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標題: Development of Subunit Vaccine against Chicken Infectious Coryza
作者: Yi-Ru Wu
引用: 1.呂榮修。傳染性可立查病。禽病診斷彩色圖譜。中華民國養雞協會。台北市。中華民國。255-263,1995。 2.Blackall PJ. Infectious Coryza: Overview of the Disease and New Diagnostic Options. Clinical Microbiology Review. 12: 627–632, 1999. 3.Blackall PJ. Vaccines against infectious coryza. World's Poultry Sciences Journal51:17–26, 1995. 4.Blackall PJ, Eaves LE, Aus G. Serotyping of Haemophilus paragallinarum by the Page scheme: comparison of the use of agglutination and hemagglutination-inhibition tests. Avian Disease: 34: 643–645, 1990a. 5.Blackall PJ, Eaves LE, Rogers DG. Proposal of a new serovar and altered nomenclature for Haemophilus paragallinarum in the Kume hemagglutinin scheme. Journal of Clinical Microbiology 28: 1185–1187, 1990b. 6.Blackall PJ, Rogers DG, Yamamoto R. Outer-membrane proteins of Haemophilus paragallinarum . Avian Disease 34: 871-877, 1990c. 7.Blackall PJ, Matsumoto M, Yamamoto R. Infectious coryza, In: B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald, and Y. M. 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傳染性可利查病又稱傳染性鼻炎,是由 Avibacterium paragallinarum 所引起之急性呼吸道疾病,在 1931 年首次為De Blieck所報告,IC主要造成的經濟損害,常為蛋雞產蛋率的下降及肉雞及種雞的生長遲緩等問題,感染此病的雞隻臨床上可見有流鼻水、呼吸困難、發出囉音,及眼窩下竇以及顏面腫脹、下痢等症狀。目前 IC的商用疫苗主要以不同血清型菌株的混合不活化全菌疫苗為主,但培養Avibacterium paragallinarum 需要添加第 V 因子,且疫苗需要多次注射才有相當的成效,造成此疫苗在商用雞上使用的成本及人力較高,故發展新的疫苗是非常重要的。在本次研究中所確認的蛋白質P17、P40 及P30,與Large adhesin蛋白被認為可能是IC的保護性抗原,且這些基因在 A. paragallinarum 中是高保留性的,因此我們利用原核表現系統,大量表現上述的重組蛋白,並製備成次單位疫苗,以鼻腔接種攻毒試驗的動物模式,來評估次單位疫苗對雞隻的保護效力,我們以54 隻SPF雞,在 6 週及 9 週齡時分別免疫不同的重組蛋白,並於 12 週齡時以血清A型野外株TW-1 攻毒,紀錄其臨床症狀至攻毒後第 6 天,其中並以西方墨點法檢測其血清抗體力價,結果免疫r-P30、r-P17 蛋白可得到平均且高度的抗體力價,此外結果也指出,P30 及 P17 在自然感染下能被表現,並可引發特異性抗體反應,且在不同血清型間能夠交互作用。而疫苗之保護力測試中,顯示在鼻腔接種 109CFU之血清A型TW-1 株攻毒條件下,在 12 週齡 SPF 雞可達到 100 % 發病率,而比較其臨床症狀指數則可發現,使用 r-P17 重組 蛋白免疫後的雞隻攻毒後臨床症狀的發生及其嚴重程度都有顯著性的下降,總合血清學及保護性測試,顯示此蛋白可能有交叉保護抗原的特性,作為 IC 的次單位疫苗是有相當的潛力的,故未來仍有必要對r-P17 蛋白做進一步的研究,包括表現系統的調整,以求大量提升疫苗內之r-P17 重組蛋白濃度或表現更多可溶性部分,及交叉保護性的攻毒測試,以求能確實保護各種血清型菌株的攻擊,此外繼續搜尋並測試新的抗原,若能添加多種具抗原性及保護性之蛋白,或與全菌疫苗合併使用,皆可能提升保護力及降低製作成本,這些研究對新一代的疫苗發展都是有益的。

Avibacterium paragallinarum is the causative agent of infectious Coryza (IC), an acute
upper respiratory tract disease of chickens. This disease was firstly reported by De Blieck in 1931, and consequently found to cause significant economic losses in poultry industry. Infectious Coryza is primarily manifested as a marked (10% to 40%) drop in egg production in layers and retardation of growth in breeders and broilers. Infected chickens usually show clinical signs including nasal discharge, facial edema, lacrimation, anorexia and diarrhea. Commercial vaccines against IC, typically based on inactivated A. paragallinarum, are wildly used around the world. However, A. paragallinarum requires 'V' factor, a special growth factor in the media to grow. Moreover, the bacterin requires multiple injections to be effective. These make the administration of the vaccine expensive and labor intensive for commercial flocks. Therefore, a novel vaccine is thought to be important for the control of IC. Three antigenic proteins, P17, P40 and P30, identified in our laboratory, together with the large adhesion, are thought to be the protective antigens against IC. They are highly conserved among different types of A. paragallinarum. These gene were cloned and expressed as recombinant P17 (r-P17), P40 (r-P40), P30 (r-P30) and r-adhesion protein in E. coli. Moreover, r-P30, r-P17, and r-adhesin recombinant proteins were evaluated for the vaccine efficacy using a nasal challenge model. Fifty-four chickens were immunized subcutaneously with these recombinant proteins with Freund's adjuvant twice at the age of 6 and 9-week-old. These animals were challenged intra-nasally with A. paragallinarum field strain TW-1 and clinical signs were measured for 6 days. To determine immune responses of the recombinant proteins, sera were collected a week prior to challenge. The serum samples were analyzed by western blot to determine antibody titers against r-P17, r-P30 and r-adhesin. We also collected the serum samples from natural infected chicken for cross-reaction test. The results of these experiments show that r-P30 and r-P17 can induce highly immune responses. P17 and P30 are able to express in hosts under natural infection. Moreover, the serum from natural infected chicken can react with r-P30 and r-P17, and the serum from vaccinated chicken also reacted with different types of A. paragallinarum. These results indicate that these two antigens may be cross-protective antigens. Moreover, intranasal challenge with 109 CFU A. paragallinarum field strain TW-1 can cause 100 % (6/6) mortality in 12-week-old SPF chicken. The use of r-P17 protein as subunit vaccine can protect 66.6% of chickens and significantly reduce the severity of clinical signs against the challenge of the field strain TW-1. These data indicate that immunized with r-P17 protein may induce systemic immunity and provide some protection against IC in chicken. However, it still needs to modify the
overexpression system to increase the productivity and solubility of r-P17. Data presented in this
study are useful for developing new vaccine be useful for developing new vaccine against IC in Taiwan.
其他識別: U0005-0807201514590000
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