Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13921
標題: Riemerella anatipestifer 菌苗之研製
The Development of Riemerella anatipestifer Bacterin
作者: 黃培峻
Huang, Pei-Jun
關鍵字: Riemerella anatipestifer
疫苗
出版社: 獸醫學系
摘要: Riemerella anatipestifer為新鴨病或稱傳染性漿膜炎、鵝傳染性感冒之病原,造成鴨隻失重、生長遲緩及不合格屠體的增加,嚴重爆發可導致高死亡率,造成養鴨業者經濟損失。因此為防制此病對水禽類的危害,疫苗的開發實為一必然之趨勢。故利用Crasta 等人(2002)所發表之cam基因,以pET表現系統表現出Cam重組蛋白,及在缺鐵環境下培養RA菌後取其外膜蛋白製成疫苗,共製作全菌菌苗、菌量減半菌苗、搭配Cam重組蛋白或外膜蛋白之菌量減半菌苗,以及僅具Cam重組蛋白或外膜蛋白之疫苗,此外由於近期研究添加Interleukin於疫苗中可促進動物之免疫反應,故製作加入IL-18之菌量減半全菌菌苗及加入三類蛋白(Cam重組蛋白、缺鐵環境下培養RA菌之外膜蛋白與IL-18)之菌量減半菌苗,總計共8種疫苗進行試驗。所有疫苗皆與油質佐劑以3:7比例混合。各疫苗製備後進行動物實驗,於雲林縣某孵化場購入土番鴨,共分A-I 9組,各組24隻,在一日齡時免疫不同疫苗,14日齡時於頸部皮下以RA野外分離菌株攻擊,依攻擊細菌菌量再將每組分4小組(攻擊菌量各為3.5×109、3.5×108、3.5×107、3.5×106 CFU/隻),以計算各組之平均致死劑量(LD50),攻擊後連續觀察14日紀錄各組死亡與發病情形。於鴨隻4週齡時將未死鴨隻犧牲進行剖檢,紀錄臟器病變,計算病變指數。實驗期間自一日齡起每週秤重。分析各項數據後,證實僅免疫Cam重組蛋白或缺鐵環境下培養的RA菌之外膜蛋白並無法提供足夠之保護力,而於菌量減半菌苗中加入各類型蛋白後,在增重、死亡率與剖檢病變等方面則可獲得相當的改善。
Riemerella anatipestifer is the pathogen of new duck diseases. It is also known as infectious serositis and goose influenza. Riemerella anatipestifer infection accounts for major economic losses to industry through death, weight loss, and condemnation. In order to control this infection, it is necessary to develop an effective vaccine. According to cam gene sequence(Crasta et al., 2002), we produced the recombinant protein Cam by use the pET System. Besides this, we cultured the Riemerella anatipestifer in an iron lacked environment, and then extracted the outer membrane protein. Furthermore, because of the recent studies showed that use the interleukin as an adjuvant can improve cell mediate immune responses. So we prepared eight types of vaccine: bacterin contained approximately 109 CFU of RA/ml; bacterin contained 5×108 CFU of RA/ml; bacterin contained 5×108 CFU of RA and 100μg Cam recombinant protein/ml; bacterin contained 5×108 CFU of RA and 0.125 ml OMP/ml; bacterin contained 5×108 CFU of RA and 2 μg IL-18/ml; bacterin contained 5×108 CFU of RA, 100 μg Cam recombinant protein, 0.125ml OMP, and 2 μg IL-18/ml; 100 μg Cam recombinant protein/ml; 0.125ml OMP/ml. The experiment was divided into A-I 9 groups, and each group immuned different vaccine individually. All vaccines were injected subcutaneously in the neck of day-old mule ducks. Ducklings were challenged subcutaneously in the neck at 2 weeks of age. According to challenged dose, each group divided 4 subgroups(3.5×109 CFU, 3.5×108 CFU, 3.5×107 CFU, and 3.5×106 CFU /duck), in order to determined each groups' LD50.Mortality was recorded for a period of 14 days after challenge. And then killed surviving ducks, recorded gross lesion. The result of experiment prove that only used Cam recombinant protein or OMP(at iron lacked environment)can't provide enough protection. And add those kinds of proteins in bacterin(contained 5×108 CFU of RA/ml)all can get quite improvement.
URI: http://hdl.handle.net/11455/13921
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