Gene expression responses to a Salmonella infection in the chicken intestine differ between lines

Poultry products are an important source of Salmonella enterica. An effective way to reduce food poisoning due to Salmonella would be to breed chickens more resistant to Salmonella. Unfortunately host responses to Salmonella are complex with many factors involved. To learn more about responses to Salmonella in young chickens, a cDNA microarray analysis was performed to compare gene expression profiles between two chicken lines under control and Salmonella infected conditions. Newly hatched chickens were orally infected with S. enterica serovar Enteritidis. Since the intestine is the first barrier the bacteria encounter after oral inoculation, intestinal gene expression was investigated at different timepoints. Differences in gene expression between the two chicken lines were found in control as well as Salmonella infected conditions. In response to the Salmonella infection a fast growing chicken broiler line induced genes that affect T-cell activation, whereas in a slow growing broiler line genes involved in macrophage activation seemed to be more affected at day 1 post-infection. At days 7 and 9 most gene expression differences between the two chicken lines were identified under control conditions, indicating a difference in the intestinal development between the two chicken lines which might be linked to the difference in Salmonella susceptibility. The findings in this study have lead to the identification of novel genes and possible cellular pathways, which are host dependent.     

Immunological and gene expression responses to a Salmonella infection in the chicken intestine

Besides infection in humans, Salmonella enteritidis can also cause serious illness in young chickens. However, the genetic and immunological parameters important for the disease in chickens are not well characterized. In this study, processes in the chicken intestine in response to a Salmonella infection were investigated in two different chicken lines. One-day-old chickens were orally infected with Salmonella. T-cell subpopulations, phagocytic properties of intestinal mononuclear cells and RNA expression levels of the jejunum were investigated. The two chicken lines differed in the amount of cfu in the liver and growth retardation after the infection. Differences in phagocytic activity of intestinal mononuclear cells were found between control and Salmonella infected chickens. The number of CD4+ T-cells of the intestine decreased after the Salmonella infection in one chicken line, while the number of CD8+ T-cells increased in both chicken lines, but the time post infection of this increase differed between the lines. In one chicken line the expression levels of the genes carboxypeptidase M and similar to ORF2 decreased after the Salmonella infection, which might be related to a decrease in the amount of macrophages. With the microarray, ten genes were found that were regulated in only one of the chicken lines, while we found six genes regulated in response to the infection in both chicken lines. So differences in genetic background of the chickens influence the intestinal host response of the Salmonella infection as observed by phagocytic activity, gene expression and changes in the number of T-cell subpopulations and macrophages.     

Immune dynamics following infection of avian macrophages and epithelial cells with typhoidal and non-typhoidal Salmonella enterica serovars; bacterial invasion and persistence, nitric oxide and oxygen production, differential host gene expression, NF-κB signalling and cell cytotoxicity

Poultry-derived food is a common source of infection of human with the non-host-adapted salmonellae while fowl typhoid and pullorum disease are serious diseases in poultry. Development of novel immune-based control strategies against Salmonella infection necessitates a better understanding of the host-pathogen interactions at the cellular level. Intestinal epithelial cells are the first line of defence against enteric infections and the role of macrophages is crucial in Salmonella infection and pathogenesis. While gene expression following Salmonella infection has been investigated, a comparison between different serovars has not been, as yet, extensively studied in poultry. In this study, chicken macrophage-like cells (HD11) and chick kidney epithelial cells (CKC) were used to study and compare the immune responses and mechanisms that develop after infection with different Salmonella serotypes. Salmonella serovars Typhimurium, Enteritidis, Hadar and Infantis showed a greater level of invasion and/or uptake characters when compared with S. Pullorum or S. Gallinarum. Nitrate and reactive oxygen species were greater in Salmonella-infected HD11 cells with the expression of iNOS and nuclear factor-κB by chicken macrophages infected with both systemic and broad host range serovars. HD11 cells revealed higher mRNA gene expression for CXCLi2, IL-6 and iNOS genes in response to S. Enteritidis infection when compared to S. Pullorum-infected cells. S. Typhimurium- and S. Hadar-infected HD11 showed higher gene expression for CXCLi2 versus S. Pullorum-infected cells. Higher mRNA gene expression levels of pro-inflammatory cytokine IL-6, chemokines CXCLi1 and CXCLi2 and iNOS genes were detected in S. Typhimurium- and S. Enteritidis-infected CKC followed by S. Hadar and S. Infantis while no significant changes were observed in S. Pullorum or S. Gallinarum-infected CKC.     

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.     

Characteristics of persistent Salmonella Enteritidis strains in two integrated broiler chicken operations of Korea

The objectives of the study were to investigate the phenotypic and genotypic characterization of the persistent Salmonella Enteritidis (S. Enteritidis) isolates in two integrated broiler chicken operations, with attention focused mainly on the epidemiological approach. In the distribution of virulence genes, Salmonella enterotoxin (stn), invading host cell (invA), and Salmonella plasmid virulence (spvC) genes were widely distributed among the S. Enteritidis irrespective of their source of isolation, and Salmonella fimbrial (sefC) and plasmid encoded fimbrial (pef) genes were present in 28 and 20 S. Enteritidis strains, respectively. A total of 5 different XbaI-PFGE types were obtained from 31 S. Enteritidis isolates. Twenty-one types were divided on the basis their PFGE pattern, phage type and antimicrobial resistance pattern determined. There was a significant difference in phenotypic and genotypic characterization by two integrated broiler operations. Also, 8 isolates shown susceptible to all antimicrobials and 11 isolates with resistance to nalidixic acid were partly classified by XbaI PFGE pattern and by the phage type.     

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.     

Oxidative and nitrosative responses of the chicken macrophage cell line MQ-NCSU to experimental Salmonella infection

Phagocytes limit replication or kill ingested organisms by producing toxic reactive oxygen and nitrogen species via NADPH oxidase and inducible nitric oxide synthase (iNOS). The present experiments were to investigate the production and the possible roles of superoxide, hydrogen peroxide (H2O2) and nitric oxide (NO) in the MQ-NCSU chicken macrophage cell line infected with Salmonella in vitro. After infection, intracellular Salmonella viable counts remained constant until 24 h post infection (PI) and started to decline from 48 h PI. Infection of cells with S. Typhimurium, S. Enteritidis and S. Gallinarum, as well as exposure to S. Enteritidis LPS induced low, but significant concentrations of superoxide 1 to 2 h PI, as determined by reduction of ferricytochrome c. There was no difference in superoxide production in infected cells and control cells after 4 h. Increased H2O2 was observed from cells infected with all the different Salmonella species between 2 and 3 h of infection. Nitrite was always greater in infected cells compared to uninfected cells at all times. However, Salmonella was not completely eliminated from the cells though these cells are capable of eliciting a noticeable oxidative burst response and great nitrosative responses, indicating that a strong oxidative burst (and other mechanism/s) is essential for the elimination of intracellular Salmonella.     

An epidemiological analysis of Salmonella enteritidis contamination in a rat-infested chicken layer farm, an egg processing facility, and liquid egg samples by pulsed-field gel electrophoresis

In order to determine the epidemiological link between the Salmonella Enteritidis contamination in a rat-infested chicken layer farm, an attached egg processing facility and liquid egg samples, several S. Enteritidis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. A total of 33 S. Enteritidis strains were isolated from a total of 4,081 samples. Similar pulsed-field patterns were generated by S. Enteritidis isolates from liquid eggs, rats and effluent water. Additionally, only two phage types were detected among the S. Enteritidis isolates, PT 1b and PT 6. These results suggest that S. Enteritidis isolates from rats, egg processing facility, and liquid eggs are genetically related. Furthermore, S. Enteritidis infection in rats in layer farms poses a serious public health concern and should be included in future epidemiological studies.     

Lysis of chicken lymphocytes by infectious bursal disease viruses

Infectious bursal disease virus types 1 and 2 were able to induce direct lysis of chicken bursal cells, thymus cells, and peripheral blood lymphocytes in chromium-release assays. These two viruses were unable to lyse two established lymphoblastoid cell lines, although IBDV-1 was capable of multiplying in MSB-1 cells.     

Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils

Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.     

Chicken gallinacin gene cluster associated with Salmonella response in advanced intercross line

Salmonella enterica serovar Enteritidis is a gram-negative bacterium that negatively affects human and animal health. Many eukaryotes use antimicrobial peptides (alpha-defensins, beta-defensins, gamma-defensins, and cathelicidins) in innate immune responses to fight bacterial infections. Poultry gallinacins are the functional equivalents of mammalian beta-defensins. Two related advanced intercross lines of chickens were analyzed for association of gallinacin genotypic variation with Salmonella Enteritidis burden levels in the cecum and spleen after infection. Thirteen genes of the chicken beta-defensin cluster (GAL1-13) were sequenced from individuals of each advanced intercross line, plus the founder broiler sire and representatives of the highly inbred Leghorn and Fayoumi founder dam lines. The mean was 17 single-nucleotide polymorphisms (SNPs) per kilobase. One single-nucleotide polymorphism per gene was genotyped with SNaPshot to test for statistical associations with Salmonella Enteritidis colonization after challenge. Among the 13 gallinacin genes evaluated, the single-nucleotide polymorphisms in all genes in a cluster of three adjacent genes (GAL11, GAL12, and GAL13) were associated with bacterial load in the cecal content in the broiler x Leghorn advanced intercross line (three-gene SNP genotype effect, P < 0.008). The results strongly suggest a role of the gallinacins in defense of poultry against enteric pathogens. The use of gallinacin single-nucleotide polymorphisms as molecular markers for genetic selection for Salmonella Enteritidis resistance might result in reduced bacterial burden via development of an enhanced innate immune response.     

Characterization of Salmonella spp. isolated from an integrated broiler chicken operation in Korea

The purpose of this study was to investigate the biological and genetic characterization of persistent Salmonella isolates in an integrated broiler chicken operation, in an attempt to elucidate the source of contamination. From the breeder farm, the hatchery, the broiler farm and the chicken slaughter house of an integrated broiler chicken operation, a total of 6 serotypes were observed. Although S. Heidelberg was not detected in the broiler farm, it was consistently found in the breeder farm, the hatchery and the chicken slaughter house. Also, S. Enteritidis and S. Senftenberg were found in the hatchery and the chicken slaughter house, and the hatchery and the broiler farm, respectively. S. Gallinarum and S. Blockley were found only in the broiler farm, and S. Virchow was only recovered in the chicken slaughter house. Isolated S. Heidelberg, S. Enteritidis and S. Senftenberg strains were divided into 3, 5 and 7 types, respectively, on the basis of all properties. Especially, S. Senftenberg isolates, divided into four types by their antimicrobial resistance patterns, were all obviously the XbaI PFGE pattern. Also, four S. Enteritidis isolates resistant to nalidixic acid showed a difference in phage type and PFGE pattern. Such a different pattern was shown despite Salmonella isolates originating from an integrated broiler operation, suggesting that further epidemiological studies on many integrated chicken companies in Korea are needed.     

Salmonella in poultry flocks and humans--S. enterica subspecies enterica serovar Enteritidis in the past

After importing of breeder lines for laying flocks from Canada into the former GDR in 1966 the egg industry in this country was completely isolated from that in Western Germany or other Western European countries until opening the border in Germany in 1989. Because of this isolation from other countries, an analysis of the clonal diversity of Salmonella (S.) Enteritidis isolates originated from humans, chickens and food in the former GDR during the 1980s would provide a unique opportunity to obtain new insight into factors that may have triggered the S. Enteritidis epidemic. While isolates had previously been typed by the phage typing scheme of Lalko and Laszlo we applied for the first time the extended phage typing scheme by Ward for the retrospective analysis of the S. Enteritidis strains. Furthermore, isolates of phage type (PT) 4/6 (Ward / Lalko and Laszlo) from different livestocks were investigated by ribotyping. Although in total the PT4/6 dominated between 1986 and 1989 in poultry, other phage types have prevailed in the early 1980s and represented a considerable fraction of isolates until 1989. For instance, PT8/7 was isolated from one large layer farm (district Halle) from 1988 until 1989. During that time in another farm (district Cottbus) only PT8/7 was detected too. PT4/6 was isolated from neither of these two laying hen farms. The strains of PT4/6 could be distinguished by ribotyping in 19 different subtypes. The strains from the northern farms were distinct from those isolated in the southern regions. As farms which were harbouring either PT4/6 or PT8/7 had obtained laying hens from the same sources (breeder lines in Deersheim and Spreenhagen) it is highly probable that S. Enteritidis infection was acquired from the environment at each individual farm. This conclusion is also supported by the presence of different PT4/6 ribotypes in different farms. The presence of different phage types or PT4/6 ribotypes at different farms of laying hens suggests that in each case the S. Enteritidis strains present in the environment were able to enter chicken flocks.     

Detection of specific antibodies against deflagellated Salmonella Enteritidis and S. Enteritidis FliC-specific 9kDa polypeptide

Specific antibody levels of laying hens and young chickens experimentally infected with Salmonella Enteritidis and vaccinated farm flocks were evaluated by enzyme-linked immunosorbent assays (ELISAs) with two different antigens, deflagellated S. Enteritidis whole cell (DEWC) and S. Enteritidis FliC-specific 9kDa polypeptide (SEP9). Infected laying hens excreted S. Enteritidis throughout the experimental period, and the specific antibody titers in DEWC-ELISA, were significantly higher than the uninfected group. It suggests that this DEWC-specific antibody will serve as an effective indicator of S. Enteritidis infection, especially for non-vaccinated laying flocks. SEP9-specific antibodies were detected in spray-inoculated young chickens but not in oral-inoculated young chickens. Compared with greatly high SEP9-specific antibody levels of vaccinated farm flocks, no response was observed in orally infected hens. These results indicate that S. Enteritidis discontinues expressing SEP9 once it has crossed the intestinal barrier, and that SEP9-ELISA will serve as a valuable monitoring tool for the status of S. Enteritidis vaccination on a flockwide basis, independent of stable S. Enteritidis infections.     

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

ABSTRACT: 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.     

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.     

Production and characterization of monoclonal antibodies reactive with the chicken interleukin-15 receptor alpha chain

We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor.     

The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.     

The distribution of Salmonella serovars in chicken and humans in Lithuania

We studied serovars of Salmonella strains isolated from chicken and humans in Lithuania over the period from 2000 to 2004. Salmonella strains were isolated and identified according to the techniques recommended by International Organisation for Standardization (Microbiology of Food and Animal Feeding Stuff--Horizontal Method for the Detection of Salmonella, 1998, ISO, Geneva). The per cent of infected flocks with Salmonella in separate years was between 1.01% and 3.2% during the period of investigation. The contamination rate of broiler legs and breasts was higher (2.36% and 4.25%) than that of wings (0.82%). Eighteen serovars of Salmonella were identified from the total 300 isolated samples. The most prevalent serovars in chicken were Salmonella Enteritidis, Salmonella Infantis and Salmonella Typhimurium. Other serovars such as Salmonella Montevideo, Salmonella Djugu, Salmonella Isangi, Salmonella Bovismorbificans, Salmonella Mbankada, Salmonella Hadar were detected only in one to two samples. In general, similar serovars of Salmonella were found in humans and chicken (S. Enteritidis and S. Typhimurium), although distinct serovars were found only in humans or only in chicken. Analysis of the distribution of Salmonella serovars in humans during the seasons of the year indicated that the highest incidence of Salmonella was in Summer and in the beginning of Autumn. Analysis of the distribution of serovars during the study period indicated that there is a shift over time in both humans and chicken.     

Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.