the pathogenic bacteria, which affects poultry production and poses a severe threat to public health. Chicken meat and eggs are the main sources of human salmonellosis. DNA methylation is involved in regulatory processes including gene expression, chromatin structure and genomic imprinting. To understand the methylation regulation in the response to SE inoculation in chicken, the genome-wide DNA methylation profile following SE inoculation was analyzed through whole-genome bisulfite sequencing in the current study. RESULTS: There were 185,362,463 clean reads and 126,098,724 unique reads in the control group, and 180,530,750 clean reads and 126,782,896 unique reads in the inoculated group. The methylation density in the gene body was higher than that in the upstream and downstream regions of the gene. There were 8946 differentially methylated genes (3639 hypo-methylated genes, 5307 hyper-methylated genes) obtained between inoculated and control groups. Methylated genes were mainly enriched in immune-related Gene Ontology (GO) terms and metabolic process terms. Cytokine-cytokine receptor interaction, TGF-beta signaling pathway, FoxO signaling pathway, Wnt signaling pathway and several metabolism-related pathways were significantly enriched. The density of differentially methylated cytosines in miRNAs was the highest. HOX genes were widely methylated. CONCLUSIONS: The genome-wide DNA methylation profile in the response to SE inoculation in chicken was analyzed. SE inoculation promoted the DNA methylation in the chicken cecum and caused methylation alteration in immune- and metabolic- related genes. Wnt signal pathway, miRNAs and HOX gene family may play crucial roles in the methylation regulation of SE inoculation in chicken. The findings herein will deepen the understanding of epigenetic regulation in the response to SE inoculation in chicken.

The correlated expression of immune and energy metabolism related genes in the response to Salmonella enterica serovar Enteritidis inoculation in chicken.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the food-borne pathogenic bacteria, which affects poultry production and poses severe threat to human health. The correlation of immune system and metabolism in chicken after SE inoculation is important but not clear. In the current study, we identified the expression of immune and energy metabolism related genes using quantitative PCR to evaluate the correlation between immune system and energy metabolism against SE inoculation in Jining Bairi chicken. RESULTS: ATP5G1, ATP5G3 and ND2 were significantly up-regulated at 1 dpi (day post inoculation), and ATP5E, ATP5G1, ATP5G3 were significantly down-regulated at 7 dpi (P < 0.05). IL-8 and IL-1ß were significantly down-regulated at 1 dpi, IL-8 and IL-18 were significantly down-regulated at 3 dpi, IL-8 and BCL10 were significantly up-regulated at 7 dpi (P < 0.05). CONCLUSIONS: These findings indicate that the correlation between immune and energy metabolism related genes gradually change with time points post SE inoculation, from one homeostasis to an opposite homeostasis with 3 dpi as a turning point. These results will pave the foundation for the relationship between immune system and energy metabolism in the response to SE inoculation in chicken.

Identification of single-nucleotide variants associated with susceptibility to Salmonella in pigs using a genome-wide association approach.

BACKGROUND: Salmonella enterica serovars are a major cause of foodborne illness and have a substantial impact on global human health. In Canada, Salmonella is commonly found on swine farms and the increasing concern about drug use and antimicrobial resistance associated with Salmonella has promoted research into alternative control methods, including selecting for pig genotypes associated with resistance to Salmonella. The objective of this study was to identify single-nucleotide variants in the pig genome associated with Salmonella susceptibility using a genome-wide association approach. Repeated blood and fecal samples were collected from 809 pigs in 14 groups on farms and tonsils and lymph nodes were collected at slaughter. Sera were analyzed for Salmonella IgG antibodies by ELISA and feces and tissues were cultured for Salmonella. Pig DNA was genotyped using a custom 54 K single-nucleotide variant oligo array and logistic mixed-models used to identify SNVs associated with IgG seropositivity, shedding, and tissue colonization. RESULTS: Variants in/near PTPRJ (p = 0.0000066), ST6GALNAC3 (p = 0.0000099), and DCDC2C (n = 3, p < 0.0000086) were associated with susceptibility to Salmonella, while variants near AKAP12 (n = 3, p < 0.0000358) and in RALGAPA2 (p = 0.0000760) may be associated with susceptibility. CONCLUSIONS: Further study of the variants and genes identified may improve our understanding of neutrophil recruitment, intracellular killing of bacteria, and/or susceptibility to Salmonella and may help future efforts to reduce Salmonella on-farm through genetic approaches.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.