Shizhong Geng

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.

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.

Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine

Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease.

An improved method to knock out the asd gene of Salmonella enterica serovar Pullorum.

An asd-deleted (Δasd) mutant of Salmonella enterica serovar Pullorum (SP) was constructed using an improved method of gene knockout by combining the π-suicide plasmid system with the Red Disruption system. The asd gene was efficiently knocked out by the recombinant suicide vector, which replaced the asd gene with the CmR gene. Based on the balanced lethal host-vector system, the phenotype of the Δasd mutant was further defined. The improved method was simpler and more effective than previously reported conventional methods.

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.

Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

Escherichia coli isolates from sick chickens in China: Changes in antimicrobial resistance between 1993 and 2013

The use of antimicrobials for the control of infectious disease has increased in recent decades. Understanding trends in antimicrobial resistance provides clues about the relationship between antimicrobial use and the emergence of resistance. We examined the resistance of 540 Escherichia coli isolates to 19 antimicrobials that represent 11 classes of antimicrobial agents. The isolates were collected from chickens between 1993 and 2013 in China. Overall, >96.7% of the isolates were resistant to at least one of the tested compounds, and 87.2% of them displayed multidrug resistance (MDR) representing five to six antimicrobial classes. A high proportion of E. coli isolates were resistant to tetracycline (90.6%), nalidixic acid (80.6%), ampicillin (77.2%), trimethoprim-sulfamethoxazole (76.9%), and streptomycin (72.8%). Only 3.0% of the isolates were resistant to nitrofurantoin, and none was resistant to meropenem. Resistance to amikacin, ampicillin, aztreonam, ceftazidime, cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, fosfomycin, levofloxacin, norfloxacin, nalidixic acid, piperacillin, and trimethoprim-sulfamethoxazole significantly increased from 1993 to 2013 (P <0.01). There was an increasing trend in MDR over the 20 year period.

Prime-Boost Immunization Using a DNA Vaccine Delivered by Attenuated Salmonella enterica Serovar Typhimurium and a Killed Vaccine Completely Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

ABSTRACT H5N1 highly pathogenic avian influenza virus (HPAIV) has posed a great threat not only for the poultry industry but also for human health. However, an effective vaccine to provide a full spectrum of protection is lacking in the poultry field. In the current study, a novel prime-boost vaccination strategy against H5N1 HPAIV was developed: chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella enterica serovar Typhimurium, and boosting with a killed vaccine followed. Chickens in the combined vaccination group but not in single vaccination and control groups were completely protected against disease following H5N1 HPAIV intranasal challenge, with no clinical signs and virus shedding. Chickens in the prime-boost group also generated significantly higher serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA titers against avian influenza virus (AIV) and higher host immune cellular responses than those from other groups before challenge. These results demonstrated that the prime-boost vaccination strategy provides an effective way to prevent and control H5N1 highly pathogenic avian influenza virus.

Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus.

A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs.