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

Abstract

水禽雷氏桿菌 (Riemerella anatipestifer，RA)感染症為水禽重要疾病之一，好發於3-4週齡雛禽，可經由呼吸道及破損皮膚感染鴨隻，特別是透過受傷的脚蹼，可造成急性或慢性敗血症和全身性漿膜炎，其感染率幾近100%，致死率可高達75%，造成嚴重經濟損失。RA共有21種血清型，其中台灣以第2血清型為最主要。不活化菌疫苗在不同血清型間無交叉保護。RA目前對於其發病機制，特別是感染後宿主的生理反應，了解甚少。本研究使用3-5月齡褐色菜鴨 (Brown Tsaiya Duck)來進行研究，本研究以3% Sephadex G-50注入鴨隻腹腔42小時後，成功誘導活化的腹腔巨噬細胞 (Peritoneal Macrophages，PMs)平均約為5x107 cells/mL。接著以MOI（Multiplicity of infection）=10、20及40等菌量攻擊1x106 PMs，並觀察1、3或5小時等時間點後打破細胞並回收胞內細菌，回收率為0~0.45%，其中MOI=10且攻菌3小時組入侵效果最佳，回收率最高。其後以MOI=10攻菌3小時及7小時並回收細胞供後續比較基因表現量高低之用。同時，在孔盤中置入玻片讓PMs貼附後以MOI=10攻菌3小時，將玻片製作成掃描式電子顯微鏡樣品觀察，成功觀察到RA菌與PMs交互作用之影像。在即時定量反轉錄聚合酶連鎖反應的基因表現結果中，包含IL-1β、IL-8、MIP-1β、TLR15等基因表現量上升，TLR4、MYD88、MAPK1等基因的表現量呈下降，代表在面對RA菌感染時，菜鴨腹腔巨噬細胞選擇與之對抗的策略可能以TLR15受RA菌所分泌的蛋白酶刺激後所活化的訊息傳導途徑為主，並進而活化NF-κB使得下游發炎前驅物質表現量上升，藉此招募更多免疫細胞前來發炎區域抵抗RA之感染。本研究以攻菌回收率、電顯拍攝細胞外部型態證實雷氏桿菌可成功進入菜鴨腹腔巨噬細胞，並以此建立雷氏桿菌入侵菜鴨巨噬細胞試驗之操作模式，同時也期待菜鴨巨噬細胞之實驗模式及相關基因表現實驗結果，能對於畜牧場防治RA感染鴨隻或後續以菜鴨巨噬細胞為試驗模式等方面能有貢獻。

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.