Claire Powers

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.

Cytokine and Chemokine Responses Associated with Clearance of a Primary Salmonella enterica Serovar Typhimurium Infection in the Chicken and in Protective Immunity to Rechallenge

ABSTRACT 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.

Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) infections cause systemic disease in the young chick, whereas in the older chicken the infection is mainly restricted to the intestine. Chickens infected orally with S. Typhimurium (F98) at 6 weeks of age and re-infected 10 weeks later were monitored for antibody production, T-cell proliferation and production of selected cytokines (interferon-γ, interleukin-1β and transforming growth factor-β4). A strong coordinated antigen-specific humoral and cellular immune response was temporally linked to resolution of the primary infection. Enhanced levels of mRNA encoding the cytokines, interleukin-1β, transforming growth factor-β4 and interferon-γ were also evident during early phases of primary infection. Secondary infection was restricted to the intestine and of shorter duration than primary infection. Splenic immune responses were not further enhanced by secondary infection; indeed, antigen-specific proliferation was significantly reduced at 1 day after secondary infection, which may be interpreted as the trafficking of reactive T cells from the spleen to the gut.

Infection of the Reproductive Tract and Eggs with Salmonella enterica Serovar Pullorum in the Chicken Is Associated with Suppression of Cellular Immunity at Sexual Maturity

ABSTRACT Salmonella enterica serovar Pullorum causes persistent infections in laying hens. Splenic macrophages are the main site of persistence. At sexual maturity, numbers of bacteria increase and spread to the reproductive tract, which may result in vertical transmission to eggs or chicks. In this study we demonstrate that both male and female chickens may develop a carrier state following infection but that the increases in bacterial numbers and spread to the reproductive tract are phenomena restricted to hens, indicating that such changes are likely to be related to the onset of egg laying. The immunological responses during the carrier state and through the onset of laying in hens were determined. These indicate that chickens produce both humoral and T-cell responses to infection, but at the onset of laying both the T-cell response to Salmonella and nonspecific responses to mitogenic stimulation fall sharply in both infected and noninfected birds. The fall in T-cell responsiveness coincided with the increase in numbers of Salmonella serovar Pullorum and its spread to the reproductive tract. Three weeks after the onset of egg laying, T-cell responsiveness began to increase and bacterial numbers declined. Specific antibody levels changed little at the onset of laying but increased following the rise in bacterial numbers in a manner reminiscent of a secondary antibody response to rechallenge. These findings indicate that a nonspecific suppression of cellular responses occurs at the onset of laying and plays a major role the ability of Salmonella serovar Pullorum to infect the reproductive tract, leading to transmission to eggs. The loss of T-cell activity at the point of laying also has implications for Salmonella enterica serovar Enteritidis infection and transmission to eggs, along with its control by vaccination offering a “window of opportunity” in which infection may occur.

Clearance of Enteric Salmonella enterica Serovar Typhimurium in Chickens Is Independent of B-Cell Function

ABSTRACT Salmonella enterica serovar Typhimurium colonizes the gut of chickens and is cleared from the intestine within about 3 weeks. Infection induces high levels of specific antibody, but B cells do not play an essential role in clearance of primary infection or in the enhanced clearance after secondary challenge.

Oral infection with the Salmonella enterica serovar Gallinarum 9R attenuated live vaccine as a model to characterise immunity to fowl typhoid in the chicken

BackgroundSalmonella enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, a severe systemic disease of chickens that results in high mortality amongst infected flocks. Due to its virulence, the immune response to S. Gallinarum is poorly characterised. In this study we have utilised infection by the live attenuated S. Gallinarum 9R vaccine strain in inbred chickens to characterise humoral, cellular and cytokine responses to systemic salmonellosis.ResultsInfection with 9R results in a mild systemic infection. Bacterial clearance at three weeks post infection coincides with increases in circulating anti-Salmonella antibodies, increased T cell proliferation to Salmonella challenge and increased expression of interferon gamma. These responses peak at four weeks post infection, then decline. Only modest increases of expression of the pro-inflammatory cytokine interleukin-1β were detected early in the infection.ConclusionInfection of chickens with the 9R vaccine strain induces a mild form of systemic salmonellosis. This induces both cellular and humoral immune responses, which peak soon after bacterial clearance. Unlike enteric-associated Salmonella infections the immune response is not prolonged, reflecting the absence of persistence of Salmonella in the gastrointestinal tract. The findings here indicate that the use of the S. Gallinarum 9R vaccine strain is an effective model to study immunity to systemic salmonellosis in the chicken and may be employed in further studies to determine which components of the immune response are needed for protection.

A Strong Antigen-Specific T-Cell Response Is Associated with Age and Genetically Dependent Resistance to Avian Enteric Salmonellosis

ABSTRACT Chicken genetics and age affect resistance to enteric infection with Salmonella enterica serovar Typhimurium and were used to identify the immune responses that may contribute to rapid clearance. When birds were infected at 40 days of age, line 61 chickens cleared the infection more effectively than line N chickens, whereas when birds were infected at 10 days of age, both chicken lines were highly susceptible to infection. Antibody levels, T-cell responsiveness, and cytokine mRNA levels were all elevated during infection. A negative correlation between resistance and antigen-specific antibody production was observed in older chickens. However, this finding was not replicated for age-related resistance; we found that older chickens exhibited a stronger and more rapid antibody response than younger chickens. The levels of interleukin-1β (IL-1β) and gamma interferon (IFN-γ) mRNA were similar in the spleens and cecal tonsils of both line 61 and line N chickens, except for higher levels of IL-1β in the spleens of line 61 chickens at 6 days postinfection. Differences in the levels of IFN-γ and IL-1β 1β mRNA between the lines were more apparent in younger chickens, but while the increases were greater than those observed in the older chickens, the clearance of enteric S. enterica serovar Typhimurium was much slower. The level of antigen-specific proliferation of splenocytes was associated with increased resistance in both experimental systems, and the strongest responses were observed in older and genetically resistant chickens. The data presented here implicate T-cell responses in the clearance of S. enterica serovar Typhimurium from the intestine of infected chickens.

Regional and global changes in TCRαβ T cell repertoires in the gut are dependent upon the complexity of the enteric microflora.

The repertoire of gut associated T cells is shaped by exposure to microbes, including the natural enteric microflora. Previous studies compared the repertoire of gut associated T cell populations in germ free (GF) and conventional mammals often focussing on intra-epithelial lymphocyte compartments. Using GF, conventional and monocolonised (gnotobiotic) chickens and chicken TCRbeta-repertoire analysis techniques, we determined the influence of microbial status on global and regional enteric TCRbeta repertoires. The gut of conventionally reared chickens exhibited non-Gaussian distributions of CDR3-lengths with some shared over-represented peaks in neighbouring gut segments. Sequence analysis revealed local clonal over-representation. Germ-free chickens exhibited a polyclonal, non-selected population of T cells in the spleen and in the gut. In contrast, gnotobiotic chickens exhibited a biased repertoire with shared clones evident throughout the gut. These data indicate the dramatic influence of enteric microflora complexity on the profile of TCRbeta repertoire in the gut at local and global levels.