Bordetella pertussis-infected innate immune cells drive the anti-pertussis response of human airway epithelium.

Pertussis is a severe respiratory tract infection caused by Bordetella pertussis. This bacterium infects the ciliated epithelium of the human airways. We investigated the epithelial cell response to B. pertussis infection in primary human airway epithelium (HAE) differentiated at air-liquid interface. Infection of the HAE cells mimicked several hallmarks of B. pertussis infection such as reduced epithelial barrier integrity and abrogation of mucociliary transport. Our data suggests mild immunological activation of HAE by B. pertussis indicated by secretion of IL-6 and CXCL8 and the enrichment of genes involved in bacterial recognition and innate immune processes. We identified IL-1ß and IFNγ, present in conditioned media derived from B. pertussis-infected macrophage and NK cells, as essential immunological factors for inducing robust chemokine secretion by HAE in response to B. pertussis. In transwell migration assays, the chemokine-containing supernatants derived from this HAE induced monocyte migration. Our data suggests that the airway epithelium on its own has a limited immunological response to B. pertussis and that for a broad immune response communication with local innate immune cells is necessary. This highlights the importance of intercellular communication in the defense against B. pertussis infection and may assist in the rational design of improved pertussis vaccines.

Chicken immune response following in ovo delivery of bacterial flagellin.

In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against several viral pathogens. We investigated the immune response of chicken embryos to purified recombinant protein. In ovo delivery of Salmonella flagellin to 18-day old embryonated eggs resulted in elevated pro-inflammatory chIL-6 and chIL-8 (CXCL8-CXCLi2) cytokine transcript levels in the intestine but not in the spleen at 24 h post-injection. Analysis of the chicken Toll-like receptor (TLR) repertoire in 19-day old embryos revealed gene transcripts in intestinal and spleen tissue for most chicken TLRs, including TLR5 which recognizes Salmonella flagellin (FliC). The in ovo administration of FliC did not alter TLR transcript levels, except for an increase in intestinal chTLR15 expression. Measurement of the antibody response in sera collected at day 11 and day 21 post-hatch demonstrated high titers of FliC-specific antibodies for the animals immunized at the late-embryonic stage in contrast to the mock-treated controls. The successful in ovo immunization with purified bacterial antigen indicates that the immune system of the chicken embryo is sufficiently mature to yield a strong humoral immune response after single exposure to purified protein. This finding strengthens the basis for the development of in ovo protein-based subunit vaccines.

Trichinella spiralis-secreted products modulate DC functionality and expand regulatory T cells in vitro.

Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-ß. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.

Differential N-glycan- and protein-directed immune responses in Dictyocaulus viviparus-infected and vaccinated calves.

Calves with naturally acquired Dictyocaulus viviparus infection mount an effective immune response. In the search for protection-inducing antigens, we found that several D. viviparus third-stage larval (L3) and adult ES products carry N-glycans. Deglycosylation of the worm antigens using PNGase F resulted in reduced IgA, IgE, IgG1 and IgG2 (but not IgM) reactivities in sera of primary infected animals, suggesting that the carbohydrate moieties contained immunodominant epitopes. Challenge infection resulted in increased specific serum antibody levels against ES and L3 in the re-infected and challenge control groups. Testing of sera by enzyme-linked immunosorbent assay (ELISA) demonstrated a significant increase in IgG1 and IgE (but not IgA or IgG2) reactivity against the deglycosylated antigens in the re-infected group compared with the challenge control group. Sera from calves vaccinated with irradiated larvae showed a strong anti-N-glycan response, but no booster response against the protein backbone after challenge infection, consistent with the absence of a memory response. Together, our results suggest that D. viviparus proteins carry immunodominant N-glycan moieties that elicit a strong but short-lived immune response during infection and after vaccination, whereas the protein backbones effectively induce a memory response which results in a long-lasting, potentially protective immune response in re-infected, but not in vaccinated calves.

Vaccine potential of recombinant Ornithobacterium rhinotracheale antigens.

Ornithobacterium rhinotracheale is a pathogen involved in respiratory infection and systemic disease in poultry. Previously, eight potential vaccine candidates were identified that induced cross-protective immunity when administered to chickens as a multi-component vaccine. In this study, we analyzed the immunogenicity of these eight recombinant proteins by subunit vaccination, and characterized the different proteins and corresponding genes more thoroughly by sequencing, in vitro expression analysis, and cellular localization experiments. We found, that all genes encoding the eight antigens were highly conserved among different O. rhinotracheale serotypes, but the different antigens were not expressed by all serotypes. Cellular fractionation experiments indicated that the majority of the antigens are predominantly located in the outer membrane fraction. Vaccination of chickens with single-antigen vaccines demonstrated that the Or77 antigen was protective against serotypes that expressed Or77 in vitro, suggesting that the protein has strong potential as a vaccine antigen. Furthermore, immunization with four-component subunit vaccines indicated the existence of immunogenic synergism between the candidate vaccine antigens.

Successful selection of cross-protective vaccine candidates for Ornithobacterium rhinotracheale infection.

Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.

Passive immunization of immune-suppressed animals: chicken antibodies protect against Ornithobacterium rhinotracheale infection.

Unravelling of the protective immunity acquired during a natural infection may contribute to vaccine development. To assess the role of antibody-mediated immunity in protection against Ornithobacterium rhinotracheale infection in chickens, a novel experimental method was applied that combined immune depletion and passive transfer of immunity within the same host. Administration of cyclophosphamide (CY) to broiler chickens successfully suppressed B lymphocyte development, and therefore humoral immunity, as confirmed by histological and serological analysis. Challenge of CY-treated birds with O. rhinotracheale revealed a significantly higher pathology score in comparison to immune-competent birds that received the same bacterial challenge. Measurement of serum immunoglobulin levels of immune-competent birds revealed a positive correlation between IgA and/or IgG production and protection against infection. Passive transfer of O. rhinotracheale-specific antiserum to the immune-suppressed birds prior to pathogen challenge significantly decreased morbidity. This protective effect was not observed after administration of control sera containing similar concentrations of immunoglobulins. Together, these results provide firm evidence that chicken humoral immunity to O. rhinotracheale is a key component in protection against infection. Our data confirm that the applied immune depletion and reconstitution approach is an attractive tool to analyse the nature of the protective immune response.

Global detection and identification of Campylobacter fetus subsp. venerealis.

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is a genital infection that threatens the cattle industry. Detection and identification of Cfv are key factors in control programmes. Trade regulations should be based on scientifically and internationally accepted methods of detection and identification of Cfv. Such methods are described in the World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. A study was conducted to determine which methods are in use in OIE Member Countries and to get an overview of new or improved tests. A questionnaire was sent to OIE Member Countries, and 26 out of 166 were returned. Globally, a diversity of methods for the detection and identification of Cfv are in use. The authors conclude that there is a lack of harmonisation that may have consequences for the description of the health status of countries and may lead to disputes with respect to trade regulations.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100.

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.