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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.

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-1β (chIL-1β) 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-1β 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.

Poultry meat and eggs contaminated withSalmonella entericaserovar Enteritidis orSalmonella entericaserovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective againstSalmonellainfection 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 withSalmonellaserovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereasSalmonellastarted 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 withSalmonellain newly hatched chickens.

Salmonella enterica serovar Enteritidis (SE) is a causative agent for human food poisoning cases throughout the world. The ovaries and the oviducts of the laying hens are the major sites of SE colonization from which vertical transmission to eggs occurs. In this study, Salmonella-induced changes in T lymphocytes, B lymphocytes and macrophages in the ovaries and oviducts were assessed after primary and secondary experimental inoculations of laying hen with SE. Statistically significant increases in the numbers of T cells (both CD4+ and CD8+) and macrophages were observed 7 to 14 days after primary inoculation, followed by a peak in B-cell numbers from the 14th day post-primary inoculation onwards in the secretory areas of the oviducts. The peak in lymphocyte numbers immediately preceded a decline in the rate of SE recovery from the reproductive tract beginning at day 14. The correlation of decreased Salmonella recovery with elevated lymphocyte and macrophage numbers strongly suggests that local cell-mediated immunity is involved in controlling SE injection in the ovaries and oviducts.

Lymphocytes expressing CD3, CD4, CD8, pan lymphocyte, IgA, IgG and IgM cell surface antigens were assessed by in the spleen and thymus of chickens following infection with Salmonella enteritidis using flow cytometric analysis. At 6 days post primary infection and 2 days post secondary infection with S. enteritidis, the percentages of IgA+ and IgM+ lymphocytes in the spleen were significantly increased (P < 0.05). At 2 days post secondary infection with S. enteritidis, the percentage of CD4+ T lymphocyte in the spleen and CD8+ T lymphocyte percentage in the thymus were significantly increased (P < 0.05). These results indicate that S. enteritidis infection induces changes in the spleen and thymus that reflect the dynamics of the host protective immune response.

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 β4 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.

The production and secretion of Salmonella enteritidis whole cell antigen-specific antibodies in the oviducts and in the serum of laying hens experimentally infected with Salmonella enteritidis, was analyzed by ELISA. The dynamics of the antibody levels in the oviducts were identical to that in the serum. Subclasses of antibodies (IgA, IgG, and IgM) in the infected hens were found to increase significantly (p < 0.01) compared to those in the control uninfected hens throughout the experiment. IgG and IgM levels in both oviducts and in sera reached to a peak by 14 days post-inoculation, and remained elevated throughout. The secretion of IgA seemed to be transient since the IgA levels increased to a peak 7 days after both primary and secondary inoculations, and declined rapidly. The elevated levels of antibodies were followed by partial clearance of Salmonella organisms from the oviducts. The present results indicate a significant local immune reaction against the Salmonella infection and suggest an association of the local antibodies with the clearance of Salmonella from the oviducts at least partially.

Distribution and the relative frequencies of subclasses of T and B lymphocytes in the 360-d-old, healthy laying hens were studied. Avidin-Biotin-Peroxidase Complex (ABC) method was used to detect T cells, whereas Indirect Immunofluorescent Technique (IFT) was applied to detect B cells. Both T and B cells were scattered throughout the ovaries and oviducts. Small lymphoid nodules were found in the upper regions of the oviducts. More T lymphocytes were found in the vagina, ovary, and infundibulum than in the other portions examined, whereas B lymphocytes were mainly found in magnum, uterus, and isthmus. The IgG-containing (B) cells were not found in the ovary. The CD8+ cells were found more close to the lining epithelium or in between epithelial cells, whereas CD4+ cells were found mainly in the lamina propria. The behavior of these cells in certain infectious diseases in which the ovaries and oviducts are major predilection sites would be very important in assessing or determining local immune responses.

Four experiments were conducted to examine the effect of Eimeria tenella infection on the recrudescence of Salmonella enteritidis in previously infected chickens. Significant recrudescence of cecal colonization in the birds challenged with E. tenella was observed at 3 wk after S. enteritidis infection in Experiment 1, at 3 and 4 wk in Experiment 2, and at 3, 4, and 5 wk in Experiments 3 and 4. The recrudescence was indicated by .81 to 6.31 logs increase in cecal S. enteritidis counts and by higher percentages of ceca that were culture-positive. The possible prolonged cecal S. enteritidis shedding from chickens infected with coccidia into the environment might be important for the perpetuation of S. enteritidis infectious cycle. Except for Experiment 1, in which a significant higher culture-positive rate of the liver was detected in the coccidia-infected group, no significant difference of culture-positive rate of liver and spleen between the treatments was observed. The recrudescence of previous S. enteritidis infection caused by E. tenella infection was obviously related to the initial S. enteritidis dose size and time of exposure to coccidia.