Dingren Bi

Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.

Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses

ABSTRACT An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.

Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia

Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates (P<0.05 or P<0.01). The levels of collagen type X (Col X), pro-alpha-1 collagen type I (Col I α1), collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1), carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3) and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization, our findings may provide new insights into understanding the pathogenesis of TD.

Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface using CTC as a fusion partner

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.

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.

Genome Sequence of Poultry Pathogen Riemerella anatipestifer Strain RA-YM

Riemerella anatipestifer is a Gram-negative, rod-shaped bacterium associated with epizootic infections in poultry. R. anatipestifer strain RA-YM, belonging to the serotype 1 prevalent in China, is a clinically isolated strain with high-level virulence. Here, we report the first genome sequence of this species.

Haemophilus parasuis induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors.

Glässers disease in pigs caused by Haemophilus parasuis is characterized by a severe membrane inflammation. In our previous study, we have identified activation of the transcription factor NF-κB after H. parasuis infection of porcine epithelial cells. In this study, we found that H. parasuis infection also contributed to the activation of p38/JNK MAPK pathway predominantly linked to inflammation, but not the ERK MAPK pathway associated with growth, differentiation and development. Inhibition of NF-κB, p38 and JNK but not ERK activity significantly reduced IL-8 and CCL4 expression by H. parasuis. We also found TLR1, TLR2, TLR4 and TLR6 were required for NF-κB, p38 and JNK MAPK activation. Furthermore, MyD88 and TRIF signaling cascades were essential for H. parasuis-induced NF-κB activation. These results provided new insights into the molecular pathways underlying the inflammatory response induced by H. parasuis.

Screening of differentially expressed genes in the growth plate of broiler chickens with Tibial Dyschondroplasia by microarray analysis

BackgroundTibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 days and determined DEGs with chicken Affymetrix GeneChip assays. The identified DEGs were verified by quantitative polymerase chain reaction (qPCR) assays.ResultsWe identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes (P < 0.05) at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-β signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6. We validated seven DEGs by qPCR.ConclusionsOur findings demonstrate previously unrecognized changes in gene transcription associated with early stage TD. The DEGs we identified by microarray analysis will be used in future studies to clarify the molecular pathogenic mechanisms of TD. From these findings, potential pathways involved in early stage TD warrant further investigation.

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.