水禽雷氏桿菌感染菜鴨腹腔巨噬細胞之模式建立及相關基因反應探討

范正一

doi:10.6342/NTU.2015.02764

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水禽雷氏桿菌感染菜鴨腹腔巨噬細胞之模式建立及相關基因反應探討

The Model for Riemerella anatipestifer infection in Tsaiya Duck Peritoneal Macrophages and Characterization of Gene Responses in Macrophages

范正一 , Masters　　Advisor：周崇熙　　

繁體中文 DOI： 10.6342/NTU.2015.02764

菜鴨；雷氏桿菌；巨噬細胞；掃描式電子顯微鏡；即時定量聚合酶鏈鎖反應； Tsaiya Duck ； RA ； Macrophages ； SEM ； qRT-PCR

Abstract │ Reference (107) │ Altmetrics

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Riemerella anatipestifer (RA) is a major cause of disease and economic loss in farm ducks worldwide, with ducklings up to 3-4 weeks of age being the most susceptible. RA infection is typically transmitted through nasal passages or broken skin on the feet. The main lesions are fibrinous serositis, caseous salpingitis and arthritis. Twenty-one serotypes have been isolated and serotype II is the most endemic in Taiwan. Vaccines based on a single serotype of inactivated bacterin or live or cell-free culture filtrate have not provided significant cross-protection among the serotypes. Research about RA pathogenesis is scant, and information about host physiological responses in RA infection is limited. We injected 3% Sephadex G-50 into duck peritoneal cavity and harvested activated peritoneal macrophages (PMs). Next, infected 1×106 PMs by RA and MOI are approximately 10, 20 and 40 separately. PMs and lysis cell counts were assessed at 1,3 and 5h to determine the recovery rates. The overall recovery rate were approximately 0-0.45% and macrophages infected by RA under MOI=10 and 3 hours condition have recovered the most RA. We next infected macrophages by MOI = 10 and recovered cells at 3 h and 7h for further detection of genes expression. Scanning Electron micrography revealed adhesion of RA to PMs within 1 h of incubation ; significant surface modification was also evident, indicating that the interaction between RA and PMs may be a potential mechanism of activation ,then the surface of PMs’- gradually changes. We used quantitative real-time PCR (qRT-PCR) to analyze target genes expression. Our results showed that IL-1β, IL-8, MIP-1β, and TLR15 are up-regulated and TLR4, MYD88, and MAPK1 are down-regulated. These findings indicate that when PMs are infected by RA, TLR15 pathway plays an important role in defense. The mechanism of TLR15 activation may be through the RA-secreting protease. Therefore, NF-κB was activated and the downstream pro-inflammatory cytokine can recruit more immune cells to fight RA infection. In conclusion, we used an RA invasion assay and SEM photography to establish experimental procedures for following Real-time PCR tests. Eventually, we will be able to determine how macrophages function in RA infection, which will facilitate the development of adequate policy and disease control strategies to help farmers prevent RA infection.

Reference ( 107 ) 〈TOP〉

[3] 陳燕萍、李淑慧。台灣水禽雷氏桿菌血清型別之調查。行政院農業委員會家畜衛生試驗所研究報告。2008 43:35-42。
連結：
[4] 陳燕萍、李淑慧、蔡向榮。2008年至2012年台灣水禽雷氏桿菌血清型別調查。行政院農業委員會家畜衛生試驗所研究報告。2013 48:21-28。
連結：
[5] Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S. Targeted disruption of the MYD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 1998 9:143-150.
連結：
[8] Baquero-Perez B, Kuchipudi SV, Ho J, Sebastian S, Puranik A, Howard W, Brookes SM, Brown IH, Chang KC. Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection. J Virol. 2015 89:2494-506
連結：
[9] Bisgaard M. Antigenic studies on Pasteurella anatipestifer, species incertae sedis, using slide and tube agglutination. Avian Pathol. 1982 11:341-350.
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