A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens

Abstract

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges

“…Recently, Maekawa et al (2021) also employed a six-color flow cytometric assay to compare the dynamic changes of CD107 + CTLs, NK cells, and regulatory T cells (Tregs) in the larynx-trachea of chickens immunized with different ILTV vaccines after challenge and found that the cellular immune responses elicited by different ILTV vaccines varied in the upper respiratory tract after challenge, and that activated CTLs rather than NK cells play a main role in vaccine protection. In addition, combining multi-color flow cytometry and gene expression profiling, macrophages and distinct DCs in chickens were characterized and shown to be activated based on upregulated expression of MHC II and costimulatory molecules CD40 and CD80 in the setting of infection ( Vu Manh et al, 2014 ; Meijerink et al, 2021 ). These studies highlighted the importance to analyze cellular immunity to avian viral infections by flow cytometry.…”

“…Classical methods for measuring T cell proliferation include 3 H-Thymidine ( 3 H-TdR) incorporation assays ( Maurer, 1981 ) and MTT assay ( Mosmann, 1983 ) but have disadvantages of generating radioactive waste and lacking of sensitivity, respectively. Therefore, new methods such as CFSE (carboxyfluorescein diacetate succinimidyl ester) and CellTrace Violet (CTV) labelling, BrdU (5'-Bromo-2'-deoxyuridine) and EdU (ethynyl-deoxyuridine) incorporation assays ( Motobu et al, 2002 ; Dalgaard et al, 2010 ; Alvarez et al, 2020 ; Meijerink et al, 2021 ) that are more sensitive and environment-friendly have been introduced for T cell proliferation assay. These methods can be combined with flow cytometry to identify the phenotype of immune cells at the same time and have been validated in chickens ( Table 2 ).…”

“…Using this dye, Lee et al (2020) found that inactivated H9N2 vaccine adjuvanted with Bacillus subtilis spores induced more proliferative and antigen-specific CD4 and CD8 T cells than did the inactivated H9N2 alone in chickens. This assay has also been used to evaluate the proliferation of CD4 and CD8 T cells following salmonella infection in combination with flow cytometry ( Meijerink et al, 2021 ).…”

“…Although antigen-specific CTLs were detected in many AVD including AIV, IBV, and MDV ( Seo and Collisson, 1997 ; Seo et al, 2002 ; Dai et al, 2019 ; Umthong et al, 2020 ), virus-specific T cell lines or clones have not been isolated and reported in chickens by repeatedly feeding exogenous antigen-pulsed APCs. Recent study showed that in vitro culture of avian T cells was feasible in X-VIVO medium in the presence of IL-2 after stimulation with antigen, with low background proliferation, raising a hope for creating CTL clones in chickens ( Meijerink et al, 2021 ). A breakthrough in the isolation of antigen-specific CTL clones may help to address the roles of T cells in anti-viral immunity in chickens.…”

Efficacy of Multivalent, Cochleate-Based Vaccine against Salmonella Infantis, S. Enteritidis and S. Typhimurium in Laying Hens

“…Recently, [35] described the changes in the innate and adaptive immune system that are generated in birds after infection with S. Enteritidis in detail. As in the control group of our study, one week after the challenge with Salmonella, there was an increase in the proportion of CD8+ T lymphocytes and other cells, but this, according to these authors, is not sufficient to prevent the progressive colonization of the cecum and spleen with Salmonella.…”