Duck Complement Factor H Binds to Outer Membrane Protein Omp24 of Riemerella anatipestifer.

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni-nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum-inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.

Artículo regular­El factor H del complemento de pato se une a la proteína de la membrana externa Omp24 de Riemerella anatipestifer La resistencia a la destrucción mediada por el complemento sérico es una propiedad vital para la virulencia de los patógenos microbianos. El factor de complemento H (FH) es un regulador negativo clave de la vía alterna del complemento (AP) que previene la formación y acelera la descomposición de la C3 convertasa de la vía alterna y actúa como cofactor en la inactivación de C3b. Los patógenos pueden reclutar factor H del huésped a través de sus proteínas de superficie para escapar de la destrucción por el sistema del complemento. Riemerella anatipestifer también pudo evadir el ataque del sistema del complemento para lograr la infección del huésped, pero el mecanismo aún no está claro. En este estudio, se seleccionaron y analizaron las proteínas de R. anatipestifer que podrían interactuar con el factor H en el suero del huésped y se determinaron las funciones. La cromatografía de afinidad con una columna de sefinosa de Ni-NTA y la espectrometría de masas identificaron tres proteínas de la membrana externa de R. anatipestifer, Omp54, Omp53 y Omp24, como posibles proteínas que se unen al factor H. Posteriormente, se llevó a cabo con éxito la expresión procariota y la preparación de anticuerpos policlonales de las tres proteínas candidatas. El ensayo de inmunofluorescencia indirecta mostró que las tres proteínas candidatas estaban presentes en R. anatipestifer. El ensayo de transferencia para afinidad, el ensayo anti-inhibidor del suero y el ensayo bactericida sérico presentaron evidencia de que la proteína Omp24 podría unirse al factor H. Además, el factor H unido a la proteína Omp24 se asoció con resistencia a la vía alterna y funcional para la supervivencia de R. anatipestifer en el suero de pato normal. Estos resultados sugirieron que la proteína Omp24 de R. anatipestifer era una proteína de unión al factor H y que la interacción con este factor bloqueaba la vía alterna del complemento. El reclutamiento de proteínas reguladoras del complemento puede facilitar una mejor resistencia de R. anatipestifer a esta línea vital de defensa del huésped.

Molecular and serological characterization of Riemerella isolates associated with poultry in Australia.

A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars.

Riemerella anatipestifer infection affects intestinal barrier structure and immune reactions in the duck caecum.

Riemerella anatipestifer (RA) infection causes high mortality and poor feed conversion, leading to great economic losses to the duck industry. This study investigated the effects of RA on the intestinal morphology and immune response of ducks. Histological examination showed that RA infection caused intestinal injury, including significantly reduced mucosal thickness on days 2, 3 and 5, significantly reduced villus height on days 1, 2, 3 and 5 (P < 0.05) and significantly reduced villus height to crypt depth ratios on days 2, 3, 5 and 9 of RA infection (P < 0.05). The expression of intestinal mucosal layer construction-associated genes and tight junction genes was significantly altered on at least one time point (day 1, 2, 3, 5, 9 or 14) after RA infection. Quantitative real-time polymerase chain reaction revealed that RA infection affected intestinal mucosal immune function. The genes encoding TLR4 (toll like receptor-4), TRAF6 (TNF receptor-associated factor 6), MYD88 (myeloid differentiation factor 88), IFN-γ (interferon-γ), IL (interleukin)-4 and IL-8 were significantly upregulated on day 2 of RA infection. Taken together, these results indicate that RA infection negatively affects intestinal barrier function in ducks due to impaired mucosal and villus-crypt structure and alters the mRNA expression of mucous layer construction-, intestinal tight junction-, and intestinal mucosal immunity-related genes.

Assessment of immunization regimens of duck Riemerella anatipestifer vaccines.

AIMS: Riemerella anatipestifer infections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes of R. anatipestifer, cross-protection is an important goal for vaccine development. METHODS AND RESULTS: In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccine vs prime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8+ T cell and higher expression levels of IFN-γ, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates of R. anatipestifer serotypes 1, 2 and 6. CONCLUSIONS: Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. SIGNIFICANCE AND IMPACT OF THE STUDY: It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit + inactivated combination an attractive option.

Evaluation of the protective immunity of Riemerella anatipestifer OmpA.

Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.

A recombinant vaccine of Riemerella anatipestifer OmpA fused with duck IgY Fc and Schisandra chinensis polysaccharide adjuvant enhance protective immune response.

Riemerella anatipestifer (R. anatipestifer) causes septicemia and infectious serositis in domestic ducks, leading to high mortality and great economic losses worldwide. Vaccination is currently considered the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we fused the duck IgY Fc gene to the outer membrane protein A (ompA) of R. anatipestifer. The eukaryotic expression plasmid carrying the fusion gene was transformed into Pichia pastoris (P. pastoris) to express the recombinant ompA and ompA-Fc proteins. Then, the effects of fused Fc on the vitality and antigen processing efficiency of duck peritoneal macrophages (PMø) were evaluated in vitro, whereas their immunogenicity was evaluated in vivo. Furthermore, Schisandra chinensis polysaccharide (SCP) was used to evaluate its immune-conditioning effects on the activation of PMø. SCP was also used as adjuvant to investigate immunomodulation on immunoresponses induced by the fused ompA-Fc in ducklings. The conventional Freund's incomplete adjuvant served as the control of SCP. Notably, ompA-Fc promoted phagocytosis of PMø and significantly increased serum antibody titers, CD4+ and CD8+ T-lymphocyte counts, lymphocyte transformation rate, and serum levels of IL-2 and IL-4. In addition, ducklings injected with the ompA-Fc vaccine exhibited considerably greater resistance to the R. anatipestifer challenge than those that received vaccines based on standalone ompA. Of note, SCP was demonstrated to boost the secretion of nitric oxide (NO), IL-1ß, IL-6, TNF-α, and IFN-ß by duck macrophages. In addition, the supplementation of SCP adjuvant to the ompA-Fc vaccines led to the further enhancement of immune response and vaccine protection. The dose of 200 µg/mL showed the most pronounced effects. This study provided valuable insights into protective strategies against R. anatipestifer infection.

Riemerella anatipestifer infection in ducks induces IL-17A production, but not IL-23p19.

R. anatipestifer (RA) is one of the most harmful bacterial pathogens affecting the duck industry, and infection is associated with the production of proinflammatory cytokines, including IL-17A. Another proinflammatory cytokine, IL-23, is critical for the development of Th17 cells, which produce IL-17. However, IL-23 roles have not been studied in this infection. Here, we describe the identification and mRNA expression analysis of duck IL-23p19 (duIL-23p19) in splenic lymphocytes and macrophages stimulated with killed RA and in spleens of RA-infected ducks. Expression of duIL-23p19 transcript identified in this study was relatively high in livers of healthy ducks and was upregulated in mitogen-activated splenic lymphocytes as well as in splenic lymphocytes and macrophages stimulated with killed RA. In spleens of RA-infected ducks, expression levels of duIL-23p19 transcript were unchanged at all time points except on days 4 and 7 post-infection; however, duIL-17A and IL-17F expression levels were upregulated in both spleens of RA-infected ducks and splenic lymphocytes and macrophages stimulated with killed RA. In sera collected at 24 h after this infection, duIL-23p19 expression levels were unchanged, whereas IL-17A significantly upregulated. These results suggest that IL-23p19 does not play a critical role in the IL-17A response in early stages of RA-infected ducks.

Genotyping of Riemerella anatipestifer by ERIC-PCR and correlation with serotypes.

Riemerella anatipestifer (RA) is a widely distributed bacterial pathogen of birds responsible for remarkable losses to poultry production, especially among waterfowl. We characterized the genomic diversity of 166 field isolates of RA, collected from geese and ducks, using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR). The field strains and five reference strains showed 17 distinct patterns consisting of five to 12 bands ranging from approximately 150-1800bp. The majority of the strains belonged to two closely related ERIC-PCR types (A and B), while the other types contained only a few isolates each. There was no association between ERIC-PCR type and host species, place, or year of isolation; however the ERIC-PCR pattern was correlated with serotype for most isolates. The majority of serotype 1 strains (101/107) belonged to ERIC-PCR type A while the remaining six strains represented five different ERIC-PCR types (D, G, L, M, and O). Serotypes 1,7 and 7 corresponded to ERIC-PCR types B and C, respectively. Serotypes 2, 4, and 10 could be subdivided by ERIC-PCR revealing two to four patterns within each serotype. These results indicate that ERIC-PCR may be a suitable technique for the molecular identification of RA serotypes, and the detection of subtypes within certain serotypes may aid further epidemiological investigations. RESEARCH HIGHLIGHTS ERIC-PCR analysis of field R. anatipestifer strains revealed 17 distinct patterns Most strains belonged to two closely related ERIC-PCR types Serotype 1 was the most prevalent serotype representing 64.5% of the strains ERIC-PCR may be suitable for molecular identification of R. anatipestifer serotypes.

Immunogenicity and safety of a live Riemerella anatipestifer vaccine and the contribution of IgA to protective efficacy in Pekin ducks.

Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry. In this study, we developed an RA vaccine to control virulent serotype 1 and 2 RA, which predominate in worldwide prevalence. We established a strategy for vaccine candidate screening, and selected strains D15-RDA-92 (serotype 1) and D14-RDA-8 (serotype 2). These strains were characterized by ≤50% embryo mortality and <3.0 serum resistance assay values in in vitro screening. We evaluated the protective efficacy of live bivalent RA vaccines against virulent homologous serotype RA. Ducklings received two oral immunizations with the bivalent vaccine and showed significant protection against two virulent strains (serotypes 1 and 2) at 21 days post-immunization. No death or clinical signs of diarrhea, tremors, or limb swelling were observed in the immunized ducks. In a safety evaluation, ducks immunized with 100 times higher doses showed no clinical signs, mortality, gross lesions, or histological lesions, and body weight of the ducks showed no significant difference compared to that of negative controls. In addition, IgA analysis showed a significant increase in secretory IgA antibodies generated in the trachea and duodenum of orally immunized ducks at 28 days of age. The IgA might be involved in one of the major immune responses to RA and contributes to protecting against virulent RA. In this study, we developed monovalent and bivalent RA vaccines that were safe in ducks and provided significant protective efficacy against virulent homologous RA strains.

Threshold level of Riemerella anatipestifer crossing blood-brain barrier and expression profiles of immune-related proteins in blood and brain tissue from infected ducks.

Riemerella anatipestifer (R. anatipestifer) is a Gram-negative bacterium that crosses the blood-brain barrier (BBB) and causes meningitis with neurological symptoms in infected ducks. A threshold level of bacteremia must be reached before the BBB can be breached. In this study, the bacterial burden and expression profiles of immune-related proteins in blood and brain tissue samples from R. anatipestifer-infected ducks were investigated. The data showed that R. anatipestifer could cross the BBB with low-level bacteremia of 7.5 × 102 CFU/ml in infected blood. Analysis of immune-related proteins revealed that IL-4, IL-17A, IL-17D, TLR3, TLR4, TLR7, and TGF-ß in blood, as well as IL-1ß, IL-2, IL-4, IL-17A, IL-17D, IL-17F, TLR3, TLR4, and TGF-ß in brain tissue were upregulated at an early stage in infection. The expression levels of Th1 and Th17-specific cytokines were significantly higher than those of Th2-specific cytokines, which indicated that mainly Th1 and Th17 immune responses were induced during R. anatipestifer infection.

Identification of duck IL-4 and its inhibitory effect on IL-17A expression in R. anatipestifer-stimulated splenic lymphocytes.

As the dysregulation of IL-17 is implicated in the pathogenesis of various autoimmune and inflammatory diseases, the suppression of IL-17 production by Th2 cytokines could alleviate the development of these diseases. Previously, we confirmed that inflammatory cytokines including IL-17A are strongly associated with R. anatipestifer infection, which is one of the most important bacterial pathogens in the duck industry. Here, we found that IL-4 treatment downregulated the expression of IL-17A and IL-17F transcripts in splenic lymphocytes stimulated with R. anatipestifer. Moreover, duck IL-4 (duIL-4) treatment in R. anatipestifer-stimulated lymphocytes suppressed the expression of IL-23p19 and IL-12p40 transcripts compared to untreated and stimulated lymphocytes. Conversely, duIL-4 increased levels of IFN-γ and IL-10. We identified a full-length duIL-4 cDNA encoding 136 amino acids from ConA-activated splenic lymphocytes that shares 49.3-50% amino acid sequence identity with chicken and quail IL-4 and 21-29.7% with mammalian and piscine homologues. Low or moderate levels of duIL-4 transcript were observed in healthy tissues, including the spleen, bursa, and thymus, whereas duIL-4 expression was higher in the kidney and lung. Levels of duIL-4 were generally upregulated in mitogen-activated splenic lymphocytes but lower in the liver and spleen of R. anatipestifer-infected ducks compared to those of infected chickens. Recombinant duIL-4 promoted nitric oxide synthesis in duck macrophages stimulated by R. anatipestifer compared to untreated and stimulated control macrophages. These results demonstrate that IL-4 is an important Th2 cytokine that inhibits inflammatory responses in splenic lymphocytes stimulated with R. anatipestifer.

Downregulation of common cytokine receptor γ chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer.

Th17-cell-mediated inflammation is affected by the soluble form of common cytokine receptor γ chain (γc). We previously suggested that inflammatory cytokines including interleukin (IL)-17A are associated with Riemerella anatipestifer infection, which a harmful bacterial pathogen in ducks. Here, the expression profiles of membrane-associated γc (duγc-a) and soluble γc (duγc-b) in R. anatipestifer-stimulated splenic lymphocytes and macrophages, and in the spleens and livers of R. anatipestifer-infected ducks, were investigated. In vitro and in vivo results indicated that the expression levels of both forms of γc were increased, showing that marked increases were detected in the expression of the duγc-b form rather than the duγc-a form. Treatment with γc-specific siRNA downregulated mRNA expression of Th17-related cytokines, including IL-17A and IL-17F, in duck splenic macrophages stimulated with R. anatipestifer, whereas the expressions of interferon (IFN)-γ and IL-2 were enhanced. The results showed that the upregulation of γc, especially the duγc-b form, was associated with expression of Th17-related cytokines during R. anatipestifer infection.

The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor.

UNLABELLED: Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate. IMPORTANCE: Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.

Identification and expression analysis of duck interleukin-17D in Riemerella anatipestifer infection.

Interleukin (IL)-17D is a proinflammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. duIL-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinflammatory cytokines such as IL-6, IL-8, and IL-1ß in duck embryo fibroblast cells. Very low levels of duIL-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The duIL-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species.

Identification of a gene in Riemerella anatipestifer CH-1 (B739-2187) that contributes to resistance to polymyxin B and evaluation of its mutant as a live attenuated vaccine.

Riemerella anatipestifer (R. anatipestifer) causes severe perihepatitis, pericarditis, airsacculitis and meningitis in the duck, leading to great economic losses worldwide. Given the increased prevalence of drug-resistance strains, vaccination is the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we identified a gene in R. anatipestifer (B739-2187) that can restore the resistance of the Salmonella phoP mutant to polymyxin B using genetic complementation. Furthermore, the deletion of B739-2187 in R. anatipestifer resulted in a mutant exhibiting increased sensitivity to polymyxin B. The R. anatipestifer B739-2187 mutant did not exhibit phenotypic defects, as indicated by its growth curve, lipopolysaccharide and outer membrane protein profiles, and attachment and invasion of duck embryo fibroblast cells. The duck animal experiments demonstrated that the deletion of B739-2187 significantly decreased the virulence of R. anatipestifer, and the B739-2187 mutant provided 100% protection against challenge with wild-type R. anatipestifer, exhibiting the characteristics of an ideal live vaccine.

Characterization and cross-protection evaluation of M949_1603 gene deletion Riemerella anatipestifer mutant RA-M1.

Riemerella anatipestifer infection causes high mortality for ducks which results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn4351 transposon mutagenesis, in which the M949_1603 gene encoding glycosyl transferase was inactivated. PCR analysis revealed that M949_1603 gene is specifically existed in R. anatipestifer serotype 1 strains. RA-M1 presented no reactivity to the anti-lipopolysaccharide (LPS) MAb in an indirect ELISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting demonstrated that RA-M1 LPS had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of RA-M1 was changed. RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, cross-protection of RA-M1 for R. anatipestifer serotypes 1, 2, and 10 strains was evaluated. Ducks that received two immunizations with inactivated RA-M1 vaccine were 100% protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2, and serotype 10 strain HXb2. No changes were observed in the liver, heart, or spleen samples from the protected ducks during autopsy and histological examination. Furthermore, vaccination generated high antibody titers of 1:12,800 against serotypes 1, 2, and 10 strains and enhanced production of interleukin 2 (IL-2) and IL-4 in ducks. These results suggested that M949_1603 gene is associated with serotype 1 O-antigen biosynthesis, and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.

Development of colloidal gold immunochromatographic strips for detection of Riemerella anatipestifer.

Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20 ± 5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1 × 10(6) colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.

Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant.

Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA+CpG ODN showed higher levels (p<0.05) of antibody titer, T cell proliferation, and percentages of CD4(+) and CD8(+) T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA+CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA+CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA+CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.

Levamisole enhances immunity in ducklings vaccinated against Riemerella anatipestifer.

Oil-adjuvant-inactivated vaccine is one of the most cost-effective vaccines used to protect ducklings against RA infection; however, it does not provide complete protection in very young ducklings with immature immune systems. In the current study, LMS was used as an immunopotentiator to improve the immune system in ducklings. Serum immunoglobulin (Ig)G titers and the secretions of both Th1-type (IFN-γ and IL-2) and Th2-type (IL-4 and IL-10) cytokines were higher in ducklings that had been vaccinated with LMS. In addition, a significantly higher T-lymphocyte proliferation rate was obtained with the addition of LMS. Furthermore, all of the ducklings vaccinated with LMS were protected against RA on the 9th day post-vaccination, whereas only 69.2% of the ducklings were protected in the group that did not receive LMS. These results suggest that LMS might be a useful adjuvant to enhance the immune response of ducklings. The use of LMS may also alleviate local injection lesions, caused by the oil-emulsion vaccine, by reducing the dose of the vaccine.

Protein expressions and their immunogenicity from Riemerella anatipestifer cultured in iron restriction medium.

Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.