Paul A. Barrow

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.

Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways

We have determined the complete genome sequences of a host-promiscuous Salmonella enterica serovar Enteritidis PT4 isolate P125109 and a chicken-restricted Salmonella enterica serovar Gallinarum isolate 287/91. Genome comparisons between these and other Salmonella isolates indicate that S. Gallinarum 287/91 is a recently evolved descendent of S. Enteritidis. Significantly, the genome of S. Gallinarum has undergone extensive degradation through deletion and pseudogene formation. Comparison of the pseudogenes in S. Gallinarum with those identified previously in other host-adapted bacteria reveals the loss of many common functional traits and provides insights into possible mechanisms of host and tissue adaptation. We propose that experimental analysis in chickens and mice of S. Enteritidis-harboring mutations in functional homologs of the pseudogenes present in S. Gallinarum could provide an experimentally tractable route toward unraveling the genetic basis of host adaptation in S. enterica.

Reduction of Experimental Salmonella and Campylobacter Contamination of Chicken Skin by Application of Lytic Bacteriophages

ABSTRACT Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10 unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10 units over 48 h. When the level of Salmonella contamination was low (< log10 2 per unit area of skin) and the MOI was 105, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 107), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.

Bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential.

Bacteriophages were discovered 82 years ago. Claims for their use in the treatment of infections were not confirmed by early controlled trials, and the success of antibiotics superseded this potential use. However, recent studies have shown interesting therapeutic effects that warrant further investigation and development.

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature‐tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI‐4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI‐4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many ‘housekeeping’ genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

ABSTRACT Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

Allelic Variation in TLR4 Is Linked to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Chickens

ABSTRACT Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection.

The immunobiology of avian systemic salmonellosis

Avian systemic salmonellosis is primarily caused by Salmonella enterica serovar Gallinarum and serovar Pullorum causing the diseases Fowl Typhoid and Pullorum Disease respectively. During infection interaction with the immune system occurs in three main phases. First is invasion via the gastrointestinal tract. Infection with S. Pullorum or S. Gallinarum does not cause substantial inflammation, unlike S. Typhimurium or S. Enteritidis. Through in vitro models it was found that S. Gallinarum does not induce expression of CXC chemokines or pro-inflammatory cytokines such as IL-1beta or IL-6, whilst in an in vivo model S. Pullorum infection leads to down-regulation of CXCLi1 and CXCLi2 in the ileum. The absence of flagella in S. Gallinarum and S. Pullorum means they are not recognised by TLR5, which is believed to play a key role in the initiation of inflammatory responses, though other pathogen-factors are likely to be involved. The second phase is establishing systemic infection. Salmonella invade macrophages and probably dendritic cells and are translocated to the spleen and liver, where replication occurs. Salmonella survival is dependent on the Salmonella pathogenicity island 2 type III secretion system, which inhibits antimicrobial activity by preventing fusion of lysosymes with the phagocytic vacuole and by modulation of MHC and cytokine expression. Studies in resistant and susceptible chicken lines have shown that the interaction with macrophages is central to the progression of infection or immunological clearance. Primary macrophages from resistant animals are more efficient in killing Salmonella through respiratory burst and by induction of cytokine expression including the initiation of protective Th1 responses that leads to the third phase. Where replication of Salmonella is not controlled the death of the animal usually results. If the innate immune system is not able to control replication then cellular and humoral responses, primarily mediated through Th1-associated cytokines, are able to clear infection. In S. Pullorum a significant number of animals develop persistent infection of splenic macrophages. Here we show preliminary evidence of modulation of adaptive immunity away from a Th1 response to facilitate the development of the carrier state. In carrier animals persistence may lead to reproductive tract and egg infection associated with a decline in CD4+ T cell numbers and function associated with the onset of sexual maturity in hens.

Rapid Expression of Chemokines and Proinflammatory Cytokines in Newly Hatched Chickens Infected with Salmonella enterica Serovar Typhimurium

ABSTRACT Poultry meat and eggs contaminated with Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective against Salmonella infection in chickens has not been characterized at the molecular level. In the present study, bacterial colonization, development of pathological lesions, and proinflammatory cytokine and chemokine gene expression were investigated in the liver, spleen, jejunum, ileum, and cecal tonsils in newly hatched chickens 6, 12, 24, and 48 h after oral infection with Salmonella serovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereas Salmonella started to appear in the liver only from 24 h postinfection. Large numbers of heterophils, equivalent to neutrophils in mammals, and inflammatory edema could be seen in the lamina propria of the intestinal villi and in the liver. Interleukin 8 (IL-8), K60 (a CXC chemokine), macrophage inflammatory protein 1 β, and IL-1β levels were significantly upregulated in the intestinal tissues and in the livers of the infected birds. However, the spleens of the infected birds show little or no change in the expression levels of these cytokines and chemokines. Increased expression of the proinflammatory cytokines and chemokines (up to several hundred-fold) correlated with the presence of inflammatory signs in those tissues. This is the first description of in vivo expression of chemokines and proinflammatory cytokines in response to oral infection with Salmonella in newly hatched chickens.

Intestinal colonisation in the chicken by food‐poisoning salmonella serotypes; Microbial characteristics associated with faecal excretion

Following oral inoculation of newly-hatched or three-week-old chickens, Salmonella organisms persisted longest in the caeca and they were also present for a shorter time in the crop. S. cholerae-suis, E. coli K12 and Saccharomyces cerevisiae did not colonise the alimentary tract when inoculated orally, but they persisted longer in the caeca than in other regions of the gut. This suggested that preferential localisation of Salmonella in the caeca may be the result of non-specific or host factors. This may include the slow rate of flow of contents through this organ which would allow greater microbial multiplication and persistence. Some Salmonella strains, particularly S. typhimurium, inoculated orally into three-week-old chickens were isolated in greater numbers from homogenates of the crop wall and, much less frequently, from the caecal wall, than from the luminal contents of these organs. None of the strains associated with the crop wall to the same extent as did lactobacilli. After oral inoculation of food-poisoning Salmonella serotypes the number of chickens excreting Salmonellae in their faeces gradually declined over a period of at least 4 weeks. In contrast, host adapted serotypes such as S. cholerae-suis and S. abortus-ovis were not excreted for longer than a few days. By testing mutants of S. typhimurium and S. infantis which were devoid of easily recognised characteristics it was concluded that neither flagellar (H) antigens, somatic (O) antigens, mannose-sensitive haemagglutinins nor the possession of the virulence-associated 85 kilobase plasmid of S. typhimurium, were essential for colonisation (persistent faecal excretion). However, a mutant of S. infantis which was created by nitroso-guanidine treatment still possessed flagellar and somatic antigens and haemagglutinins and yet did not colonise the alimentary tract, indicating that an unidentified microbial factor was essential for colonisation.