Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

Monophasic expression of FliC by Salmonella 4,[5],12:i:- DT193 does not alter its pathogenicity during infection of porcine intestinal epithelial cells.

Non-typhoidal serotypes of Salmonella enterica remain important food-borne pathogens worldwide and the frequent emergence of epidemic strains in food-producing animals is a risk to public health. In recent years, Salmonella 4,[5],12:i:- isolates, expressing only phase 1 (FliC) of the two flagellar antigens, have emerged and increased in prevalence worldwide. In Europe, the majority of 4,[5],12:i:- isolates belong to phage types DT193 and DT120 of Salmonella Typhimurium and pigs have been identified as the reservoir species. In this study we investigated the ability of pig-derived monophasic (4,[5],12:i:-) and biphasic DT193 isolates to invade a porcine intestinal epithelial cell line (IPEC-1) and activate TLR-5, IL-8 and caspases. We found that the 4,[5],12:i:- isolates exhibited comparable adhesion and invasion to that of the virulent S. Typhimurium isolate 4/74, suggesting that these strains could be capable of colonizing the small intestine of pigs in vivo. Infection with 4,[5],12:i:- and biphasic DT193 isolates resulted in approximately the same level of TLR-5 (a flagellin receptor) and IL-8 (a proinflammatory chemokine) mRNA upregulation. The monophasic variants also elicited similar levels of caspase activation and cytotoxicity to the phase-variable DT193 isolates. These findings suggest that failure of 4,[5],12:i:- DT193 isolates to express a second phase of flagellar antigen (FljB) is unlikely to hamper their pathogenicity during colonization of the porcine intestinal tract.

Dietary supplementation with soluble plantain non-starch polysaccharides inhibits intestinal invasion of Salmonella Typhimurium in the chicken.

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1-99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5-10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64-81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75-90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis.

Immunity to bacterial infection in the chicken.

Bacterial infections remain important to the poultry industry both in terms of animal and public health, the latter due to the importance of poultry as a source of foodborne bacterial zoonoses such as Salmonella and Campylobacter. As such, much focus of research to the immune response to bacterial infection has been to Salmonella. In this review we will focus on how research on avian salmonellosis has developed our understanding of immunity to bacteria in the chicken from understanding the role of TLRs in recognition of bacterial pathogens, through the role of heterophils, macrophages and γδ lymphocytes in innate immunity and activation of adaptive responses to the role of cellular and humoral immunity in immune clearance and protection. What is known of the immune response to other bacterial infections and in particular infections that have emerged recently as major problems in poultry production including Campylobacter jejuni, Avian Pathogenic Escherichia coli, Ornithobacterium rhinotracheale and Clostridium perfringens are discussed.

Immunological changes at point-of-lay increase susceptibility to Salmonella enterica Serovar enteritidis infection in vaccinated chickens.

Chicken eggs are the main source of human Salmonella enterica serovar Enteritidis infection. S. Enteritidis infects the oviduct and ovary of the chicken leading to infection of developing eggs. Therefore, control in poultry production is a major public health priority. Vaccination of hens has proved successful in control strategies in United Kingdom leading to a 70% drop in human cases since introduced. However, as hens reach sexual maturity they become immunosuppressed and it has been postulated this leads to increased susceptibility to Salmonella infection. In this study we define the changes to the systemic and reproductive tract-associated immune system of hens throughout sexual development by flow cytometry and histology and determine changes in susceptibility to experimental S. Enteritidis challenge in naive and vaccinated hens. Changes to both systemic and local immune systems occur in chickens at sexual development around 140 days of age. The population of several leukocyte classes drop, with the greatest fall in CD4+ lymphocyte numbers. Within the developing reproductive tract there an organised structure of lymphocytic aggregates with γδ-T lymphocytes associated with the mucosa. At point-of-lay, this organised structure disappears and only scattered lymphocytes remain. Protection against Salmonella challenge is significantly reduced in vaccinated birds at point-of-lay, coinciding with the drop in CD4+ lymphocytes. Susceptibility to reproductive tract infection by Salmonella increased in vaccinated and naïve animals at 140 and 148 days of age. We hypothesise that the drop in γδ-T lymphocytes in the tract leads to decreased innate protection of the mucosa to infection. These findings indicate that systemic and local changes to the immune system increase the susceptibility of hens to S. Enteritidis infection. The loss of protective immunity in vaccinated birds demonstrates that Salmonella control should not rely on vaccination alone, but as part of an integrated control strategy including biosecurity and improved animal welfare.

The Salmonella pathogenicity island 2-encoded type III secretion system is essential for the survival of Salmonella enterica serovar Typhimurium in free-living amoebae.

Free-living amoebae represent a potential reservoir and predator of Salmonella enterica. Through the use of type III secretion system (T3SS) mutants and analysis of transcription of selected T3SS genes, we demonstrated that the Salmonella pathogenicity island 2 is highly induced during S. enterica serovar Typhimurium infection of Acanthamoeba polyphaga and is essential for survival within amoebae.

Characterisation of Salmonella enterica serotype Typhimurium isolates from wild birds in northern England from 2005 - 2006.

BACKGROUND: Several studies have shown that a number of serovars of Salmonella enterica may be isolated from wild birds, and it has been suggested that wild birds may play a role in the epidemiology of human and livestock salmonellosis. However, little is known about the relationship between wild bird S. enterica strains and human- and livestock- associated strains in the United Kingdom. Given the zoonotic potential of salmonellosis, the main aim of this study was to investigate the molecular epidemiology of S. enterica infections in wild birds in the north of England and, in particular, to determine if wild bird isolates were similar to those associated with disease in livestock or humans. RESULTS: Thirty two Salmonella enterica isolates were collected from wild birds in northern England between February 2005 and October 2006, of which 29 were S. enterica serovar Typhimurium (S. Typhimurium); one S. Newport, one S. Senftenberg, and one isolate could not be classified by serotyping. Further analysis through phage typing and macro-restriction pulsed-field gel electrophoresis indicated that wild passerine deaths associated with salmonellosis were caused by closely-related S. Typhimurium isolates, some of which were clonal. These isolates were susceptible to all antimicrobials tested, capable of invading and persisting within avian macrophage-like HD11 cells in vitro, and contained a range of virulence factors associated with both systemic and enteric infections of birds and mammals. However, all the isolates lacked the sopE gene associated with some human and livestock disease outbreaks caused by S. Typhimurium. CONCLUSION: The wild bird isolates of S. enterica characterised in this investigation may not represent a large zoonotic risk. Molecular characterisation of isolates suggested that S. Typhimurium infection in wild passerines is maintained within wild bird populations and the causative strains may be host-adapted.

Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge.

Macrophages from inbred chickens that are resistant to salmonellosis show greater and more rapid expression of proinflammatory chemokines and cytokines, including the key Th1-inducing cytokine interleukin-18, upon Salmonella challenge than those from susceptible birds. This suggests the possibility that salmonellosis resistant-line macrophages signal more effectively and rapidly and are more able to induce protective Th1 adaptive responses.

Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge.

Infection of poultry with Salmonella enterica serovar Typhimurium poses a significant risk to public health through contamination of meat from infected animals. Vaccination has been proposed to control infections in chickens. However, the vaccines are currently largely empirical, and our understanding of the mechanisms that underpin immune clearance and protection in avian salmonellosis is not complete. In this study we describe the cytokine, chemokine, and antibody responses and cellular changes in primary and secondary infections of chickens with Salmonella serovar Typhimurium. Infection of 1-week-old chickens induced early expression of a macrophage inflammatory protein (MIP) family chemokine in the spleen and liver, followed by increased expression of gamma interferon accompanied by increased numbers of both CD4(+) and CD8(+) T cells and the formation of granuloma-like follicular lesions. This response correlated with a Th1-mediated clearance of the systemic infection. Primary infection also induced specific immunoglobulin M (IgM), IgG, and IgA antibody responses. In contrast to previously published studies performed with newly hatched chicks, the expression levels of proinflammatory cytokines in the gastrointestinal tract were not greatly increased following infection. However, significant expression of the anti-inflammatory cytokine transforming growth factor beta4 was detected in the gut early in infection. Following secondary challenge, the birds were fully protected against systemic infection and showed a high level of protection against gastrointestinal colonization. Rapid expression of the MIP family chemokine and interleukin-6 was detected in the guts of these birds and was accompanied by an influx of lymphocytes. Increased levels of serum IgA-specific antibodies were also found following rechallenge. These findings suggest that cellular responses, particularly Th1 responses, play a crucial role in immune clearance in avian salmonellosis and that protection against rechallenge involves the rapid recruitment of cells to the gastrointestinal tract. Additionally, the high levels of inflammatory response found following Salmonella serovar Typhimurium infection of newly hatched chicks were not observed following infection of older birds (1 week old), in which the expression of regulatory cytokines appeared to limit inflammation.

Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium.

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5+ cells to flagellin induced elevated levels of chicken interleukin-1beta (chIL-1beta) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1beta mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken.

In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus.

A number of inbred lines of chickens have been shown to be resistant or susceptible to systemic salmonellosis caused by Salmonella enterica serovar Gallinarum in adult birds, or by S. enterica serovar Enteritidis and S. enterica serovar Typhimurium in young chicks. Resistant lines show only moderate pathology and low mortality rates, whereas susceptible lines display extensive pathological changes and higher levels of mortality following Salmonella infection. Genetic resistance to salmonellosis is dominant and not linked to sex, MHC or Slc11a1 (formerly known as Nramp1), which leads to resistance in mice and other species. A novel locus encoding resistance to salmonellosis has been identified on chicken chromosome 5, and designated SAL1. The nature of the differences in pathology found between resistant and susceptible chicken lines in vivo indicates that resistance is expressed at the level of the mononuclear phagocyte system. Macrophages from adult resistant line birds cleared Salmonella serovar Gallinarum from infected macrophages within 24 h, whereas Salmonella bacteria persisted within macrophages from susceptible line birds for at least 48 h. Clearance of Salmonella by macrophages was accompanied by a strong and reproducible respiratory burst response in resistant lines, but little or no response in susceptible lines. Macrophages from an outbred chicken line showed variable responses. No differences were seen in macrophage nitric oxide production in cells from resistant or susceptible lines. These differences suggest that increased macrophage antimicrobial activity correlates with resistance and that macrophage activity plays an important role in genetic resistance to systemic salmonellosis in the chicken.

Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum.

Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)-1ss, IL-2, IL-6 and interferon (IFN)-gamma were measured by quantitative RT-PCR, and production of IL-6 and IFN-gamma was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-gamma compared with non-infected cells; S. enteritidis invasion caused a slight down-regulation of IL-2 production. For IL-1ss production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1ss mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.