Heterogeneity of avian gammadelta T cells Pieper, Jana; Methner, Ulrich; Berndt, Angela
Veterinary immunology and immunopathology,
2008-Aug-15, 20080815, Letnik:
124, Številka:
3-4
Journal Article
Recenzirano
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.
There is a strong interest to reduce the expenditure for the detection of Salmonella spp. from animal faeces and environmental samples from primary production according to ISO 6579:2002 Annex D by ...including a rapid and effective method to detect Salmonella spp. already after pre-enrichment in BPW. It has been shown that real-time PCR methods are very effective to detect Salmonella organisms after pre-enrichment of foods. However, materials from primary animal production compose of much higher amounts of substances which might inhibit the sensitivity of real-time PCR. Different techniques of DNA isolation after pre-enrichment of artificially inoculated bovine faecal material were used to compare their detection limit and detection probability using an invA 5' nuclease real-time PCR approach. A detection probability of 100% was shown at 10(5) cfu/ml using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), at 10(4) cfu/ml using the High Pure PCR Template Preparation Kit (Roche, Germany) and at 10(3) cfu/ml using thermal cell lysis or an in-house lab protocol, respectively. In comparison DNA isolation by thermal cell lysis revealed a very good detection limit, low costs and almost no risks of contamination. Furthermore, caecal contents from pigs were analysed by ISO 6579:2002 Annex D and the invA real-time PCR using thermal cell lysis for DNA extraction. As a result neither false positive nor false negative findings were obtained. Inclusion of the real-time PCR after pre-enrichment of samples in BPW followed by bacterial detection of Salmonella only with samples positive with real-time PCR might be a valuable tool to fulfil the international standard of ISO 6579:2002 Annex D but also to diminish the expenditures. However, it must be stated that the modification of an international standard method and its use in routine diagnostic requires the validation and registration of national and/or international competent authorities.
Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused bySalmonella(S.), the immune mechanisms responsible for a longer lasting protection ...againstSalmonellainfection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial liveS. Enteritidis vaccine and challenged with wild-typeS. 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 theSalmonellawild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number ofSalmonellawild-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-γ. In contrast, immunised birds exhibited higher amounts of CD8+T cells as well as IgA than the non-immunised chickens afterS. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens uponS. Enteritidis wild-type challenge.
gammadelta T cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterize gammadelta (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 gammadelta T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1(+) cell populations were found to display considerable variation regarding CD8alpha antigen expression: (i) CD8alpha(+high) TCR1(+) cells, (ii) CD8alpha(+dim) TCR1(+) cells, and (iii) CD8alpha(-) TCR1(+) cells. While most of the CD8alpha(+high) TCR1(+) cells expressed the CD8alphabeta heterodimeric antigen, the majority of the CD8alpha(+dim) TCR1(+) cells were found to express the CD8alphaalpha homodimeric form. After immunization, a significant increase of CD8alphaalpha(+high) gammadelta T cells was observed within the CD8alpha(+high) TCR1(+) cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor alpha (IL-7Ralpha) and Bcl-x expression and elevated IL-2Ralpha mRNA expression of the CD8alphaalpha(+high) gammadelta T cells. Immunohistochemical analysis demonstrated a significant increase of CD8alpha(+) and TCR1(+) cells in the cecum and spleen and a decreased percentage of CD8beta(+) 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 CD8alphaalpha(+high) gammadelta 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.
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
+high
γδ 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.
(.) subspecies serovar Choleraesuis, the swine-adapted serovar is found rarely in Western European countries including Germany. However, the regional laboratory of the federal state Thuringia in ...Germany examined diseased wild boars routinely also for the occurrence of organisms. Between 2006 and 2008, only the serovar Choleraesuis was islolated from 24 animals, three strains isolated from domestic pigs were included. In order to detect a possible epidemiological context, the strains of Choleraesuis were characterised by macrorestriction and plasmid analysis, repetitive sequence PCR, antimicrobial testing and determining the biochemical profile. A combination of all methods enabled the identification of five epidemiological groups. Two groups were detected in the same territory but three other discriminative groups were predominant in different regions. Choleraesuis strains of the different epidemiological groups circulate in wild boar populations in the corresponding regions. However, it could be concluded that both natural barriers like mountains and artificial barriers like arterial roads may cause the separation of wild boar populations and as a result also the respective Choleraesuis organisms. The occurrence of the identical epidemiological groups in wild boars and domestic pigs indicates the possible mutual exposure of the pathogen. To avoid risks for human and domestic pig health regular inspection of meat from wildlife by official veterinarians and advice of hunters and persons who prepare and consume wild boar meat should be enhanced.
Interactions of
Salmonella (
S.) outer membrane structures with extracellular matrix (ECM) of host tissues seem to be crucial for bacterial adhesion and invasion. To evaluate the relationship between ...the ECM and bacterial invasiveness, the reorganisation of fibronectin, tenascin-C and laminin after
Salmonella exposure
in vivo, the
Salmonella adhesiveness to ECM proteins
in vitro and the virulence gene expression upon co-cultivation of salmonellae and ECM proteins were elucidated for two
Salmonella strains with different capabilities to enter the intestinal mucosa. Immunohistochemistry and confocal microscopy showed that the infection of day-old chicks using either the highly invasive
S. Enteritidis (SE) or the nearly non-invasive
S. Infantis (SINF) strain was associated with an invasion-dependent reorganisation of fibronectin and tenascin-C in the caecal wall. Compared to SINF, clustered formations of SE were localised within and attached to the fibronectin and tenascin-C scaffold in the lamina propria indicating a relevance of ECM for bacterial dissemination in lower regions of the mucosa. In adhesion assays, SE was, indeed, significantly more adhesive to the matrix proteins than SINF. The attachment was accompanied by an increased
fliC mRNA expression in SE demonstrated by microarray analysis as well as quantitative real-time RT-PCR. The data suggest a relationship between the capability of
Salmonella serovars to interact with matrix proteins and to disseminate in gut mucosa perhaps in consequence of a matrix-mediated upregulation of the
Salmonella motility gene
fliC.
Despite the fact that, in a number of countries, vaccination programmes are extensively used to control
Salmonella infection in poultry, information on the immune mechanisms, especially the cellular ...response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA
+, IgM
+, IgG
+ cells, macrophages), bursa of Fabricius (IgM
+ cells, macrophages), and spleen (IgM
+ cells) of chicks after oral administration of a non-attenuated
Salmonella (
S.)
typhimurium wild-type strain (infection) or an attenuated commercial live
S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis.
In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA
+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM
+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against
Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against
Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against
Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential
Salmonella live vaccine candidates.
Quand bien même, dans de nombreux pays, des programmes de vaccination sont utilisés à large échelle pour contrôler l'infection
Salmonella pour la volaille, on a encore besoin d'information concernant les mécanismes immunitaires, en particulier la réponse cellulaire. Cette étude a pour but de caractériser la cellule B et la réponse des macrophages dans le cæcum (cellules IgA
+, IgM
+, IgC
+, macrophages), dans la bourse de Fabricius (cellules IgM
+, macrophages) et dans la rate (cellules IgM
+) des poussins après qu'on leur a administré oralement une souche sauvage de
Salmonella (
S.)
typhimurium non atténuée (infection) ou un vaccin vivant commercial atténué de
S. typhimurium (immunisation) administré à des poussins d'un jour, en comparaison avec des oiseaux de contrôle non traités, en utilisant l'immuno-histo chimie et l'analyse de l'image.
Dans le cæcum, on a détecté un nombre plus élevé de cellules secrétant des IgM, à la fois dans les animaux infectés comparés avec les contrôles menés le 5è et le 12è jour de vie. Par contraste, dans les groupes traités, les cellules secrétant des IgA ont été détectées en nombre plus élevé seulement entre le 8è et le 12è jour de vie. Dans la bourse de Fabricius des oiseaux infectés, on a établi une diminution des cellules IgM
+ fortement affaiblies et des macrophages entre le 5è et le 9è jour, ce qui indique que cet organe joue peut-être un rôle particulier et indépendant pendant la réaction immunologique contre les organismes
Salmonella. Les résultats portent à penser que les cellules secrétant IgM et IgA ont de l'importance dans la réponse immunitaire du cæcum des poussins contre les souches de
Salmonella. Les poussins immunisés ont toujours présenté une réaction L'examen immuno-histochimique de la réponse cellulaire dans les organes compétents du poulet peut étre instrument important pour l'évaluation des caractéristiques immunogéniques des candidates potentials à un vaccin vivant anti-salmonella.
The objective of this study was the comparative evaluation of four indirect Salmonella ELISA tests at study time approved in Germany to detect Salmonella infection in pigs.Three tests are based on a ...LPS-antigen mix and directed against specific IgG antibodies. The fourth test is based on a purified S. Typhimurium whole-cell lysate antigen and discriminates between Salmonella-specific IgM-, IgA-, and IgG- antibodies. In a longitudinal study, two groups of six weeks old hybrid piglets were orally infected with a porcine S. Infantis or S. Derby strain. Clinical and bacteriological parameters were monitored weekly during an observation period of 130 days after infection and serum samples were investigated in parallel with the respective ELISAs. Apparently, the LPS-based ELISA systems used in this study failed to recognize S. Infantis-infected pigs although those animals shed the pathogen in high amounts throughout the study until day 81 post infection (p. i.). In contrast, the isotype-specific Salmonella Typhimurium whole-cell-lysate based ELISA was capable of detecting Salmonella-infected pigs from day ten p. i. at all tested serotypes and revealed the highest sensitivity in detection of S. Infantis-infected pigs. Furthermore, it became apparent that the often used surveillance cut-off value of 40 OD% is not appropriate for intra-vitam detection of S. Infantis- and S. Derby-infected pigs. In contrast, the cut-off values of the ELISAs given by the suppliers result in considerable higher detection rates.
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