Ulrich Methner

Chicken Cecum Immune Response to Salmonella enterica Serovars of Different Levels of Invasiveness

ABSTRACT Day-old chicks are very susceptible to infections with Salmonella enterica subspecies. The gut mucosa is the initial site of host invasion and provides the first line of defense against the bacteria. To study the potential of different S. enterica serovars to invade the gut mucosa and trigger an immune response, day-old chicks were infected orally with Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, S. enterica serovar Hadar, or S. enterica serovar Infantis, respectively. The localization of Salmonella organisms in gut mucosa and the number of immune cells in cecum were determined by immunohistochemistry in the period between 4 h and 9 days after infection. Using quantitative real-time reverse transcription (RT)-PCR, mRNA expression of various cytokines, chemokines, and inducible nitric oxide synthase (iNOS) was examined in cecum. As a result, all S. enterica serovars were able to infect epithelial cells and the lamina propria. Notably, serovar Enteritidis showed the highest invasiveness of lamina propria tissue, whereas serovars Typhimurium and Hadar displayed moderate invasiveness and serovar Infantis hardly any invasion capabilities. Only a limited number of bacteria of all serovars were found within intestinal macrophages. Elevated numbers of granulocytes, CD8+ cells, and TCR1+ cells and mRNA expression rates for interleukin 12 (IL-12), IL-18, tumor necrosis factor alpha factor, and iNOS in cecum correlated well with the invasiveness of serovars in the lamina propria. In contrast, changes in numbers of TCR2+ and CD4+ cells and IL-2 mRNA expression seemed to be more dependent on infection of epithelial cells. The data indicate that the capability of Salmonella serovars to enter the cecal mucosa and invade lower regions affects both the level and character of the immune response in tissue.

Vaccination and early protection against non-host-specific Salmonella serotypes in poultry: exploitation of innate immunity and microbial activity

A recent European Union Directive required member states to put monitoring and control programmes in place, of which vaccination is a central component. Live Salmonella vaccines generally confer better protection than killed vaccines, because the former stimulate both cell-mediated and humoral immunity. Administering Salmonella bacteria orally to newly hatched chickens results in extensive gut colonization and a strong adaptive immune stimulus but broiler chickens are immunologically immature. However, colonization exerts a variety of rapid (within 24 h) protective effects. These include specific colonization-inhibition (competitive exclusion) in which the protective bacteria exert a profound resistance to establishment and colonization by other related bacteria. This is thought to be primarily a metabolic attribute of the vaccinating bacteria but may also involve competition for attachment sites. The presence of large numbers of bacteria originating from a live Salmonella vaccine in the intestine can also induce infiltration of polymorphonuclear cells into the intestinal wall, which confers resistance to invasion and systemic spread by virulent Salmonella strains. This opens new perspectives for vaccine usage in broilers, layers and breeding poultry but also in other animals which show increased susceptibility to infection because of their young age or for other reasons, such as oral chemoprophylaxis or chemotherapy, where the lack of established normal gut flora is an issue. We recommend that all live vaccines considered for oral administration should be tested for their ability to induce the two protective effects described above. Further developments in live Salmonella vaccines are, however, currently hindered by fears associated with the use and release of live vaccines which may be genetically modified.

Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains

Poultry represents an important source of Salmonella infection in man. Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars. In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S. typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry. Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically. Blood samples and tissues were examined between days 1 and 12 of age. Chicks inoculated with S. typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals. The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age. In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen. Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5. As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen. In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level. The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.

Comparative study of the protective effect against Salmonella colonisation in newly hatched SPF chickens using live, attenuated Salmonella vaccine strains, wild-type Salmonella strains or a competitive exclusion product

There is a need to prevent intestinal colonisation by Salmonella enteritidis and S. typhimurium in newly hatched chicks. Treatment with an undefined bacterial flora is not acceptable to regulatory agencies in some countries because of the potential risk of transmitting pathogens. A defined culture with a potency and stability equivalent to those of an undefined culture has not yet been developed. Since attenuated Salmonella vaccine strains could possess the colonisation characteristics but not the virulence of Salmonella wild-type strains, they could inhibit colonisation of the challenge organism. S. typhimurium live vaccines registered in Germany (Zoosaloral H, Salmonella vac T), S. enteritidis aroA and S. typhimurium aroA strains, S. enteritidis, S. typhimurium and S. infantis wild-type strains or a competitive exclusion product (Broilact) were used as pretreatment cultures and evaluated for their inhibitory effects against S. enteritidis and S. typhimurium colonisation in newly hatched SPF chickens. Day-old chicks were administered a pretreatment culture and infected orally with variants of S. enteritidis or S. typhimurium wild type-strains resistant to nalidixic acid or rifampicin 1 day after pretreatment. On days 2 and 6 after infection, viable numbers of the challenge strain in liver and caeca were determined. The results for birds pretreated with Broilact showed a distinct protective effect against both S. enteritidis and S. typhimurium at a challenge dose of 10(4) cfu/bird. After pretreatment of chicks with S. enteritidis and S. typhimurium wild-type strains, the greatest degree of inhibition of caecal colonisation was produced using isogenic strains. Colonisation after infection with non-isogenic strains could not be prevented but only reduced for a brief period. These effects were also observed after administration of aroA strains of S. enteritidis and S. typhimurium but the protective effect was considerably lower than after pretreatment with wild-type Salmonella strains. Inoculation with attenuated S. typhimurium vaccines resulted in a weak but significantly reduced colonisation by S. typhimurium. Colonisation by S. enteritidis could not be diminished by either of the S. typhimurium vaccine strains. The results indicate in principle the potency of Salmonella vaccine strains to inhibit Salmonella wild-type colonisation in newly hatched chicks. Potential vaccine candidates should be tested for their capacity to prevent intestinal colonisation in newly hatched chicks.

Circulating γδ T Cells in Response to Salmonella enterica Serovar Enteritidis Exposure in Chickens

ABSTRACT γδT cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterizeγδ (T-cell receptor 1+ [TCR1+]) T cells phenotypically and functionally in avian immune response. Day-old chicks were orally immunized with Salmonella enterica serovar Enteritidis live vaccine or S. enterica serovar Enteritidis wild-type strain and infected using the S. enterica serovar Enteritidis wild-type strain on day 44 of life. Between days 3 and 71, peripheral blood was examined flow cytometrically for the occurrence of γδ T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1+ cell populations were found to display considerable variation regarding CD8α antigen expression: (i) CD8α+high TCR1+ cells, (ii) CD8α+dim TCR1+ cells, and (iii) CD8α− TCR1+ cells. While most of the CD8α+high TCR1+ cells expressed the CD8αβ heterodimeric antigen, the majority of the CD8α+dim TCR1+ cells were found to express the CD8αα homodimeric form. After immunization, a significant increase of CD8αα+high γδ T cells was observed within the CD8α+high TCR1+ cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor α (IL-7Rα) and Bcl-x expression and elevated IL-2Rα mRNA expression of the CD8αα+highγδ T cells. Immunohistochemical analysis demonstrated a significant increase of CD8α+ and TCR1+ cells in the cecum and spleen and a decreased percentage of CD8β+ T cells in the spleen after Salmonella immunization. After infection of immunized animals, immune reactions were restricted to intestinal tissue. The study showed that Salmonella immunization of very young chicks is accompanied by an increase of CD8αα+high γδ T cells in peripheral blood, which are probably activated, and thus represent an important factor for the development of a protective immune response to Salmonella organisms in chickens.

Combination of vaccination and competitive exclusion to prevent Salmonella colonization in chickens: experimental studies

Vaccination and competitive exclusion (CE) represent accepted prophylactic measures to control Salmonella infections in chickens. To use the advantages of both the CE technique and vaccination with live Salmonella vaccines the combination of these methods was studied. In three experiments, SPF chickens were pre-treated using combined or unique administration of CE and vaccination with a live Salmonella typhimurium strain on days 1 and 2 of life and challenged with the antibiotic resistant, but otherwise isogenic mutant of this Salmonella typhimurium strain on days 3, 15 or 40 of life. The caecal colonization of both the vaccine and the challenge strain and the antibody response after infection were examined to evaluate the protective effects of the different combinations. The exclusion effect of CE cultures against Salmonella infection could be seen in very young chicks and was still considerable on day 40 of life of the birds. The Salmonella wild-type strain used as vaccine alone also resulted in a substantial protective effect against homologous challenge. The combined administration of competitive exclusion and immunization using the Salmonella wild-type strain as vaccine resulted in a considerable additional protective effect above the level of the respective exclusive application of these prophylactic measures. Administration of the Salmonella vaccine strain prior to or simultaneously with the CE culture produced the best protective effect, because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of colonization inhibition effects and a strong immune response. The full exploitation of this potential using attenuated live Salmonella vaccines will require the presence of high inhibitory and immunogenic properties in the vaccine strain after attenuation of a selected parent strain. The combination of competitive exclusion and vaccination as a new measure in integrated control programmes against Salmonella infection in poultry could result in a considerable increase of protection in both very young and older chickens.

Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens

Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused by Salmonella (S.), the immune mechanisms responsible for a longer lasting protection against Salmonella infection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial live S. Enteritidis vaccine and challenged with wild-type S. Enteritidis 147N at day 56 of life. The bacterial cell count was determined in gut and liver, while the immune cell composition and cytokine gene expression patterns were analysed by immunohistochemistry and quantitative real-time RT-PCR in caecum samples. The presented data suggest that the vaccine-elicited immune protection against the Salmonella wild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number of Salmonella wild-type organisms found in caecal wall and liver of the non-immunised compared to immunised birds after challenge correlated with a more pronounced gene expression rate for IL-8, LITAF, iNOS, IL-12 and IFN-gamma. In contrast, immunised birds exhibited higher amounts of CD8(+) T cells as well as IgA than the non-immunised chickens after S. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens upon S. Enteritidis wild-type challenge.

Heterogeneity of avian γδ T cells

gammadelta T cells are distinct with respect to tissue localisation, phenotype and biological functions and similarities between species are not very apparent. To elucidate local and functional heterogeneity of non-stimulated avian gammadelta T cells, the CD8-characterised gammadelta T cell subsets [CD8alpha(+high) (CD8alphaalpha(+) and CD8alphabeta(+)); CD8alpha(+dim); CD8(-)] of blood, spleen and caecum were flow cytometrically quantified and analysed for proliferation state as well as sorted for determination of immune-relevant gene expression by quantitative real-time RT-PCR. The number of avian CD8-characterised gammadelta T cell subsets differed in dependence on tissue and age of bird. Compared to blood and spleen, caecum showed the highest percentage of gammadelta T cells as well as of the CD8alpha(+high) gammadelta T cell subset in 7-week-old birds. Generally, the CD8alphabeta(+) cells significantly outnumbered the CD8alphaalpha(+) lymphocytes within the CD8alpha(+high) gammadelta T cell population of all organs. Additionally, the splenic CD8alphabeta(+) subpopulation revealed the highest proliferation activity. By RT-PCR, mRNA expression of immune-relevant genes was proved in non-stimulated gammadelta T cell subsets, but on different levels. Generally, both CD8alpha(+high) cell subsets (CD8alphaalpha(+) and CD8alphabeta(+)) of blood and spleen showed elevated expression levels for Fas ligand (FasL), XCL1 (lymphotactin) and interferon-gamma (IFNgamma) compared to the CD8alpha(-) gammadelta T cell subset. In contrast, all caecal gammadelta T cell subsets showed similar high levels of these transcripts. Notably, the CD8alphaalpha(+) cells of all locations showed unique expression of TLR4 and interleukin (IL)-2. The results demonstrated that avian gammadelta T cells are not only heterogeneous concerning their CD8 antigen characteristics and tissue localisation, but also with regard to functional features such as proliferation and mRNA expression.

Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of Salmonella enterica with an Oligonucleotide Microarray-Based Assay

Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.

Exploitation of Intestinal Colonization‐Inhibition Between Salmonella Organisms for Live Vaccines in Poultry – Potential and Limitations

Immunization represents one of the most important methods to increase the resistance of chickens against Salmonella infection. In addition to the development of an adaptive immune response, oral administration of live Salmonella strains to day‐old chicks provides protection against infection within hours by intestinal colonization‐inhibition. For the exploitation of this phenomenon, practical information on colonization‐inhibition between Salmonella organisms is needed. Colonization‐inhibition capacity between Salmonella strains from serogroups B, C1, C2, D and G was assessed in chickens. The most profound level of intestinal colonization‐inhibition occurred between isogenic strains. Inhibition between strains of the same serovar was greater than that between strains of different serovars. The degree of inhibition between different serovars was not sufficiently high to identify a single strain which might inhibit a wide range of other Salmonella organisms. However, as Salmonella Enteritidis is the dominant serovar in poultry in many countries and because of the profound colonization‐inhibition within this serovar there is a considerable potential to exploit this phenomenon in the development of novel live S. Enteritidis vaccines. Treatment of young chicks with mixtures of different Salmonella serovars resulted not only in a very strong growth inhibition of the isogenic strains but also in a substantial inhibition of heterologous serovars. The potential of mixtures of heterologous Salmonella strains as a ‘Salmonella Inhibition Culture’ and as a ‘live Salmonella vaccine’ should be further explored.