Wenjie Jin

Development of an impedimetric immunosensor for the determination of 3-amino-2-oxazolidone residue in food samples

The use of furazolidone in food animals has been banned in European Union (EU) because of its carcinogenicity and mutagenicity on human health, but its continued misuse is widespread. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in food products. In this regard, a sensitive and reliable electrochemical method was presented to detect AOZ based on a novel label-free electrochemical impedimetric immunosensor to address this need. The immobilization of monoclonal antibody against AOZ (denoted as AOZ-McAb) on the gold electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies on the self-assembled monolayer (SAM). The detection of AOZ was performed by measuring the relative change in charge transfer resistance before and after AOZ and AOZ-McAb immunoreaction by electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the relative change in charge transfer resistance was proportional to the logarithmic value of AOZ concentrations in the range of 20.0 to 1.0×10(4) ng mL(-1) (r=0.9987). Moreover, the proposed immunosensor has a high selectivity to AOZ alone with no significant response to the metabolites of other nitrofuran antibiotics, such as 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SEM), and 1-aminohydantoin hydrochloride (AHD). This protocol has been applied to detect AOZ in food samples with satisfactory results.

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

ABSTRACT H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.

Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

216 avian pathogenic Escherichia coli (APEC) isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as O78, O88, and O93. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD (70.8%), iss (58.8%), and tsh (51.4%) in APEC isolates. The detection rate of high pathogenicity islands (HPI)-associated fyuA and irp2 genes were both 44.9%, with no LEE (the locus of enterocyte effacement) island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained only fimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% O78 isolates had a gene distribution patterns of fimC+iucD+irp2+fyuA+iss+colV+tsh+.

Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells

To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design.

[Construction and immunological characterization of recombinant Marek's disease virus expressing IBDV VP2 fusion protein].

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Mareks disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.

Monoclonal antibodies directed against chicken β2-microglobulin developed with a synthesized peptide.

We developed a panel of monoclonal antibodies (MAb) against chicken β2-microglobulin (chβ2M) by fusions between SP2/0 myeloma cells and spleen cells from mice immunized with a synthesized peptide corresponding to positions 91-119 of the COOH domain of chβ2M. Two of them, 6E7 and 3D1, identified as IgG1/κ, could react with chβ2M protein from avian macrophage HD11 cells and human 293T cells transfected with pcDNA3.1-chβ2M in immunofluorescence assays. Only a 12 kDa protein band of chβ2M could be detected in the HD11 and 293T/chβ2M cell lysates by Western blot analysis. Chicken β2M in serum and plasma could be found in Western blot by MAb 3D1. Moreover, MAb 3D1 also recognized the chβ2M antigen on the cell membranes in flow cytometry. Immunohistochemical staining with these MAbs revealed that chβ2M was present in chicken thymus, spleen, and bursa. These MAbs will be good tools for analyzing the mechanism of the chicken immune system.

Fosfomycin Resistance in Avian Pathogenic Escherichia coli Isolates

Abstract Fosfomycin, a broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, is very important in the clinic but many fosfomycin-resistant bacteria have been isolated from patients. In this study, the resistance mechanism of three fosfomycin-resistant avian pathogenic Escherichia coli (APEC) strains (JE1, IF7 and CD11) isolated from septicemic chickens were analyzed. The results showed that their fosfomycin-resistance mechanisms were different. An alteration in the glpT transport system was the main reason of the fosfomycin-resistance mechanisms of strain IF7. Compared with the control stain BL21, the capacity of fosfomycin-uptake was low in all these three stains (JE1>IF7>CD11). Sequence results of murA showed that there were more than 10 sites of nucleotide mutation, but only one amino acid mutation T116A showed in CD11. Real-time detection test showed that the expression level of the murA gene of the three stains was significantly increased (four times increase in strain CD11 and two times increase in strains JE1 and IF7). The transformation and recombinant test showed that the recombinant bacteria with the murA of JE1 and CD11 showed high minimal inhibitory concentration (MIC) against fosfomycin. From the results of this research, it showed that most of the fosfomycin-resistance mechanisms once showed in patient bacteria have appeared in the APEC strains and the fosfomycin-resistance mechanism of the three APEC isolates was different.