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Chicken genetics and age affect resistance to enteric infection withSalmonella entericaserovar 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 61chickens 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 61and line N chickens, except for higher levels of IL-1β in the spleens of line 61chickens 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 entericS. entericaserovar 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 ofS. entericaserovar Typhimurium from the intestine of infected chickens.

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.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both “public” and “private” CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation., Author Summary Many viral infections target the immune system, making use of the long lived, highly proliferative lymphocytes to propagate and survive within the host. This characteristic has led to an association between some viruses such as Epstein Barr Virus (EBV), Human T cell Lymphotrophic Virus-1 (HTLV-1) and Mareks Disease Virus (MDV) and lymphoid tumors. We employed methods for identifying the T cell receptor repertoire as a molecular bar-code to study the biology of MDV-induced tumors and the anti-tumor response. Each individual contained a small number of large (high frequency) tumor clones alongside some smaller (lower frequency) clones in the CD4+ T cell population. The tumor infiltrating CD8+ T cell response was highly focused with a small number of large clones, with one representing a public CDR3 sequence. This data is consistent with the recognition of a small number of dominant antigens and understanding the relationship between these and protective immunity is important to improve development of new vaccination strategies. Collectively, our results provide insights into the clonal structure of MDV driven tumors and in the responding CD8+ T cell compartment. These studies advance our understanding of MDV biology, an important poultry disease and a natural infection model of virus-induced tumor formation.

Human enteritis resulting from the consumption of poultry products contaminated with serovars of Salmonella enterica remains a major public-health concern. Reducing food contamination by preventing or controlling infection in the chicken during rearing is an attractive solution. An accurate understanding of the mechanisms of immunity to Salmonella infection in the chicken will help to focus the development of vaccines for birds and prevent contaminated products from entering the human food chain. Infection is primarily restricted to the intestinal lumen when chickens are infected with S. enterica serovars Typhimurium or Enteritidis, where they persist for many weeks. High titers of Salmonella-specific antibodies are observed following infection and demonstrate a high degree of cross-reactivity against other serovars. However, depletion of B cells and, therefore, removal of the capacity for antibody production in the chicken does not exacerbate the infection following either primary or secondary challenge.

Chickens infected with Salmonella enterica serovars Typhimurium (ST) and Enteritidis (SE) still represent a major source of human food poisoning via consumption of contaminated meat and eggs. Vaccination represents a sustainable approach to control Salmonella in the chicken and the serovar specificity of immunity has the potential to impact on the need for multivalent vaccines. The issue of cross-reactive immune responses and cross-serovar protection was examined in these experiments. Cellular and humoral immune responses were measured by antigen-specific ELISA and splenocyte proliferation assays during primary infections (with ST and SE) and during a second challenge with homologous or heterologous serovars. Primary infection with ST or SE induced strong lymphocyte proliferation and high levels of specific antibody (IgM, IgG and IgA) responses with substantial serovar cross-reactivity. The occurrence of high levels of splenocyte proliferation and strong antibody responses corresponded to the initiation of clearance with both ST and SE. Re-challenge of ST and SE infection-primed chickens with either serovar resulted in significant levels of protection (assessed by bacterial numbers and rate of clearance) with little difference between homologous or heterologous challenge schedules. Relatively low levels of antigen-specific splenocyte proliferation were detected during secondary infection, which may be caused by splenic T cells exiting to the gut. In contrast, the more rapid specific antibody responses (compared with primary infection controls) indicate the development of a secondary antigen-specific adaptive response. The substantial level of cross-protection between serovars and the level of antigenic cross-reactivity indicates the potential for single serovar live vaccines to protect against both group B and D salmonellae.

Background Salmonella 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. Results Infection 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. Conclusion Infection 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.

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.

Salmonella enterica remains one of the most important food-borne pathogens of humans and is often acquired through consumption of infected poultry meat or eggs. Control of Salmonella infections in chicken is therefore an important public health issue. Infection with S. enterica serovar Typhimurium results in a persistent enteric infection without clinical disease in chickens of more than 3 days of age, and represents a source for contamination of carcass at slaughter and entry into the human food chain. Data presented indicate a profound effect of age at initial exposure on the persistence of infection and a lesser effect on the development of effective immunity to re-challenge. The percentage of birds positive for Salmonella was high until 8-9 weeks of age, regardless of the age at which the birds were infected (1, 3 or 6 weeks). The birds infected at 3 and 6 weeks of age produced a more rapid and higher antibody response (IgY and IgA) than those infected at 1 week of age, but in all cases infection persisted for a considerable period despite the presence of high antibody levels. Following a re-challenge infection with S. Typhimurium, all three previously-infected groups had fewer bacteria in the gut, spleen and liver compared with age-matched birds receiving a parallel primary infection. However, the birds primary infected at 3 and 6 weeks of age cleared infection more rapidly than those infected at a younger age. Interestingly older-primed birds had higher specific T lymphocyte proliferative responses and specific circulating levels of IgY antibody at time of re-challenge. Although birds initially infected at 1 week of age and those that were previously uninfected produced a stronger antibody response following re-challenge, they were slower to clear Salmonella from the gut than the older-primed groups which expressed a stronger T lymphocyte response. The data presented indicate that clearance of Salmonella from the gut is age-dependent and we propose that this relates to the increased competence of the enteric T cell response. The findings that Salmonella persists beyond 8-9 weeks, irrespective of age at exposure, has implications for the broiler sector and indicates the need to remain Salmonella free throughout the rearing period. Moreover, the re-challenge data demonstrates that infection at a young age is less effective in producing protective immunity than in older chickens. This feature of the development of protective immunity needs to be considered when developing vaccines for the broiler sector of the poultry industry.