Summary We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, HYPE (Huntingtin yeast-interacting protein E), also adenylylates Rho GTPases in vitro. Thus, Fic domain containing proteins are a new class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic post-translational modification that may regulate key signaling events.

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Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3 end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain 8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10 9 CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.

ABSTRACTIn this work, we investigated theCampylobacter jejuni dps(DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry.In vitrobiofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by theC. jejuni dpsmutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of thedpsgene in host colonization byC. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-typeC. jejuniindicated that thedpsgene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with thedpsmutant than chicks infected with the wild-typeC. jejunistrain. Moreover, thedpsmutant failed to colonize 75% of the chicks following challenge with 105CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuatedSalmonella entericastrain synthesizing theC. jejuniDps protein. No reduction inC. jejuniwas noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction ofC. jejuniwas achieved in chicks vaccinated with the attenuatedSalmonellavector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through aSalmonellavaccine vector.

-Genetic resistance to diseases is a multigenic trait governed mainly by the immune system and its interactions with many physiologic and environmental factors. In the adaptive immunity, T cell and B cell responses, the specific recognition of antigens and interactions between antigen presenting cells, T cells and B cells are crucial. It occurs through a network of mediator proteins such as the molecules of the major histocompatibility complex (MHC), T cell receptors, immunoglobulins and secreted proteins such as the cytokines and antibodies. The diversity of these proteins that mainly is due to an intrinsic polymorphism of the genes causes phenotypic variation in disease resistance. The well-known linkage of MHC polymorphism and Marek's disease resistance difference represents a classic model revealing immunological factors in resistance differences and diversity of mediator molecules. The molecular bases in any resistance variation to infectious pathogens are vaguely understood. This paper presents a review of the major immune mediators involved in resistance and susceptibility to infectious diseases and their functional mechanisms in the chicken. The genetic interaction of disease resistance with production traits and the environment is mentioned. et les interactions entre les cellules présentant des antigènes, les cellules T et B, sont cruciales. Cela se produit à travers un réseau de protéines médiatrices telles que les molécules du complexe majeur d'histocompatibilité (CMH), les récepteurs des cellules T, les immunoglobulines et les protéines sé-crétées telles que les cytokines et les anticorps. La diversité de ces protéines, qui est surtout due au polymorphisme intrinsèque des gènes, entraîne une variation phénotypique dans la résistance aux maladies. Le lien bien connu entre le polymorphisme du CMH et les différences dans la résistance à la maladie de Marek représente un modèle classique révélant les facteurs immunologiques dans les différences de résistance et la diversité dans les molécules médiatrices. Les bases moléculaires impliquées dans toute variation de résistance aux pathogènes infectieux sont à peu près comprises. Cet article présente une synthèse sur les principaux médiateurs de l'immunité impliqués dans la résis-tance et la sensibilité aux maladies infectieuses chez le poulet et leurs mécanismes fonctionnels. L'interaction génétique entre la résistance aux maladies et les traits de production et l'environnement est mentionnée.

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cOur previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSVplus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection. R espiratory infections are characteristically polymicrobial. We previously investigated the mechanisms of viral and bacterial synergy in bovine respiratory disease (BRD) (1) by infecting calves with bovine respiratory syncytial virus (BRSV) followed by infection with Histophilus somni or with either pathogen alone. The calves dually infected with BRSV and H. somni had the most severe disease and the highest serum IgE antibody responses to H. somni (1). Duration of pneumonia and persistence of H. somni in the lungs were also greatest in dually infected animals (1). Our earlier immunohistochemistry studies of experimental H. somni pneumonia showed that the bacteria are detected primarily in the alveoli at 24 h after intrabronchial inoculation (2). Since H. somni causes septicemia and its sequelae (3), it is likely that it crosses into the circulation over the alveolar barrier. We also showed that the toxic Fic (filamentation induced by cyclic AMP [cAMP]) motifs of the direct repeat domains (DR1 and DR2) of the immunoglobulin binding protein A (IbpA) cause bovine alveolar type 2 (BAT2) cells to retract in vitro, allowing paracellular migration (4). This cytotoxicity is due to adenylylation of host cell Rho GTPases by the Fic motif (5). Conservation of IbpA and Fic motifs in all tested H. somni disease isolates was c...

Malabsorption syndrome (MAS) is a multifactorial disease that causes intestinal disorders in broilers due