| H9N2 subtype avian influenza virus(H9N2 AIV) is a low pathogenic avian influenza virus with the mild clinical symptoms, and it causes secondary infection of bacterium or some virus easily, which causing serious economic losses to livestock in China. Broilers infected with H9N2 AIV causing immune suppression, and if suffering secondary bacterial infection, it could produce inflammation in the gastrointestinal tract.However, the impact on the intestinal flora of broilers infected with H9N2 AIV is still unknown. This study investigated changes of the structure of intestinal flora and intestinal tight junctions on broilers infected with H9N2 AIV by Illumina the Hiseq 2000 sequencing technology and Real-time Quantitative polymerase chain reaction(q PCR) technology,providing theoretical guidance for the H9N2 AIV secondary infection prevention and control, and provide a new theoretical basis for the further development of anti-influenza virus probiotics.In this study, the strain of H9N2 AIV, named HN/SH01/15, was isolated from a chicken farm in Henan Province in January, 2015, and its titer was 106.13 EID50 /0.1 m L.The result of HA gene phylogenetic tree analysis showed that this isolate belongs to the H9.4.2.1sub-lineage viruses, and the homology with domestic vaccine strains SH/F/98, GD/SS/94 and SD/6/96 is 89.4%, 90.4% and 89.8%.This study used Illumina the Hiseq 2000 sequencing technology to obtain the 16 S r DNA of the ileal and ceaca mucosa microflora of broilers infected the SH01 isolates, to assess the influence of intestinal flora in broiler infected with H9N2 AIV. The study showed the following consequences: 1) Depth analysis of the sample showed the depth of the intestinal flora sequence substantially cover all species in the sample to meet the test needs analysis;2) Both the infection group and the mock group had specific Operational Taxonomic Unit(OTU); 3) The composition and relative abundance of intestinal flora in the infection group were changed. The flora imbalance appeared at 3 days post-infection on ileal mucosa, and the most obvious was at 5 days post-infection. However, on the cecal mucosa, the imbalance was appear more obvious at 3 days post-infection and 5 days post-infection, and the cecal mucosa flora of boilers infected with H9N2 AIV existed Sutterella but Phascolarctobacterium.In this study, the quantity of Escherichia at ileal and cecal mucosa flora in boilers infecting with H9N2 AIV was detected by q PCR to further verify the result of 16 S r DNA sequencing metagenomics. Compared with the mock group, at 5 days post-infection, Escherichia of the infection group increased significantly(p<0.05) in the ileal mucosa, increase in cecal mucosa; at day 12 post-infection, Escherichia the infection group decreased significantly(p<0.01) in the ileal mucosa, reduceing in cecal mucosa. Its trends consistented with Illumina met agenomic sequencing.This study explored the effect of the ileal and cecal mucosal tight junction in boiler infected with H9N2 AIV by relative fluorescence quantitative PCR. The results showed that at day 5post-infection, the ileal intestinal villus length decreased significantly(p<0.05); Cecal mucosa structural protein ZO-1 and Occludin decreased very significantly(p<0.01); at day12 post-infection, ileal crypt reduced significantly(p<0.05), the ratro of villus length and crypt depth was significantly reduced(p<0.05), Occludin m RNA expression quantity decreased significantly(p<0.05); cecal mucosa Occludin m RNA expression quantity decreased very significantly(p<0.01); After H9N2 AIV infected biolers, the mucosal tight junction reduced,and increased the intestinal mucosa permeability.This study indicated that H9N2 AIV infected boilers could cause intestinal flora disorder,and the infection caused intestinal mucosal tight junction reduced, which increase the chance of secondary infection. |
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