Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins

Valid publication of the names Caecibacterium and Caecibacterium sporoformans

Descriptions of the genus Caecibacterium and its proposed type species Caecibacterium sporoformans were published in the IJSEM by Onrust et al. (Int J Syst Evol Microbiol 2017;67:4589-4594). The type strain was deposited as LMG 27730 and DSM 26959. DSM 26959 is a patent strain, and therefore the names were effectively, but not validly, published based on Rule 30 (4) of the International Code of Nomenclature of Prokaryotes. The type strain of C. sporoformans is now available from the Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 103070 and no restrictions have been placed on its distribution. We here present new descriptions of the genus and its type species so that the names can be validly published

Infection of Differentiated Porcine Airway Epithelial Cells by Influenza Virus: Differential Susceptibility to Infection by Porcine and Avian Viruses

BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine

Bioactivities of a tumour necrosis like factor released by chicken rnacrophages

To test for tumour necrosis-like factor (TNF) of chickens, supernatants of a lipopolysaccharide (LPS)-stimulated chicken macrophage cell line MQ-NCSU were analysed. A sequence of ion-exchange and gel-permeation chromatography was utilised to isolate TNF-like activity from the culture supernatant. The peak of TNF-like cytotoxic activity corresponded to the fractions with a nlolecular weight of 81 kDa or higher. Polyclonal anti-human TNF-a antiserum cross-reacted by Western blotting with a 17 kDa protein in the TNF-containing fraction ~ ~ n d e denaturing conditions. This result indicated that chicken TNF-like factor in the biologically active form may be a protein multimer of monomers of about 17 kDa. The molecular weight of these monomers is similar to the molecular weight of mammalian TNF-CLC. hicken TNF-like factor stimulated macrophages by inducing morphological changes, enhancing Ia-expression, nitric oxide (NO) production and by synergising with interferon (1FN)-y in the induction of NO release from macrophages. The biological activities were not neutralised by anti-human TNF antiserum. These data suggest that LPS-stimulated chicken macrophages produced a functional homologue to mammalian TNF-CL. This may be structurally quite different from the mammalian TNF molecule. Other factors may have been co-purified with the chicken TNF-like factor having overlapping functions and molecular weight. However, CO-purification of chemokines and interleukin-l, major macrophage derived factors, with the chicken TNF-like factor can be excluded based on the purification strategies

Bioactivities of a tumour necrosis like factor released by chicken macrophages

To test for tumour necrosis-like factor (TNF) of chickens, supernatants of a lipopolysaccharide (LPS)-stimulated chicken macrophage cell line MQ-NCSU were analysed. A sequence of ion-exchange and gel-permeation chromatography was utilised to isolate TNF-like activity from the culture supernatant. The peak of TNF-like cytotoxic activity corresponded to the fractions with a nlolecular weight of 81 kDa or higher. Polyclonal anti-human TNF-a antiserum cross-reacted by Western blotting with a 17 kDa protein in the TNF-containing fraction ~ ~ n d e denaturing conditions. This result indicated that chicken TNF-like factor in the biologically active form may be a protein multimer of monomers of about 17 kDa. The molecular weight of these monomers is similar to the molecular weight of mammalian TNF-CLC. hicken TNF-like factor stimulated macrophages by inducing morphological changes, enhancing Ia-expression, nitric oxide (NO) production and by synergising with interferon (1FN)-y in the induction of NO release from macrophages. The biological activities were not neutralised by anti-human TNF antiserum. These data suggest that LPS-stimulated chicken macrophages produced a functional homologue to mammalian TNF-CL. This may be structurally quite different from the mammalian TNF molecule. Other factors may have been co-purified with the chicken TNF-like factor having overlapping functions and molecular weight. However, CO-purification of chemokines and interleukin-l, major macrophage derived factors, with the chicken TNF-like factor can be excluded based on the purification strategies

Investigations of Histomonosis-Favouring Conditions: A Hypotheses-Generating Case-Series-Study

Since the ban of effective feed additives and therapeutics, histomonosis has become an important disease and, subsequently, a welfare issue for turkey production. We conducted an interview-based case series study to generate hypotheses about possible disease-favouring conditions in 31 H. meleagridis-infected flocks. The determined parameters were related to the general farm (flock management, biosecurity measures, etc.) as well as the histomonosis-specific disease management. Some inadequate biosecurity measures were observed. An inappropriate usage of the hygiene lock and cleaning as well as the disinfection frequency of equipment, clothes, and the hygiene lock could possibly be histomonosis-favouring conditions. These factors could increase the risk for the introduction of H. meleagridis and the risk of a pathogen spread on an affected farm. Insects, wild birds, litter materials, and contaminated dung could be potential vectors of H. meleagridis. Predisposing gastrointestinal diseases were observed in 71% of the affected flocks. Additionally, stress events related to higher temperature, movement of birds, and vaccination were documented in association with clinical histomonosis. The results emphasise the need for both good disease control and health management to ensure sustainable animal health and welfare

Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species.

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants

Colonization patterns of Enterococcus cecorum in two different broiler production cycles detected with a newly developed quantitative real-time PCR

Abstract Background Although Enterococcus cecorum (EC) infection is one of the most important bacterial diseases in modern broiler chickens today, many aspects of epidemiology and pathogenesis are still unknown. There is a need for better detection methods for EC than classical cultivation. In the present study, we describe the validation and application of a newly developed quantitative TaqMan real-time PCR (qPCR) assay based on the 16S–rRNA-gene for the detection of EC. Results Fifty EC strains isolated from 12 different animal species were detected with the assay, while none of the other 26 examined bacterial species were tested positive during validation procedure. The detection limit of the PCR was 6.25 CFU/ml PBS. The qPCR assay was also considerably more sensitive using intestine and organ samples than the classical cultivation method. Field application of the PCR setup was tested comparing two different broiler production cycles on one farm: in cycle I broilers showed signs of enterococcal spondylitis (ES) from day 24 post hatch onwards while broilers in cycle II developed no ES. Two totally different colonization patterns were found in the two cycles with the qPCR using cloacal swabs. Animals in cycle I showed significantly (P ≤ 0.05) higher detection rates of EC at the day of placement and throughout the cycle than broilers of cycle II. Additionally, significantly higher detection rates were found in the cecum compared to duodenum, jejunum and ileum within one cycle. Conclusions The new qPCR for EC is highly specific, more sensitive than classical cultivation and was able to show differences in colonization in a broiler cycle with later EC disease outbreak compared to a healthy cycle. These findings may be explained by infection with different strains, pathogenic EC isolates are probably more effective in colonization than commensal isolates. A high correlation was found between qPCR results from cecum and cloacal swabs in this study, indicating that cloacal swabs can be used to examine intestinal colonization of broilers with EC. The new qPCR significantly improves the diagnostic of EC infections and may help to answer open questions concerning epidemiology and pathogenesis

Interaction of Influenza A Viruses with Oviduct Explants of Different Avian Species

Infection of poultry with low pathogenic avian influenza viruses (LPAIV) is often associated with mild respiratory symptoms but may also lead to loss in egg production in laying birds. In vivo susceptibility of the reproductive tract for LPAIV infection was reported for turkeys and chickens, but virus-interaction with epithelial cells of the oviduct and possible stimulation of the local antiviral immune responses have not been characterized. In this study, we wanted to investigate the suitability of magnum organ cultures (MOC) as an in vitro model to study virus-host interactions. We compared the susceptibility of duck (Du), chicken (Ch), and turkey (Tu) MOC for three different influenza A viruses (IAV). Overall, the course of infection and the antiviral immune response varied between strains as well as host cell origin, but MOC gave reproducible results for all investigated parameters within each species. While pandemic (p) H1N1 and H9N2 efficiently replicated in MOC-Ch and MOC-Tu, MOC-Du were significantly less susceptible to infection as indicated by a reduced replication level for both viruses (p < 0.05). Overall, virus replication levels did not correlate with interferonα (IFNα) mRNA-expression levels in neither species. H9N2-infection led to a significant upregulation of interferonλ (IFNλ) mRNA expression in MOC of all species compared to the non-infected controls (p < 0.05), while a correlation with replication levels was only seen for MOC-Tu. pH1N1-infection induced only significant upregulation of IFNλ mRNA expression in MOC-Tu at 48 hours post infection (p < 0.05), but the expression pattern did not correlate with replication levels. Our results show that MOC are a suitable model to study IAV-interaction with the mucosal surface of the avian reproductive tract. The data suggest that the reproductive tract may play a role in the pathobiology of IAV in poultry