Zhiming Pan

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.

Prevalence of qnr, aac(6′)-Ib-cr, qepA, and oqxAB in Escherichia coli Isolates from Humans, Animals, and the Environment

ABSTRACT qnr, aac(6′)-Ib-cr, qepA, and oqxAB genes were detected in 5.7%, 4.9%, 2.6%, and 20.2% of 1,022 Escherichia coli isolates from humans, animals, and the environment, respectively, collected between 1993 and 2010 in China. The prevalence of oqxAB in porcine isolates (51.0%) was significantly higher than that in other isolates. This is the first report of oqxAB-positive isolates from ducks and geese and as early as 1994 from chickens.

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.

Phenotypic characteristics and genotypic correlation between Salmonella isolates from a slaughterhouse and retail markets in Yangzhou, China.

An epidemiological investigation of Salmonella spp. in pig and pork samples from one slaughterhouse and its downstream retail markets in Yangzhou, Jiangsu Province, China, was conducted from October 2013 to March 2014. A total of 71.8% (155/216) and 70.9% (78/110), respectively, of the slaughterhouse and retail market samples were recovered positive for Salmonella. All Salmonella isolates were characterized using serotyping, antimicrobial resistance detection, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Seven serotypes were shared by isolates from the two sources, with the most common serotypes being Salmonella Derby, Typhimurium, and Uganda. Antimicrobial sensitivity testing revealed that the highest antimicrobial resistance rate was against tetracycline (49.7% and 37.2% in isolates from the slaughterhouse and retail market, respectively) with many multidrug-resistant (MDR) isolates in both sources. MLST analysis showed that eight sequence type (ST) patterns were shared, and ST40 occupied an absolute superiority among isolates from both sources. PFGE permitted the resolution of XbaI macrorestriction fragments of the selected 31 Salmonella Derby and 19 Salmonella Typhimurium into 30 and 10 distinct pulsotypes, displaying the high similarity between the isolates from the two sources. Our findings indicated that Salmonella isolates from a slaughterhouse and its downstream retail markets were phenotypically and genetically homologous. Additionally, Salmonella may propagate along the slaughter line and pork production chain from the slaughterhouse to retail markets.

Changes in antimicrobial resistance among Salmonella enterica subspecies enterica serovar Pullorum isolates in China from 1962 to 2007.

There are few data available for the trends of antimicrobial resistance of Salmonella enterica subspecies enterica serovar Pullorum (S. Pullorum) in China and other parts of the world. Thus, the objective of this study was to evaluate the changes in antimicrobial resistance of S. Pullorum isolated from diseased chickens in China from 1962 to 2007. A total of 450 S. Pullorum isolates were tested for their susceptibility to 17 antimicrobials in a disk diffusion method. 39-95% of the isolates displayed a high level of resistance, particularly against ampicillin, carbenicillin, streptomycin, tetracycline, trimethoprim and sulfafurazole. Isolates exhibited increased resistance to carbenicillin, spectinomycin, trimethoprim, trimethoprim/sulfamethoxazole and nalidixic acid during the study period. Moreover, 56.2% of the isolates exhibited multiple drug resistance (MDR; resistance> or =4 antimicrobials) and showed an increasing trend between 1970-1979 and 2000-2007. Therefore, the results suggest that certain measures, including continued surveillance of antimicrobial resistance and the rational use of antimicrobials, are necessary and important in order to control the rapid increase in antimicrobial resistance in S. Pullorum.

Oral and nasal DNA vaccines delivered by attenuated Salmonella enterica serovar Typhimurium induce a protective immune response against infectious bronchitis in chickens.

ABSTRACT Several studies have reported that intramuscular injection of DNA vaccines against infectious bronchitis virus (IBV) induces protective immune responses. In the present study, we developed oral and nasal DNA vaccines that carried the S1 gene and N gene of IBV delivered by attenuated Salmonella enterica serovar Typhimurium strains SL/pV-S1 and SL/pV-N, respectively. The safety and stability of recombinant Salmonella vaccine were evaluated. Following oral and nasal administration to chickens, the serum and mucosal samples were collected and antibodies against IBV were measured. Chickens were then challenged with IBV strain M41 by the nasal-ocular route 3 weeks after boosting. The results showed that oral and nasal immunization with coadministered SL/pV-S1 and SL/pV-N elicited significant IBV-specific humoral and mucosal immune responses and conferred protective efficacy against IBV challenge higher than that in chickens immunized only with SL/pV-S1. The current study shows that novel DNA vaccines delivered by attenuated S. Typhimurium may be promising candidates for the prevention of infectious bronchitis (IB).These vaccines are efficacious, easily produced economically, and able to be delivered orally and nasally rather than injected. Coadministration of SL/pV-S1 and SL/pV-N may represent an effective mucosal vaccination regimen.