Table_1_A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum.DOCX

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.</p

Mast cells supported productive replication of influenza A viruses.

<p>P815 cells were mock-treated or infected with the three subtypes of influenza viruses at an equal MOI of 0.1 for the periods specified. Culture supernatants were collected, and total viral protein quantification and viral titers were determined using a (A) hemagglutination (HA) assay and (B) standard plaque assay. (C) Cells were homogenized in Trizol and relative viral NS1 gene quantification was determined using real time PCR. The results shown here were pooled from three independent replicates.</p

Inhibition of apoptosis suppressed the viral replication and release of pro-inflammatory cytokines and chemokines.

<p>P815 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100109#pone-0100109-g004" target="_blank">Figure 4</a>. Cell supernatants were harvested 24 h after infection. (A) Viral titers were determined using a standard plaque assay. Results shown were pooled from three independent repeats. (B) Expression levels of IL-1β, IL-6, IL-18, TNF-α, TGF-β1, IFN-γ, and MCP-1 were analyzed using ELISA. Statistically significant differences between the inhibitor-treated groups and corresponding DMSO controls are indicated by * (<i>P</i><0.05) and ** (<i>P</i><0.01). Results shown are pooled from three independent repeats. ND, not detectable.</p

Apoptosis of P815 cells was further measured at specified times after influenza A virus infection.

<p>P815 cells were treated or infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100109#pone-0100109-g001" target="_blank">Figure 1</a> and harvested at 6 h, 12 h, and 24 h post-infection. (A) Quantification of the total number of apoptotic cells (early and late-stage apoptosis) by flow cytometric analysis with Taxol as a positive control. Asterisks indicate statistically significantly higher values than among mock-treated cells (*<i>P</i><0.05 and **<i>P</i><0.01). (B) Immunoblotting analysis of the expression levels of apoptosis-associated proteins, caspase 3, caspase 8, caspase 9, Apaf-1, Bcl-2, and Bcl-xL, with β-actin as a loading control. (C) Immunoblotting analysis of the cytosolic and mitochondrial Cyt C expression after fractionation. α-Tubulin and cytochrome oxidase subunit IV (Cox IV) were used as specific markers for the cytosolic and mitochondrial fractionations, respectively. Shown here are representative results of three separate experiments.</p

Caspase inhibitors suppressed the apoptosis in P815 cells infected with influenza A virus.

<p>VAD (abbreviation of Z-VAD-fmk, pan-caspase inhibitor), IETD (abbreviation of Z-IETD-fmk, caspase-8 inhibitor), and LEHD (abbreviation of Z-LEHD-fmk, caspase-9 inhibitor) were added before and after infection with influenza A viruses, and P815 cells were harvested 24 h after infection. (A) Apoptosis was quantified using flow cytometric analysis. Asterisks indicate that the inhibitor-incubated groups were statistically significantly different from the DMSO-incubated groups, as indicated by ANOVA. (B) The expression of full-length and cleaved PARP in P815 cells was measured using Western blot analysis. Graphs show results of one representative case (out of three).</p

Apoptosis was induced in influenza A viruses infected P815 cells.

<p>P815 cells were mock-treated or infected with H1N1, H5N1, or H7N2 at an MOI of 0.1 for 12 h. (A) Transmission electron microscopy showed ultrastructure of mock-treated and virus-infected cells. (B) A TUNEL assay was used to measure apoptosis in P815 cells using DNase as a positive control. Blue indicates nuclear staining and green indicates positive TUNEL staining due to the presence of apoptosis. The results shown here are representative of three different donors.</p

Percentage survival of chickens after inoculation with SD09 or GD09-2 velogenic NDVs.

<p>Percentage survival of chickens after inoculation with SD09 or GD09-2 velogenic NDVs.</p

Histopathology on tissues from 1-week-old chickens infected with NDV GD09-2 or SD09 (H&E).

<p>B and C: amalgamation of collapsed cell and inflammatory exudates created the homogeneous and pink-staining appearance in the white pulps of spleens (black arrow); E and F: dropout and necrosis of the mucosal epithelia in the proventriculus (black arrow); H and I: dropout of epithelium and numerous inflammatory cell infiltration in the small intestine (black arrow); K and L: congestion (black arrow) or glomerulus atrophy (white arrow) in the kidneys; N and O: dropout and necrosis of mucous epithelial cells in the trachea (black arrow); Q and R: congestion and hemorrhage in the lung (black arrow); T and U: venous congestion in the cerebrum (black arrow); W and X: venous congestion in the cerebellum (black arrow); A, D, G, J, M, P, S and V: Corresponding control tissues. Scale bar = 50 µm in cerebellum or 100 µm in other tissues.</p

NDV strains and their accession numbers used for phylogenetic analysis.

<p>NDV strains and their accession numbers used for phylogenetic analysis.</p

Phylogenetic tree based on the nucleotide sequences of the fusion gene (A) (nt 1–1662) and hemagglutinin-neuraminidase gene (B) (nt 1–1713 or 1731) of NDV.

<p>The phylogenetic tree was constructed by the Neighbor-Joining method with 1000 bootstrap replicates (bootstrap values are shown on the tree).</p