Shaowen Li

Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose DNA in meat products

Identifying the origin of animal species in manufactured meat products is of considerable economic, religious and sanitary importance. In this study, we developed a multiplex PCR method to simultaneously detect chicken, duck and goose DNA in meat products derived from beef, pork, mutton or quail. The PCR primers were designed based on the sequence of mitochondrial genes of each avian species, and the amplicon sizes were 131, 283 and 387bp for chicken, duck and goose, respectively. The method had no cross-reaction with DNA isolated from beef, mutton, pork or quail, and generated products at a target DNA content as low as 0.05ng, or a target meat content of 1% of total meat weight. Moreover, screening of 24 commercial meat samples using this method indicated that six, two and one samples were contaminated with chicken, duck, or both, respectively, suggesting its usefulness for the simultaneous identification of chicken, duck and goose DNA in commercial meat products.

Multiple amino acid substitutions involved in enhanced pathogenicity of LPAI H9N2 in mice.

Human infection of avian influenza H9N2 virus highlighted the need to better understand the mechanism of interspecies transmission. In this study, we generated mouse-adapted influenza virus (ma01) through serial lung-to-lung passages of a wild-type H9N2 (A/chicken/Hubei/01/1999). Ma01 caused highly lethal infection in mice with severe lung pathology and extended tissue tropism. Nine amino acid substitutions of ma01 were observed in five viral genes (those for PB2, PA, NA, M1, and NS1). Of these mutations, substitutes of PB2(627), PA(349), PA(605), NA(88), and NA(356) were absent in influenza H9N2. Furthermore, the targets of wild-type virus responding to mouse microRNA mmu-mir-1940 and mmu-mir-1904 were eliminated in ma01. The mutation PB2(627) of ma01 confirmed as a key virulence determinant of influenza H5N1 was responsible for the altered recognition of mmu-mir-1904. In addition, induction of IL-1β, IL-6, TNF-α, and IFN-β was found in significantly higher levels in ma01 infected mouse peripheral blood than parental strain. These results demonstrate that multiple amino acid substitutions and avoidance of microRNA recognitions may be essential for lethal infection and high speed of virus growth can outcompete the antiviral response of infected host.

Haemophilus parasuis infection activates the NF-κB pathway in PK-15 cells through IκB degradation.

Haemophilus parasuis is the causative agent inducing a severe inflammation of the serous membranes in pigs, which contribute to the great economic losses in the pig industry in China in the recent years. In this study, it was demonstrated that H. parasuis could activate the inflammatory transcription factor nuclear factor-kappaB (NF-κB) in a bacteria time- and dose-dependent manner in PK-15 cells, and inactivated H. parasuis significantly reduced the level of NF-κB activation in PK-15 cells compared with the counterpart especially in the later stage. After H. parasuis infection, the degradation of IκBα and phosphorylation of p65 was detected in PK-15 cells. Furthermore, the subcellular localization analyzed using confocal laser microscopy showed that p65-GFP rapidly translocated to the nucleus when PK-15 cells were stimulated with H. parasuis. In addition, real-time RT-PCR showed that the key inflammatory mediators including IL-8, CCL4 and CCL5, regulated by nuclear factor-kappaB (NF-κB) were up-regulated dramatically by the infection of H. parasuis in PK-15 cells. This was the first time to report that H. parasuis infection activated the NF-κB pathway in vitro through IκB degradation.

TolC Promotes ExPEC Biofilm Formation and Curli Production in Response to Medium Osmolarity

While a high osmolarity medium activates Cpx signaling and causes CpxR to repress csgD expression, and efflux protein TolC protein plays an important role in biofilm formation in Escherichia coli, whether TolC also responds to an osmolarity change to regulate biofilm formation in extraintestinal pathogenic E. coli (ExPEC) remains unknown. In this study, we constructed ΔtolC mutant and complement ExPEC strains to investigate the role of TolC in the retention of biofilm formation and curli production capability under different osmotic conditions. The ΔtolC mutant showed significantly decreased biofilm formation and lost the ability to produce curli fimbriae compared to its parent ExPEC strain PPECC42 when cultured in M9 medium or 1/2 M9 medium of increased osmolarity with NaCl or sucrose at 28°C. However, biofilm formation and curli production levels were restored to wild-type levels in the ΔtolC mutant in 1/2 M9 medium. We propose for the first time that TolC protein is able to form biofilm even under high osmotic stress. Our findings reveal an interplay between the role of TolC in ExPEC biofilm formation and the osmolarity of the surrounding environment, thus providing guidance for the development of a treatment for ExPEC biofilm formation.

Draft Genome Sequence of Listeria monocytogenes LM201, Isolated from Foodstuff

ABSTRACT Listeria monocytogenes is a facultative intracellular foodborne pathogen that can cause listeriosis in humans and animals. L. monocytogenes LM201 was isolated from foodstuff. The draft genome sequence of strain LM201 provides the genetic basis for the application of this strain in biotechnological vaccine production.

Phenotypic and Genotypic Resistance of Salmonella Isolates from Healthy and Diseased Pigs in China During 2008-2015.

The antimicrobial resistance of Salmonella strains is rapidly increasing worldwide, which poses significant threats to animal and public health. In this study, a total of 249 porcine Salmonella isolates collected in China during 2008-2015 were examined, including 155 clinical isolates from diseased pigs and 94 nonclinical isolates from healthy pigs. Based on the minimum inhibitory concentration of seven antimicrobial agents, 96.4% of the isolates were resistant to at least one of the tested antibiotics and 81.0% of them showed multidrug resistance. The highest antimicrobial resistance was observed for tetracycline (85.9%), and the lowest was found for cefotaxime (13.3%). The isolates from diseased pigs exhibited significantly higher levels of antimicrobial resistance than those from healthy pigs. Twenty-two isolates from healthy pigs were resistant to ciprofloxacin, which may inhibit the curative effectiveness of fluoroquinolones on bacterial food-borne poisoning and infections in humans caused by contaminated food. Moreover, cefotaxime resistance of the strains isolated from diseased pigs during 2013-2015 was significantly higher compared with the strains isolated during 2008-2010. Further study showed that the correlation between phenotypic and genotypic resistance varied among the isolates from different sources, and in many cases, the presence of resistance genes was not consistent with the resistance to the corresponding antimicrobials. These results are very significant for veterinary practice and public health.

TGEV infection up-regulates FcRn expression via activation of NF-κB signaling.

It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling.

Neonatal Fc Receptor-Mediated IgG Transport Across Porcine Intestinal Epithelial Cells: Potentially Provide the Mucosal Protection

It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces.

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer

The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YMΔRS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YMΔRS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YMΔRS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YMΔRS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer.

Riemerella anatipestifer Type IX Secretion System Is Required for Virulence and Gelatinase Secretion

Riemerella anatipestifer (RA), a major causative agent of septicemia anserum exsudativa in domesticated ducklings, has a protein secretion system known as the type IX secretion system (T9SS). It is unknown whether the T9SS contributes to the virulence of RA through secretion of factors associated with pathogenesis. To answer this question, we constructed an RA mutant deficient in sprT, which encodes a core protein of the T9SS. Deletion of sprT yielded cells that failed to digest gelatin, an effect that was rescued via complementation by a plasmid encoding wild-type sprT. Complement-mediated killing was significantly increased in the deletion mutant, suggesting that proteins secreted by the T9SS are necessary for complement evasion in RA. Liquid chromatography-tandem mass spectrometry analysis revealed that RAYM_01812 and RAYM_04099 proteins containing a subtilisin-like serine protease domain and exhibiting extracellular gelatinase activity were secreted by the T9SS. Animal experiments demonstrated that the virulence of mutant strain ΔsprT strain was attenuated by 42,000-fold relative to wild-type RA-YM. Immunization with the ΔsprT protected ducks from challenge with RA-YM, suggesting that the former can be used as a live attenuated vaccine. These results indicate that the T9SS is functional in RA and contributes to its virulence by exporting key proteins. In addition, subtilisin-like serine proteases which are important virulence factors that interact with complement proteins may enable RA to evade immune surveillance in the avian innate immune system.