Mono ADP-ribosylation is a posttranslational protein modification that has been implicated in the regulation of key biological functions in bacteria as well as in animals. Recently, the first cDNAs for eucaryotic mono-(ADPribosyl)transferases were cloned and found to exhibit significant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6. In this paper we describe secondary structure analyses of Rt6 and related proteins and show conserved structure motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreover, we have expressed soluble mouse Rt6 -1 and Rt6 -2 gene products in which C-terminal tags (FLAG-His 6 ) replace the native glycosylphosphatidylinositol anchor signal sequences. Purified recombinant Rt6 -2, but not Rt6 -1, shows NAD ؉ glycohydrolase activity, which is inhibited by the arginine analogue agmatine. Immunoprecipitation of recombinant Rt6 -1 and Rt6 -2 with anti-FLAG M2 antibody followed by incubation with [ P]NAD؉ leads to rapid and covalent incorporation of radioactivity into the light chain of the M2 antibody. The bound label is resistant to treatment with HgCl 2 but sensitive to NH 2 OH, characteristic of arginine-linked ADP-ribosylation. These results demonstrate that Rt6 -1 and Rt6 -2 possess the enzymatic activities typical for NAD ؉ -dependent arginine/ protein mono(ADPribosyl)transferases (EC 2.4.2.31). They are the first such enzymes to be molecularly characterized in the immune system.

The Campylobacter jejuni-host interaction may be affected by the host's gut microbiota through competitive exclusion, metabolites, or modification of the immune response. To understand this interaction, C. jejuni colonization and local immune responses were compared in chickens with different gut microbiota compositions. Birds were treated with an antibiotic cocktail (AT) (experiments 1 and 2) or raised under germfree (GF) conditions (experiment 3). At 18 days posthatch (dph), they were orally inoculated either with 104 CFU of C. jejuni or with diluent. Cecal as well as systemic C. jejuni colonization, T- and B-cell numbers in the gut, and gut-associated tissue were compared between the different groups. Significantly higher numbers of CFU of C. jejuni were detected in the cecal contents of AT and GF birds, with higher colonization rates in spleen, liver, and ileum, than in birds with a conventional gut microbiota (P < 0.05). Significant upregulation of T and B lymphocyte numbers was detected in cecum, cecal tonsils, and bursa of Fabricius of AT or GF birds after C. jejuni inoculation compared to the respective controls (P < 0.05). This difference was less clear in birds with a conventional gut microbiota. Histopathological gut lesions were observed only in C. jejuni-inoculated AT and GF birds but not in microbiota-colonized C. jejuni-inoculated hatchmates. These results demonstrate that the gut microbiota may contribute to the control of C. jejuni colonization and prevent lesion development. Further studies are needed to identify key players of the gut microbiota and the mechanisms behind their protective role.

BackgroundChickens are regarded as the main reservoir for human campylobacteriosis. Little is known about the interaction between Campylobacter jejuni (C. jejuni) and chickens. This interaction may be influenced by the stage of maturation of the immune system, developing gut microbiota composition and other factors including breed and diet. Our aim was to investigate the impact of breed, and diet on C. jejuni colonization and host immune responses in chickens. Birds were inoculated with 104 colony forming units (CFU) of C. jejuni or diluent at one (Exp. 1) or 22 (Exp. 2) days post hatch. We compared local immune cell subpopulations, cytokine expression levels, and gut microbiota composition between broiler-type (BT) and layer-type (LT) birds fed with either commercial broiler feed (bf) or layer feed (lf).ResultsLower colonization rates were observed in the older age group independent of breed and diet. Independent of breed, birds fed with bf showed higher CFU of C. jejuni compared to lf-fed groups. Campylobacter jejuni-inoculation had a significant effect on lymphocyte numbers and cytokine expression levels in BT birds independent of feeding strategy (p < 0.05). These effects were not detected in LT birds, only LT birds fed with bf showed a significant increase in IL-8-expression at 7 days post C. jejuni inoculation compared to LT-control birds (p < 0.05). Diet influenced gut microbiota composition in a comparable manner between BT and LT birds, but changes in microbiota composition associated with C. jejuni inoculation varied between breeds.ConclusionsDiet and breed influenced C. jejuni colonization, immune responses and microbiota composition to a different extent comparing between LT and BT birds. The mechanisms behind these differences have to be elucidated further. Our results suggest that selection for more resistant breeds in combination with adapted feeding strategies may help to reduce Campylobacter colonization levels in commercial poultry in the future.

The role of intestinal epithelial cells (IECs) in the physiology of the gastrointestinal tract (GIT) of chickens and pathogenesis of various diseases in chickens is still poorly understood. IECs line the GIT and represent the border between the unsterile environment and the sterile internal tissues. Bacterial, viral, fungal, or parasitic pathogens are able to invade or pass IECs under certain circumstances and cause various diseases. Pathogen-host interactions in the chicken gut are poorly understood because of the lack of suitable in vitro and ex vivo models. In this context, there is a need to optimize the cell isolation and culture conditions to be able to provide reproducible IEC cultures with defined epithelial characteristics. We compared different mechanical IEC isolation protocols and cell culture media and established a reproducible primary intestinal epithelial cell culture model from specific-pathogen-free layer-type chickens. By using isolated crypts from the duodenum of 5- to 12-wk-old birds to create the starting material, we were able to culture replicating cells between 7 and 10 days. Cells built an almost closed monolayer and showed epithelial-like characteristics, such as the expression of cytokeratin and epithelial cadherin. The primary IEC cultures described in this study represent a suitable model with which to investigate in vitro pathogen-host interactions relevant to the chicken gut.

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