Xilong Kang

Analysis of immune-related gene expression in chicken peripheral blood mononuclear cells following Salmonella enterica serovar Enteritidis infection in vitro.

We examined mRNA expression of eight genes, TLR4, TLR5, TLR15, interleukin (IL)-1β, IL-6, transforming growth factor-β4 (TGF-β4), CXCLi2, and a macrophage inflammatory protein (MIP) family chemokine called CCLi2, in peripheral blood mononuclear cells (PBMCs) isolated from the blood of chickens after in vitro exposure to Salmonella enterica serovar Enteritidis (SE). The chickens of four Chinese native lines, Qingjiaoma, Sanhuang, Wugu, and Xueshanma, were evaluated for mRNA expression levels at 2 and 4h post-infection using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). TLR4 and TLR15 mRNA were in particular highly expressed in PBMCs of Wugu and Xueshanma chickens exposed to SE, while TLR5 was expressed less in the Sanhuang chickens than in others. Breed effect was significant (P<0.05) for IL-1β, IL-6, CXCLi2, and CCLi2 mRNA expression, all of which were expressed to a greater extent in Wugu and Xueshanma than in the other two lines. These findings demonstrate the difference of mRNA expression profiles for innate immune molecules in PBMCs infected to SE among different lines.

Molecular cloning, characterization and expression of goose Toll-like receptor 5.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs and the spleen. These results imply that the expression of goose TLR5 is differentially regulated in various tissues and may participate in the immune response against bacterial pathogens.

Immunopotentiation of Different Adjuvants on Humoral and Cellular Immune Responses Induced by HA1-2 Subunit Vaccines of H7N9 Influenza in Mice

In spring 2013, human infections with a novel avian influenza A (H7N9) virus were reported in China. The number of cases has increased with over 200 mortalities reported to date. However, there is currently no vaccine available for the H7 subtype of influenza A virus. Virus-specific cellular immune responses play a critical role in virus clearance during influenza infection. In this study, we undertook a side-by-side evaluation of two different adjuvants, Salmonella typhimurium flagellin (fliC) and polyethyleneimine (PEI), through intraperitoneal administration to assess their effects on the immunogenicity of the recombinant HA1-2 subunit vaccine of H7N9 influenza. The fusion protein HA1-2-fliC and HA1-2 combined with PEI could induce significantly higher HA1-2-specific IgG and hemagglutination inhibition titers than HA1-2 alone at 12 days post-boost, with superior HA1-2 specific IgG titers in the HA1-2-fliC group compared with the PEI adjuvanted group. The PEI adjuvanted vaccine induced higher IgG1/IgG2a ratio and significantly increased numbers of IFN-γ- and IL-4-producing cells than HA1-2 alone, suggesting a mixed Th1/Th2-type cellular immune response with a Th2 bias. Meanwhile, the HA1-2-fliC induced higher IgG2a and IgG1 levels, which is indicative of a mixed Th1/Th2-type profile. Consistent with this, significant levels, and equal numbers, of IFN-γ- and IL-4-producing cells were detected after HA1-2-fliC vaccination. Moreover, the marked increase in CD69 expression and the proliferative index with the HA1-2-fliC and PEI adjuvanted vaccines indicated that both adjuvanted vaccine candidates effectively induced antigen-specific cellular immune responses. Taken together, our findings indicate that the two adjuvanted vaccine candidates elicit effective and HA1-2-specific humoral and cellular immune responses, offering significant promise for the development of a successful recombinant HA1-2 subunit vaccine for H7N9 influenza.

Amino acids 89-96 of Salmonella typhimurium flagellin represent the major domain responsible for TLR5-independent adjuvanticity in the humoral immune response

Toll-like receptor 5 (TLR5) signaling in response to flagellin is dispensable for inducing humoral immunity, but alterations of aa 89–96, the TLR5 binding site, significantly reduced the adjuvanticity of flagellin. These observations indicate that the underlying mechanism remains incompletely understood. Here, we found that the native form of Salmonella typhimurium aa 89–96-mutant flagellin extracted from flagella retains some TLR5 recognition activity, indicating that aa 89–96 is the primary, but not the only site that imparts TLR5 activity. Additionally, this mutation impaired the production of IL-1β and IL-18. Using TLR5KO mice, we found that aa 89–96 is critical for the humoral adjuvant effect, but this effect was independent of TLR5 activation triggered by this region of flagellin. In summary, our findings suggest that aa 89–96 of flagellin is not only the crucial site responsible for TLR5 recognition, but is also important for humoral immune adjuvanticity through a TLR5-independent pathway.

Prevalence, Characteristics, and Antimicrobial Resistance Patterns of Salmonella in Retail Pork in Jiangsu Province, Eastern China

Salmonella is commonly isolated from raw pork and is a leading cause of foodborne illness. Because China has the highest rate of pork consumption and the largest number of pig breeding facilities in the world, an epidemiological analysis of Salmonella species from pork in China is warranted. In this study, pork samples (n = 1,096) were collected from 20 major free markets in four cities of Jiangsu province from August 2010 to December 2012. A total of 163 Salmonella isolates were recovered from 154 Salmonella-positive samples. Among 14 Salmonella serovars identified, Derby (47.9%) was most prevalent, followed by Typhimurium (10.4%), Meleagridis (9.2%), Anatum (8.6%), and London (6.7%). Antimicrobial sensitivity testing revealed that 134 (82.2%) of the isolates were resistant to at least one antimicrobial agent, and 41 (25.2%) were resistant to more than three antimicrobials. The highest resistance was to tetracycline (66.3% of isolates) followed by ampicillin (39.9%), trimethoprim-sulfamethoxazole (31.3%), and nalidixic acid (30.1%). Multilocus sequence typing analysis revealed 14 sequence type (ST) patterns; ST40 was the most common (77 isolates) followed by ST64 (19 isolates). Our research revealed a high prevalence of Salmonella in retail pork. Diversity among the Salmonella isolates was high in terms of serovar and genotype, and multidrug resistance was prevalent. Multilocus sequence type was generally associated with serovar and provided a reliable prediction of the most common Salmonella serovars.

Flagellin from recombinant attenuated Salmonella enterica serovar Typhimurium reveals a fundamental role in chicken innate immunity.

ABSTRACT Recombinant attenuated Salmonella vaccines have been extensively studied, with a focus on eliciting specific immune responses against foreign antigens. However, very little is known about the innate immune responses, particularly the role of flagellin, in the induction of innate immunity triggered by recombinant attenuated Salmonella in chickens. In the present report, we describe two Salmonella enterica serovar Typhimurium vaccine strains, wild-type (WT) or flagellin-deficient (flhD) Salmonella, both expressing the fusion protein (F) gene of Newcastle disease virus. We examined the bacterial load and spatiotemporal kinetics of expression of inflammatory cytokine, chemokine, and Toll-like receptor 5 (TLR5) genes in the cecum, spleen, liver, and heterophils following oral immunization of chickens with the two Salmonella strains. The flhD mutant exhibited an enhanced ability to establish systemic infection compared to the WT. In contrast, the WT strain induced higher levels of interleukin-1β (IL-1β), CXCLi2, and TLR5 mRNAs in cecum, the spleen, and the heterophils than the flhD mutant at different times postinfection. Collectively, the present data reveal a fundamental role of flagellin in the innate immune responses induced by recombinant attenuated Salmonella vaccines in chickens that should be considered for the rational design of novel vaccines for poultry.

Molecular cloning and functional analysis of duck Toll-like receptor 5.

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.

Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1–2 Co-Delivered Intranasally with Flagellin or Polyethyleneimine in Mice and Chickens

Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1–2-fliC fusion protein consisting of the globular head domain (HA1–2; amino acids 62–284) of HA fused with Salmonella typhimurium fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1–2-fliC and HA1–2-PEI showed higher HA1–2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1–2-specific interferon (IFN)-γ- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-γ, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1–2. These findings provide a basis for the development of H7N9 influenza HA1–2 mucosal subunit vaccines.

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3.

ABSTRACT Tumor necrosis factor receptor‐associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704‐bp full‐length open reading frame encodes a 567‐amino acid protein. One Ring finger, two TRAF‐type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT‐PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over‐expression of wild‐type, ▵Ring, ▵Zinc finger, and ▵Coiled coil PiTRAF3, but not a ▵MATH form, significantly increased IFN‐&bgr; promoter activity. Zinc finger and Coiled coil domains were essential for NF‐&kgr;B activation. In chicken HD11 cells, PiTRAF3 increased IFN‐&bgr; promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL‐8, and IL‐10. These data demonstrate TRAF3s innate immune function and improve understanding of its involvement in poultry antiviral defense. HighlightsPigeon TRAF3 was expressed in all tested tissues.The MATH domain is necessary for IFN‐&bgr; promoter activation.The zinc finger and coiled coil are essential for NF‐&kgr;B activation.R848 induces TRAF3 and cytokine up‐regulation in pigeon PBMCs and splenocytes.

Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway

Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway