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.

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.

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.

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.

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.

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.

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.

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.

Identification of genes related to resistance against S. Typhimurium in ovine macrophages.

Salmonella enteric serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen causing public health hazards. Identification of genes related to macrophages resistance to S. Typhimurium and their immune mechanisms can provide a theoretical basis for disease resistance. In this study, sixty significant differentially expressed genes were screened between susceptible and resistant sheep macrophages by transcriptome RNA-seq. Eight significantly enriched GO terms and six canonical pathways were involved by GO and KEGG enrichment analysis. Furthermore, knockdown of HMOX1 and SLPI increased remarkably the clearance of S. typhimurium, but SPP1 had little effect on the clearance of S. Typhimurium within sheep macrophages. Altogether, these results suggest that many genes of macrophages were reprogrammed via S. Typhimurium infection, some of which may facilitate host defence against Salmonella, while others allow Salmonella to escape.

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.

T cell fate and clonality inference from single-cell transcriptomes.

We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.

A genome-wide association study explores the genetic determinism of host resistance to Salmonella pullorum infection in chickens.

BACKGROUND: Salmonella infection is a serious concern in poultry farming because of its impact on both economic loss and human health. Chicks aged 20 days or less are extremely vulnerable to Salmonella pullorum (SP), which causes high mortality. Furthermore, an outbreak of SP infection can result in a considerable number of carriers that become potential transmitters, thus, threatening fellow chickens and offspring. In this study, we conducted a genome-wide association study (GWAS) to detect potential genomic loci and candidate genes associated with two disease-related traits: death and carrier state. METHODS: In total, 818 birds were phenotyped for death and carrier state traits through a SP challenge experiment, and genotyped by using a 600 K high-density single nucleotide polymorphism (SNP) array. A GWAS using a single-marker linear mixed model was performed with the GEMMA software. RNA-sequencing on spleen samples was carried out for further identification of candidate genes. RESULTS: We detected a region that was located between 33.48 and 34.03 Mb on chicken chromosome 4 and was significantly associated with death, with the most significant SNP (rs314483802) accounting for 11.73% of the phenotypic variation. Two candidate genes, FBXW7 and LRBA, were identified as the most promising genes involved in resistance to SP. The expression levels of FBXW7 and LRBA were significantly downregulated after SP infection, which suggests that they may have a role in controlling SP infections. Two other significant loci and related genes (TRAF3 and gga-mir-489) were associated with carrier state, which indicates a different polygenic determinism compared with that of death. In addition, genomic inbreeding coefficients showed no correlation with resistance to SP within each breed in our study. CONCLUSIONS: The results of this GWAS with a carefully organized Salmonella challenge experiment represent an important milestone in understanding the genetics of infectious disease resistance, offer a theoretical basis for breeding SP-resistant chicken lines using marker-assisted selection, and provide new information for salmonellosis research in humans and other animals.

Genome-wide association study identified genes in the response to Salmonella pullorum infection in chickens.

Pullorum is a bacterial disease that threatens the modern poultry industry. Over the years, research on this topic has focused mainly on its epidemiology, whereas the hosts' genetic basis of infection is still vague. In order to identify chickens' genes associated with pullorum, we sequenced 300 New Pudong chicken by double digest genotyping-by-sequencing. We obtained 1 527 953 SNPs for a genome-wide association analysis, which identified 43 genome-wide significant markers. Most of the significant SNPs were in the interval of 57.7-59.0 Mb on chromosome 5. The gene set enrichment analysis suggests a potential manner for bacterial infection and remaining inside the host. This work provides basic data for the purification, prevention and treatment of pullorum disease.

Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice.

BACKGROUND: Salmonella is a Gram-negative bacterium causing a wide range of clinical syndromes ranging from typhoid fever to diarrheic disease. Non-typhoidal Salmonella (NTS) serovars infect humans and animals, causing important health burden in the world. Susceptibility to salmonellosis varies between individuals under the control of host genes, as demonstrated by the identification of over 20 genetic loci in various mouse crosses. We have investigated the host response to S. Typhimurium infection in 35 Collaborative Cross (CC) strains, a genetic population which involves wild-derived strains that had not been previously assessed. RESULTS: One hundred and forty-eight mice from 35 CC strains were challenged intravenously with 1000 colony-forming units (CFUs) of S. Typhimurium. Bacterial load was measured in spleen and liver at day 4 post-infection. CC strains differed significantly (P < 0.0001) in spleen and liver bacterial loads, while sex and age had no effect. Two significant quantitative trait loci (QTLs) on chromosomes 8 and 10 and one suggestive QTL on chromosome 1 were found for spleen bacterial load, while two suggestive QTLs on chromosomes 6 and 17 were found for liver bacterial load. These QTLs are caused by distinct allelic patterns, principally involving alleles originating from the wild-derived founders. Using sequence variations between the eight CC founder strains combined with database mining for expression in target organs and known immune phenotypes, we were able to refine the QTLs intervals and establish a list of the most promising candidate genes. Furthermore, we identified one strain, CC042/GeniUnc (CC042), as highly susceptible to S. Typhimurium infection. CONCLUSIONS: By exploring a broader genetic variation, the Collaborative Cross population has revealed novel loci of resistance to Salmonella Typhimurium. It also led to the identification of CC042 as an extremely susceptible strain.

Regulators of Salmonella-host interaction identified by peripheral blood transcriptome profiling: roles of TGFB1 and TRP53 in intracellular Salmonella replication in pigs.

Peripheral blood transcriptome is an important intermediate data source for investigating the mechanism of Salmonella invasion, proliferation, and transmission. We challenged 4-week old piglets with Salmonella enterica serovar Typhimurium LT2 and investigated the peripheral blood gene expression profile before treatment (d0) and at 2 and 7 days post-inoculation (dpi) using deep sequencing. Regulator pathways were first predicted in silico and validated by wet-lab experiments. In total, 1255, 765, and 853 genes were differentially expressed between 2 dpi/d0, 7 dpi/d0, and 7 dpi/2 dpi, respectively. Additionally, 1333 genes showed a time effect during the investigated Salmonella infection period. Clustering analysis showed that the differentially expressed genes fell into six distinct expression clusters. Pathway annotation of these gene clusters showed that the innate immune system was first significantly upregulated at 2 dpi and then attenuated at 7 dpi. Toll-like receptor cascades, MyD88 cascade, phagosome pathway, cytokine signaling pathway, and lysosome pathway showed a similar expression pattern. Interestingly, we found that the ribosome pathway was significantly inhibited at 2 and 7 dpi. Gene expression regulation network enrichment analysis identified several candidate factors controlling the expression clusters. Further in vitro study showed that TGFB1 can inhibit Salmonella replication whereas TRP53 can promote Salmonella replication in porcine peripheral blood mononuclear cells and murine macrophages. These results provide new insights into the molecular mechanism of Salmonella-host interactions and clues for the genetic improvement of Salmonella infection resistance in pigs.

Silent but deadly: IS200 promotes pathogenicity in Salmonella Typhimurium.

Bacterial transposons were long thought of as selfish mobile genetic elements that propagate at the expense of 'host' bacterium fitness. However, limited transposition can benefit the host organism by promoting DNA rearrangements and facilitating horizontal gene transfer. Here we discuss and provide context for our recently published work which reported the surprising finding that an otherwise dormant transposon, IS200, encodes a regulatory RNA in Salmonella Typhimurium. This previous work identified a trans-acting sRNA that is encoded in the 5'UTR of IS200 transposase mRNA (tnpA). This sRNA represses expression of genes encoded within Salmonella Pathogenicity Island 1 (SPI-1), and accordingly limits invasion into non-phagocytic cells in vitro. We present new data here that shows IS200 elements are important for colonization of the mouse gastrointestinal tract. We discuss our previous and current findings in the context of transposon biology and suggest that otherwise 'silent' transposons may in fact play an important role in controlling host gene expression.

Occurrence, antimicrobial susceptibility patterns and genotypic relatedness of Salmonella spp. isolates from captive wildlife, their caretakers, feed and water in India.

Occurrence of Salmonella spp. in captive wild animal species in India is largely unknown. The purpose of this study was to determine the occurrence of different Salmonella serotypes, antimicrobial resistance patterns and genotypic relatedness of recovered isolates. A total of 370 samples including faecal (n = 314), feed and water (n = 26) and caretakers stool swabs (n = 30) were collected from 40 different wild animal species in captivity, their caretakers, feed and water in four zoological gardens and wildlife enclosures in India. Salmonellae were isolated using conventional culture methods and tested for antimicrobial susceptibility with the Kirby-Bauer disc diffusion method. Salmonella isolates were serotyped and genotyping was performed using enterobacterial repetitive intergenic consensus (ERIC) PCR and 16S rRNA sequencing. Animal faecal samples were also subjected to direct PCR assay. Salmonella was detected in 10 of 314 (3.1%) faecal samples by isolation and 18 of 314 (5.7%) samples by direct PCR assay; one of 26 (3.8%) feed and water samples and five of 30 (16.7%) caretakers stool swabs by isolation. Salmonella was more commonly isolated in faecal samples from golden pheasants (25%; 2/8) and leopard (10%; 2/20). Salmonella enterica serotypes of known public health significance including S. Typhimurium (37.5%; 6/14), S. Kentucky (28.5%; 4/14) and S. Enteritidis (14.3%; 2/14) were identified. While the majority of the Salmonella isolates were pan-susceptible to the commonly used antibiotics. Seven (43.7%; 7/16) of the isolates were resistant to at least one antibiotic and one isolate each among them exhibited penta and tetra multidrug-resistant types. Three S. Kentucky serotype were identified in a same golden pheasants cage, two from the birds and one from the feed. This serotype was also isolated from its caretaker. Similarly, one isolate each of S. Typhimurium were recovered from ostrich and its caretaker. These isolates were found to be clonally related suggesting that wildlife may serve as reservoir for infections to humans and vice versa. These results emphasise the transmission of Salmonella among hosts via environmental contamination of feces to workers, visitors and other wildlife.

Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1.

Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon-ß production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

Cecal MicroRNAome response to Salmonella enterica serovar Enteritidis infection in White Leghorn Layer.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is a food-borne pathogen and of great threat to human health through consuming the contaminated poultry products. MicroRNAs (miRNAs) play an important role in different biological activities and have been shown to regulate the innate immunity in the bacterial infection. The objective of this study is to identify miRNAs associated with SE infection in laying chicken cecum. RESULTS: Average number of reads of three libraries constructed from infected and non-infected chickens was 12,476,156 and 10,866,976, respectively. There were 598 miRNAs including 194 potential novel miRNAs identified in which 37 miRNAs were significantly differentially expressed between infected and non-infected chickens. In total, 2897 unique target genes regulated by differentially expressed miRNAs were predicted, in which, 841 genes were uniquely regulated by up-regulated miRNAs (G1), 636 genes were uniquely regulated by down-regulated miRNAs (G2), and 1420 were co-regulated by both up and down- regulated miRNAs (G3). There were 118, 73 and 178 GO (Gene ontology) BP (Biological process) terms significantly enriched in G1, G2 and G3 groups, respectively. More immune-related GO BP terms than metabolism-related terms were found in G1. Expression of 12 immune-related genes of four differentially expressed miRNAs was detected through qRT-PCR. The regulatory direction of gga-miR-1416-5p, gga-miR-1662, and gga-miR-34a-5p were opposite with the target genes of TLR21, BCL10, TLR1LA, NOTCH2 and THBS1, respectively. CONCLUSION: The miRNAs contribute to the response to SE infection at the onset of egg laying through regulating the homeostasis between metabolism and immunity. The gga-miR-125b-5p, gga-miR-34a-5p, gga-miR-1416-5p and gga-miR-1662 could play an important role in SE infection through regulating their target genes. The finding herein will pave the foundation for the studies of microRNA regulation in SE infection in laying hens.