Huhu Wang

Effect of intranasal immunization with inactivated avian influenza virus on local and systemic immune responses in ducks

To evaluate the effects of co-administration of inactivated avian influenza H9N2 virus and adjuvants in waterfowls, 10-d-old ducks were immunized intranasally with inactivated avian influenza virus (IAIV) combined with CpG DNA and sodium cholate. Immunoglobulin A and IgG antibody levels in throat and tracheal tissues increased significantly, as did specific IgA and IgG antibody levels in the serum after intranasal immunization with IAIV combined with CpG DNA and sodium cholate, compared with immunization with IAIV only. Furthermore, enhanced hemagglutination inhibition titers were also detected in serum samples taken between the third and seventh weeks after immunization with IAIV and both adjuvants compared with IAIV alone. The expression of IL-2 and IL-6 in tracheal and lung tissues increased significantly in the early period after booster immunization. However, the enhancement induced by a single adjuvant was insignificant, and no significant change was detected in the antibody titers or cytokine levels between the ducks that received IAIV alone or saline. In the viral challenge study, prior administration of both CpG DNA and sodium cholate with IAIV reduced the viral titers in the oropharynx and cloaca swabs. Our study suggests that the combination of CpG DNA and sodium cholate could be beneficial to immunization with inactivated H9N2 virus by enhancing the local and systemic immune responses.

Development of interspecific competition models for the growth of Listeria monocytogenes and Lactobacillus on vacuum-packaged chilled pork by quantitative real-time PCR

The competitive growth of Listeria monocytogenes and Lactobacillus on vacuum-packaged chilled pork (VPCP) was studied by the use of Real-time PCR assay and selective culture counting method, where the Jameson-effect model and modified Lotka-Volterra model were chosen for the fitting of growth data. Real-time PCR assay was successfully applied to model the growth of the L.monocytogenes and Lactobacillus respectively on VPCP. The Jameson effect was occurred between L.monocytogenes and Lactobacillus when they co-existed in the same culture, and Lactobacillus clearly induced an early stationary phase of L. monocytogenes. The Jameson-effect model could be used to fit the growth curves of L. monocytogenes and Lactobacillus on VPCP. Thus the modified Lotka-Volterra competition model could be used to evaluate the interaction of two bacterial populations during the growth process, and found that the influence from Lactobacillus on L. monocytogenes was much higher than L. monocytogenes on Lactobacillus through the interaction coefficients. In conclusion, bacterial interactions need to be considered in microbial predictive models, which will provide a more practical assessment in predictive microbiology, and the model for the prediction based on the Real-time PCR assay can be used in future studies, especially for research of interaction models.

Impact of High Hydrostatic Pressure Treatment on Microbial Communities in Chinese Water-Boiled Salted Duck

The objective of this study was to determine the impact of high hydrostatic pressure (HHP) treatment on microbial communities in Chinese water-boiled salted duck (CWBSD) and the synergistic effect of HHP and mild heat treatment. In this work, the bacterial diversity was evaluated by using both a culture-dependent method and denaturing gradient gel electrophoresis. The total aerobic bacterial counts in pressure-treated samples were significantly lower than those in controls, which indicated that HHP could extend the shelf life of CWBSD. Weissella hellenica and Enterobacteriaceae, the predominant bacteria found in CWBSD, were not detected after HHP treatment. On the other hand, Staphylococcus spp. and Bacillus spp. became the predominant bacteria in HHP-treated samples. Moreover, the inhibitory effect was greater at 400 MPa than at 200 MPa combined with mild heat at 40°C. This study investigated the diversity of bacteria in HHP-treated CWBSD, and the information derived from this research may help to understand the bacterial ecology and develop effective HHP treatments to extend the shelf life of CWBSD.

Identification and characterization of the proteins in broth of stewed traditional Chinese yellow-feathered chickens

&NA; Soups and broths are popular in the world due to their nutrition and flavor, and flavor compounds tend to be bound by the proteins in the soups and broth, influencing the flavor perception. Thus, identification of the major proteins in meat‐based broth may present a basis for understanding protein adsorption of flavor compounds. The present study aimed to identify the major proteins in traditional Chinese chicken broth and to describe the structural changes of proteins during stewing (1, 2, or 3 h). As stewing time increased, protein content in the broth significantly increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) indicated that the macro‐molecule proteins (>10 kDa) in the broth were mainly gelatin and actin and that the micro‐molecule proteins fractions (<10 kDa) increased substantially. The gelatin had an ordered structure even after 3 h of stewing, as seen by circular dichroism (CD) spectroscopy. The presence of reactive sulfhydryl groups increased remarkably with stewing time. The surface hydrophobicity of the proteins significantly increased within 2 h then deceased slightly after 3 h. The intermolecular crosslinks, as indicated by dispersion index, increased remarkably, consistent with the result of atomic force microscopy (AFM), which together suggested that protein aggregation increased during stewing. These findings suggested that gelatin was the structural protein in the broth system and that intermolecular crosslinks functioned to maintain the broth system.

Characterization of Extracellular Polymeric Substances Produced by Pseudomonas fragi Under Air and Modified Atmosphere Packaging

Extracellular polymeric substances (EPS) play an important role in bacterial biochemical properties. The characteristics of EPS from 2 strains of Pseudomonas fragi cultured in meat aerobically (control) and in modified atmosphere packaging (MAP) were studied. The amount and components of EPS, the surface properties, and the effect on biofilm formation of several spoilage organisms were evaluated. The results showed that MAP inhibited the growth of the P. fragi strains. Compared with the control, more loose and less bound EPS (containing protein and carbohydrate) were produced by P. fragi in MAP samples. MAP also caused increased cell autoaggregation and surface hydrophobicity. After the removal of the EPS, the surface property changes were strain-dependent, suggesting that membrane compositions were also changed. In addition, the EPS displayed significant antibiofilm activity on Pseudomonas fluorescens and Serratia liquefaciens. In conclusion, P. fragi strains not only modified the amount, components, and surface properties of EPS but also changed the cell membrane compositions to adapt to MAP stress. Moreover, EPS may play an important role in microbial community competitions.

Preliminary Transcriptome Analysis of Mature Biofilm and Planktonic Cells of Salmonella Enteritidis Exposure to Acid Stress

Salmonella has emerged as a well-recognized food-borne pathogen, with many strains able to form biofilms and thus cause cross-contamination in food processing environments where acid-based disinfectants are widely encountered. In the present study, RNA sequencing was employed to establish complete transcriptome profiles of Salmonella Enteritidis in the forms of planktonic and biofilm-associated cells cultured in Tryptic Soytone Broth (TSB) and acidic TSB (aTSB). The gene expression patterns of S. Enteritidis significantly differed between biofilm-associated and planktonic cells cultivated under the same conditions. The assembled transcriptome of S. Enteritidis in this study contained 5,442 assembled transcripts, including 3,877 differentially expressed genes (DEGs) identified in biofilm and planktonic cells. These DEGs were enriched in terms such as regulation of biological process, metabolic process, macromolecular complex, binding and transferase activity, which may play crucial roles in the biofilm formation of S. Enteritidis cultivated in aTSB. Three significant pathways were observed to be enriched under acidic conditions: bacterial chemotaxis, porphyrin-chlorophyll metabolism and sulfur metabolism. In addition, 15 differentially expressed novel non-coding small RNAs (sRNAs) were identified, and only one was found to be up-regulated in mature biofilms. This preliminary study of the S. Enteritidis transcriptome serves as a basis for future investigations examining the complex network systems that regulate Salmonella biofilm in acidic environments, which provide information on biofilm formation and acid stress interaction that may facilitate the development of novel disinfection procedures in the food processing industry.

High-pressure effects on the molecular aggregation and physicochemical properties of myosin in relation to heat gelation

Myosin was extracted from the M. psoas muscle of rabbits, and dissolved in 0.6M KCl buffer (pH6.5). Effects of high-pressure (HP, 100 to 300MPa, 9min, 25°C) treatment on myosin solubility, molecular traits (molecular weight and morphology), flow behavior and strength of heat-induced myosin gels were studied and compared with the untreated controls. Myosin subjected to 200MPa HP treatment had lower solubility than samples treated at other pressures (P<0.05). Molecular dimerization and morphological swelling of myosin was observed using gel-permeation chromatography and atomic-force microscopy. Additionally, the shear-thinning behavior of myosin solutions (10mg/mL) was improved by HP treatment (≥200MPa), and a positive trend in gel-strength enhancement was inferred. It is postulated that significant morphological changes in myosin accounted for changes in its functional properties, by the influence of HP treatment on protein-protein and/or protein-water interactions. There is a relationship between molecular morphology and the coalescing behavior of myosin, since significant changes of both attributes were observed at pressures ≥200MPa.

Physicochemical properties of Pseudomonas fragi isolates response to modified atmosphere packaging

Pseudomonas spp., in particular Pseudomonas fragi, are dominant in aerobically stored chilled meats. This work isolated P. fragi isolates from spoiled chicken and investigated the effect of modified atmosphere packaging (MAP, CO2/N2 30%/70%) on physicochemical properties of P. fragi isolates compared to the corresponding controls (air). A total of six P. fragi isolates were isolated from aerobically stored spoiled chicken. MAP inhibited the growth of the isolates primarily in the exponential phase without inducing cell death and weakened the isolates ability to form biofilms. Isolates 2 and 25, which readily form biofilms, exhibited improved stationary-phase growth in MAP compared to the control packaging. Changes in the surface properties, including reduced motility, increased aggregation and hydrophobicity, were also induced by MAP. The surface property results were then confirmed via scanning electron microscopy. In addition, significant differences were observed in cell phenotypic characterization between individual isolates tested, which indicated that the responses to MAP exposure were strain-dependent. These cell responses allowed P. fragi to adapt to MAP. The study provides practical information regarding microbial responses to MAP stresses, which will help further understanding the bacteriostatic mechanism of MAP.

Full-length genome sequencing analysis of avian infectious bronchitis virus isolate associated with nephropathogenic infection

Abstract Infectious bronchitis virus (IBV) produces infectious bronchitis (IB) disease in poultry worldwide. In spite of proper vaccinations against the IBV, new IBV strains are continually emerging worldwide. In this study, a new highly virulent nephropathogenic IBV strain named CK/CH/XDC-2/2013 was identified from a vaccinated flock with clinical signs of IB in the Jiangsu province of China. The full-length genome sequence of the isolate was 27,714 nucleotides long, and the genome was organized similarly to classical IBV strains. Minimum divergence, phylogenetic analysis, and distance matrix of the genome showed that the CK/CH/XDC-2/2013 isolate had the highest similarity to the IBV BJ strain. The spike glycoprotein (S) gene had the greatest similarity to the nephropathogenic BJ strain and showed an 8 amino acid insertion (YSNGNSDV) at 73 to 80 sites and 3 amino acid deletion at sites 126 to 128 compared to the IBV vaccine strains. A recombination analysis of the S gene showed that the new isolate evolved from the IBV BJ strain and the KM91 vaccine strain. An animal challenge experiment showed a mortality of 60 to 80% in early-age chickens by different inoculation routes. Pathological examinations of the kidneys revealed inflammation, distention with uric acid deposits, and tubular degeneration. It indicated that the CK/CH/XDC-2/2013 isolate has robust kidney tissue tropism, and new nephropathogenic IBV strains are continuously evolving in China.

Response of Formed-Biofilm of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter freundii to Chlorite-Based Disinfectants: Response of formed-biofilm…

Bacterial biofilms formed on equipment surfaces are potential sources of cross-contamination and can be responsible for the spread of bacteria involved in food spoilage, such as some Enterobacteriaceae family members. In this study, the effect of chlorite-based disinfectants, including sodium hypochlorite (SH), chlorine dioxide (CD), strongly acidic electrolyzed water (StAEW), and neutral electrolyzed water (NEW), on inactivation of mono-biofilms of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter freundii was evaluated separately. All the strains were enumerated by the viable plate-count method after disinfection for 30 min. A comparison of the surviving cells after disinfection indicated that E. cloacae biofilms were more resistant to disinfectants than the biofilms of the other two strains, and treatment with all the disinfectants improved sanitizing. SH (200 mg/L) was the most effective in the reduction of cell number in the biofilms of all strains. Considering the safety of use and environmental protection, electrolyzed oxidizing water, especially StAEW, was a good suggestion for the inactivation of cells in K. oxytoca or C. freundii biofilms. These results suggest that the cells in biofilm of E. cloacae, K. oxytoca, and C. freundii were highly sensitive to chlorite-based disinfectants and provide insights into the efficacy of disinfectants in killing bacteria. PRACTICAL APPLICATION The Enterobacteriaceae biofilms formed on equipment surfaces, which can cause cross-contamination and food spoilage, are greatly challenging bacterial contaminants of food products. Electrolyzed oxidizing water is a novel, environmentally friendly disinfectant that can effectively treat Enterobacteriaceae biofilms. The results of this study may be used to design effective measures to disinfect biofilms on equipment contact surfaces.