SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression

Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression

High resolution parallel sequencing reveals multistrain Campylobacter in broiler chicken flocks testing 'negative' by conventional culture methods: implications for control of Campylobacter infection.

Contaminated chicken meat is a major source of human Campylobacteriosis and rates of infection remain high, despite efforts to limit the colonisation of broiler (meat) chicken flocks on farms. Using conventional testing methods of culture or qPCR, Campylobacter is typically detected amongst broiler flocks from 3 wk of age, leading to the assumption that infection is introduced horizontally into chicken rearing houses at this time. In this study, we use parallel sequencing of a fragment of the Campylobacter outer membrane protein, encoded by the porA gene, to test for presence of Campylobacter DNA amongst fresh fecal samples collected from broiler flocks aged 23 to 28 d. Campylobacter DNA was detected in all of the 290 samples tested using the porA target, and in 48% of samples using 16S bacterial profiling, irrespective of whether or not Campylobacter could be detected using conventional qPCR thresholds. A single porAf2 variant was predominant among flocks that would be determined to be Campylobacter 'positive' by conventional means, but a diverse pattern was seen among flocks that were Campylobacter 'negative'. The ability to routinely detect low levels of Campylobacter amongst broiler flocks at a much earlier age than would conventionally be identified requires a re-examination of how and when biosecurity measures are best applied for live birds. In addition, it may be useful to investigate why single Campylobacter variants proliferate in some broiler flocks and not others

Experimental infection of chickens by a flagellated motile strain of Salmonella enterica serovar Gallinarum biovar Gallinarum

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT), a septicaemic disease which can result in high mortality in poultry flocks. The absence of flagella in SG is thought to favour systemic invasion, since bacterial recognition via Toll-like receptor (TLR)-5 does not take place during the early stages of FT. In the present study, chicks susceptible to FT were inoculated with a wild type SG (SG) or its flagellated motile derivative (SG Fla+). In experiment 1, mortality and clinical signs were assessed, whereas in experiment 2, gross pathology, histopathology, systemic invasion and immune responses were evaluated. SG Fla+ infection resulted in later development of clinical signs, lower mortality, lower bacterial numbers in the liver and spleen, and less severe pathological changes compared to SG. The CD8+ T lymphocyte population was higher in the livers of chicks infected with SG at 4 days post-inoculation (dpi). Chicks infected with SG had increased expression of interleukin (IL)-6 mRNA in the caecal tonsil at 1 dpi and increased expression of IL-18 mRNA in the spleen at 4 dpi. In contrast, the CD4+ T lymphocyte population was higher at 6 dpi in the livers of birds infected with SG Fla+. Therefore, flagella appeared to modulate the chicken immune response towards a CD4+ T profile, resulting in more efficient bacterial clearance from systemic sites and milder infection

Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria

Highly variable microbiota development in the chicken gastrointestinal tract

Studies investigating the role that complex microbiotas associated with animals and humans play in health and wellbeing have been greatly facilitated by advances in DNA sequencing technology. Due to the still relatively high sequencing costs and the expense of establishing and running animal trials and collecting clinical samples, most of the studies reported in the literature are limited to a single trial and relatively small numbers of samples. Results from different laboratories, investigating similar trials and samples, have often produced quite different pictures of microbiota composition. This study investigated batch to batch variations in chicken cecal microbiota across three similar trials, represented by individually analysed samples from 207 birds. Very different microbiota profiles were found across the three flocks. The flocks also differed in the efficiency of nutrient use as indicated by feed conversion ratios. In addition, large variations in the microbiota of birds within a single trial were noted. It is postulated that the large variability in microbiota composition is due, at least in part, to the lack of colonisation of the chicks by maternally derived bacteria. The high hygiene levels maintained in modern commercial hatcheries, although effective in reducing the burden of specific diseases, may have the undesirable effect of causing highly variable bacterial colonization of the gut. Studies in humans and other animals have previously demonstrated large variations in microbiota composition when comparing individuals from different populations and from different environments but this study shows that even under carefully controlled conditions large variations in microbiota composition still occur.Dragana Stanley, Mark S. Geier, Robert J. Hughes, Stuart E. Denman and Robert J. Moor

Effects of dietary supplementation with a laminarin-rich extract on the growth performance and gastrointestinal health in broilers

Restriction in antimicrobial use in broiler chicken production is driving the exploration of alternative feed additives that will support growth through the promotion of gastrointestinal health and development. The objective of this study was to determine the effects of dietary inclusion of laminarin on growth performance, the expression of nutrient transporters, markers of inflammation and intestinal integrity in the small intestine and composition of the caecal microbiota in broiler chickens. Two-hundred-and-forty day-old male Ross 308 broiler chicks (40.64 (3.43 SD) g) were randomly assigned to: (T1) basal diet (control); (T2) basal diet + 150 ppm laminarin; (T3) basal diet + 300 ppm laminarin (5 bird/pen; 16 pens/treatment). The basal diet was supplemented with a laminarin-rich Laminaria spp. extract (65% laminarin) to achieve the two laminarin inclusion levels (150 and 300 ppm). Chick weights and feed intake was recorded weekly. After 35 days of supplementation, one bird per pen from the control and best performing (300 ppm) laminarin groups were euthanized. Duodenal, jejunal and ileal tissues were collected for gene expression analysis. Caecal digesta was collected for microbiota analysis (high-throughput sequencing and QPCR). Dietary supplementation with 300 ppm laminarin increased both final body weight (2033 vs. 1906 ± 30.4, P < 0.05) and average daily gain (62.3 vs. 58.2 ± 0.95, P < 0.05) compared to the control group and average daily feed intake (114.1 vs. 106.0 and 104.5 ± 1.77, P < 0.05) compared to all other groups. Laminarin supplementation at 300 ppm increased the relative and absolute abundance of Bifidobacterium (P < 0.05) in the caecum. Laminarin supplementation increased the expression of interleukin 17A (IL17A) in the duodenum, claudin 1 (CLDN1) and toll-like receptor 2 (TLR2) in the jejunum and IL17A, CLDN1 and SLC15A1/peptide transporter 1 (SLC15A1/PepT1) in the ileum (P < 0.05). In conclusion, supplementation with laminarin is a promising dietary strategy to enhance growth performance and 300 ppm was the optimal inclusion level with which to promote a beneficial profile of the gastrointestinal microbiota in broiler chickens

Evolution of the avian β-defensin and cathelicidin genes

Background: β-defensins and cathelicidins are two families of cationic antimicrobial peptides (AMPs) with a broad range of antimicrobial activities that are key components of the innate immune system. Due to their important roles in host defense against rapidly evolving pathogens, the two gene families provide an ideal system for studying adaptive gene evolution. In this study we performed phylogenetic and selection analyses on β-defensins and cathelicidins from 53 avian species representing 32 orders to examine the evolutionary dynamics of these peptides in birds. Results and conclusions: Avian β-defensins are found in a gene cluster consisting of 13 subfamiles. Nine of these are conserved as one to one orthologs in all birds, while the others (AvBD1, AvBD3, AvBD7 and AvBD14) are more subject to gene duplication or pseudogenisation events in specific avian lineages. Avian cathelicidins are found in a gene cluster consisting of three subfamilies with species-specific duplications and gene loss. Evidence suggested that the propiece and mature peptide domains of avian cathelicidins are possibly co-evolving in such a way that the cationicity of the mature peptide is partially neutralised by the negative charge of the propiece prior to peptide secretion (further evidence obtained by repeating the analyses on primate cathelicidins). Negative selection (overall mean d