Jinlin Huang

Intranasal Immunization with Chitosan/pCAGGS-flaA Nanoparticles Inhibits Campylobacter jejuni in a White Leghorn Model

Campylobacter jejuni is the most common zoonotic bacterium associated with human diarrhea, and chickens are considered to be one of the most important sources for human infection, with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce specific immune responses. The chitosan used for intranasal administration is a natural mucus absorption enhancer, which results in transgenic DNA expression in chicken nasopharynx. Chickens immunized with chitosan-DNA nanoparticles, which carried a gene for the major structural protein FlaA, produced significantly increased levels of serum anti-Campylobacter jejuni IgG and intestinal mucosal antibody (IgA), compared to those treated with chitosan-DNA (pCAGGS). Chitosan-pCAGGS-flaA intranasal immunization induced reductions of bacterial expellation by 2-3 log10 and 2 log10 in large intestine and cecum of chickens, respectively, when administered with the isolated C. jejuni strain. This study demonstrated that intranasal delivery of chitosan-DNA vaccine successfully induced effective immune response and might be a promising vaccine candidate against C. jejuni infection.

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 degrees C to 35 degrees C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R2 value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (mu) was modeled by square root type equation, and the relationship between mu and lag time (lambda) was described by a rule of mu x lambda = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (degrees C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R2), bias factor (Bf) and accuracy factor (Af), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R2, Bf and Af values were 0.958, 1.019 and 1.035, respectively.

Serodiversity, Pandemic O3:K6 Clone, Molecular Typing, and Antibiotic Susceptibility of Foodborne and Clinical Vibrio parahaemolyticus Isolates in Jiangsu, China

Vibrio parahaemolyticus is a major foodborne pathogen in China, Japan, and other Asian countries. In this study, we collected 437 strains of V. parahaemolyticus and investigated their serotypes, distribution of virulence genes, and presence of pandemic O3:K6 clone strains. A total of 327 strains were isolated from food and 110 strains were isolated from active surveillance hospitals or food outbreaks during 2005 to 2008. Presence of the tdh and trh genes is the key characteristic of virulent strains. Positive for both the tdh gene and group-specific polymerase chain reaction is the key characteristic of pandemic strains. A total of 9 O serogroups and 62 serovars were identified in all strains. Nine O serogroups and 56 serovars existed in 327 foodborne strains, and 6 O serogroups and 20 serovars existed in 110 clinical strains. Among the 327 food isolates, 6 isolates belonged to the pandemic clone with the orf8 gene (1 isolate was O1:KUT (untyped) and 5 isolates were O3:K6) and 4 isolates carried the trh gene (2 isolates belonged to O1:KUT and 2 isolates belonged to O5:KUT and O5:K17). Seventy-nine percent of the clinical isolates were pandemic strains, 9.4% of which lacked the orf8 gene. O3:K6 was the main serovar of the pandemic strains accounting for 83.5% of the clinical pandemic strains. Pandemic clonal serovars included O3:K6, O1:KUT, O1:K25, O1:K26, and O4:K68, and the newly emerging serovars O1:K36, O3:K25, and O3:K68 identified in the current study. O3:K6 was the dominant serovar in pandemic strains. All pandemic isolates had identical arbitrarily primed polymerase chain reaction fragment patterns, but did not share similar antibiotic sensitivity patterns. These results suggest that high serodiversity of V. parahaemolyticus was present in foodborne strains. Pathogenic isolates, especially pandemic isolates, were present in high-priced iced seafood and became the potential risk factor in food.

Distribution of Genes Encoding Four Pathogenicity Islands (VPaIs), T6SS, Biofilm, and Type I Pilus in Food and Clinical Strains of Vibrio parahaemolyticus in China

Vibrio parahaemolyticus is a major cause of foodborne gastroenteritis in China, Japan, and other countries. The pandemic O3:K6 clone, which harbors thermostable direct hemolysin [tdh] gene and toxRS/new gene, is mainly responsible for the foodborne outbreaks after 1995. Previous studies showed that genes in the pathogenicity island-1 (VPaI-1) and VPaI-5 are harbored only by pandemic strains, whereas genes in VPaI-7 and type III secretion system 2 are closely associated with tdh-positive strains of V. parahaemolyticus. In this study, we examined the distribution of genes encoding VPaI-2, VPaI-3, VPaI-4, VPaI-6, type VI secretion systems (T6SS), biofilm, and type I pilus in 71 food and 116 clinical strains of V. parahaemolyticus. The results showed that most of the pandemic strains of V. parahaemolyticus harbored the complete genes of VPaI-2, T6SS, and type I pilus. In contrast, most of the pathogenic strains (harboring tdh gene or TDH-related hemolysin [trh] gene) and nonpathogenic strains (harboring neither tdh gene nor trh gene) contained partial genes of VPaI-2, T6SS, and type I pilus. Genes of VPaI-4 were exclusively present in the pandemic strains. Genes of VPaI-3 were present in most of the pandemic strains and a small percentage of nonpathogenic strains, mainly O3:K6 strains. VPaI-6 and biofilm-associated genes were harbored by almost all the strains, irrespective of their pandemic, pathogenic, or nonpathogenic traits.

Epidemiological surveillance of Campylobacter jejuni in chicken, dairy cattle and diarrhoea patients.

Campylobacter jejuni of different subtypes were identified in chicken, dairy cattle and diarrhoea patients in China from 2005 to 2006, using multiplex PCR and RFLP. The results indicated that, of the three types of samples, C. jejuni was most frequently detected in poultry of the three types of samples, with an average isolation rate of up to 18.61% and a flock contamination rate of 86.67%. The average incidence of C. jejuni in overall cattle and environmental samples, milk cows, heifers and diarrhoea patients was 7.77, 5.02, 8.70 and 4.84%, respectively. A higher prevalence was detected in outpatients than ward patients (P<0.01), and in patients aged <7 years than in older patients (P<0.01). The 265 isolates of C. jejuni were classified into 20 distinct types by PCR-RFLP analysis of the flaA gene, with the genotype distribution in humans overlapping that in poultry and cattle. This suggests that certain C. jejuni strains circulate between humans and domestic animals such as cattle and poultry.

Priming with a DNA vaccine delivered by attenuated Salmonella typhimurium and boosting with a killed vaccine confers protection of chickens against infection with the H9 subtype of avian influenza virus.

Control of the circulation of H9 low-pathogenic avian influenza virus (LPAIV) is a major concern for both animal and public health. To improve vaccine efficacy against H9 LPAIV, we have utilized a novel prime-boost vaccination strategy. Specific-pathogen free (SPF) chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella typhimurium, followed by boosting with a killed avian influenza (AI) vaccine. Chickens in this combined vaccination group were completely protected against both oropharyngeal and cloacal virus shedding after intranasal challenge with H9N2 AIV, while viruses were detected from these sites in other vaccination groups. Prior to challenge, chickens in the prime-boost group also had higher (P<0.05) serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA ELISA titers against AIV, and higher lymphoproliferation stimulation indices than those from other groups. Thus, we have demonstrated the efficacy of a novel prime-boost vaccination strategy against H9N2 avian influenza virus, which could be also applied for the development of vaccines against other mucosally infectious pathogens.

Origin of Vibrio parahaemolyticus O3:K6 pandemic clone.

O3:K6 pandemic clone of Vibrio parahaemolyticus has caused outbreaks in coastal countries since 1996. Mutilocus sequence typing (MLST) is an important tool to trace the source and analysis the evolution of bacteria. Based on MLST, the first pandemic clonal complex (CC) of V. parahaemolyticus has been confirmed. In this study, 57 pandemic strains, 27 pathogenic strains (tdh or trh positive) and 36 nonpathogenic strains isolated from China were analyzed with MLST. Forty-seven unique sequence types, one clonal complex (CC) and one doublet (D) were identified by eBURST and Mega4 analyses. CC corresponded to not only the known O3:K6 pandemic clone (including ST-3, ST-192, ST-227) but nonpathogenic clone (including ST-3, S-T2, ST-196, ST-220, ST-226). ST-3 was the founder of the complex. STs of the isolates were not inevitably associated with the presence or number of the accessory genes or the serotypes of the isolates. The ancestor strain of O3:K6 pandemic clone was originated from an environmental nonpathogenic O3:K6, ST-3 strain. The pandemic O3:K6 clone was developed from this strain in approximately 1996 by laterally transferring large fragments of genes including systematic functional genes and genomic islands.

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.

Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th1-type immune response and CTL effects following intranasal immunization.

The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses. Thus, we constructed an attenuated Salmonella strain SL5928(fliC/esat) expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi. The chimeric flagellin functioned normally, as demonstrated using a flagella swarming assay and electron microscopy. To analyze the effects of chimeric flagellin, the cell-mediated immune response and cytotoxic T lymphocyte (CTL) effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat). The results showed that SL5928(fliC/esat) intranasal immunization can strongly elicit an ESAT-6-specific T helper (Th) 1-type immune response in mucosal lymphoid tissues, such as nasopharynx-associated lymph nodes, lung and Peyers patches, and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes. Furthermore, intranasal immunization of SL5928(fliC/esat) produced efficient CTL effects, as demonstrated using a 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Thus, our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization. Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

BACKGROUND Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.