Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14156
標題: 豬萎縮性鼻炎重組巴氏桿菌毒素次單位疫苗之免疫效力試驗
Efficacy Study of Recombinant Subunit Pasteurella multocida toxin (PMT) Vaccine to Swine Atrophic Rhinitis
作者: 陳有得
Der, Chen You
關鍵字: Swine atrophic rhinitis
豬萎縮性鼻炎
Pasteurella multocida toxin
重組巴氏桿菌毒素
次單位疫苗
出版社: 獸醫學系
摘要: 豬萎縮性鼻炎(atrophic rhinitis;AR)為豬隻重要之上呼吸道細菌性疾病。本實驗首先針對中部四縣市的53個豬場共164個送檢病例進行AR與肺炎之疫學調查,結果發現共有123頭豬隻罹患程度不一的AR (75%),而有135頭豬隻則至少感染一種或呈現複合性肺炎之病變(82%),而豬隻罹患AR且同時併發感染肺炎的比率更高達95﹪(117/123),顯示AR與呼吸道疾病的關係應為絕對的正性相關。選取構築的重組Pasteurella multocida toxin(PMT)毒素基因選殖株,分成不同的免疫實驗組,並比較國外進口商品化AR疫苗進行免疫效力評估,實驗母豬於分娩前五週、三週時各進行一次免疫,而其分娩的仔豬則於18日齡時分別以不同劑量PMT毒素,以肌肉注射方式進行攻毒。在移行抗體保護效力試驗中,母豬及仔豬之移行抗體經由vero cell測試得知,免疫Tox4+5、Tox1+2+7、Tox1+2+7+P. multocida type A cell lysate和傳統類毒素疫苗的懷孕母豬,經兩次免疫後所產生的抗體及所生下之仔豬移行抗體力價皆大於64倍以上,但免疫商品化AR疫苗組則低於32倍。而各組母豬所生仔豬於18日齡時,以致死劑量(1 mg)的PMT毒素進行攻毒,發現經免疫商品化AR疫苗的母豬和未經任何處理的母豬所生下之仔豬於攻毒後4天內全部死亡,但免疫Tox1+2+7、Tox1+2+7+P. multocida type A cell lysate和傳統類毒素組所生之仔豬,其移行抗體在攻毒後則具有顯著的保護效力。而在增重試驗結果顯示,免疫Tox4+5、類毒素和未免疫的懷孕母豬其所生的仔豬於18日齡時,以0.2 mg的PMT毒素攻毒後發現,只有未免疫組母豬所生的仔豬呈現鼻甲介骨萎縮之臨床症狀,且攻毒後2週內的平均增重則明顯少於同胎未攻毒的實驗仔豬。至於重組毒素疫苗對仔豬的免疫效力試驗中,將60頭離乳仔豬,分別利用傳統PMT類毒素、Tox4、Tox5、Tox7的表現產物進行免疫,並以只免疫competent cells及未經任何處理的仔豬做為對照組。仔豬於五、七週齡時進行免疫,11週齡時再分別以0.5 mg或高於20倍的致死劑量(20 mg)PMT毒素進行攻毒,一週後所有免疫實驗組豬隻均能存活,且抗體力價最高能揚升至1,024倍,但兩對照組豬隻於攻毒後其抗體力價均不能揚升而低於4倍,更無法耐過致死劑量PMT的攻毒,其鼻甲介骨也呈現嚴重萎縮的臨床症狀。在嘗試探討仔豬鼻道黏膜免疫的前置試驗中,以Tox4+5的表現產物經鼻腔噴霧的免疫方式,測試對仔豬之保護效力,結果顯示豬隻除可耐過5倍致死劑量PMT攻毒,且中和抗體力價亦上升至128倍。所有結果顯示AR重組PMT疫苗經免疫母豬或仔豬後,確實能夠產生有效的中和抗體,並且母豬可藉由初乳將移行抗體傳給仔豬,使仔豬對早期AR感染具有良好之保護效力。
The result in evaluation of protective immunity in colostrum showed that sows and the offspring allotted to groups of Tox1+2+7, Tox1+2+7+toxin-producing strain of P. multocida type A cell lysate, and toxoid could mount good protective immunity after immunization and the neutralization antibody titer markedly increased up to over 64 x with v Swine atrophic rhinitis(AR)is an important bacterial disease in upper respiratory of swine. In a survey of nursing pigs from herds in the middle of Taiwan in 1998/99 showed that 123 of 164(75﹪)pigs examined were infected with AR, while 135 of 164(82﹪)pigs got pneumonia. Of 123 AR-infected pigs, 117 pigs were complicated with pneumonia, indicating a positive correlation between AR and other respiratory disease. Five recombinant PMT gene clones were allotted to groups for efficacy of vaccine trials. All allotted groups were compared with commercial AR vaccine. Vaccination was performed twice 5 and 3 weeks before farrowing in sows, and the offspring were challenged intramuscularly at 18 days old with varied dose of PMT. ero cell toxicity test, while in commercial AR vaccine-immunized and nonvaccination control groups, the neutralization antibody titer down to 32 x. Piglets from all groups were challenged with lethal dose of PMT(1 mg)at the age of 18 days. All piglets of commercial AR vaccine-immunized and nonvaccination control groups die within 4 days after challenge, while maternal antibody could mount protective immunity in Tox1+2+7, Tox1+2+7+toxin-producing strain of P. multocida type A cell lysate, and toxoid groups. Moreover, piglets of Tox4+5, toxoid, and nonvaccination control groups were challenged with 0.2 mg PMT at the age of 18 days, it was found that only nonvaccination control piglets present turbinate atrophy and poorest weight gain(p<0.05). To evaluate the protective immunity of recombinant PMT in piglet, the native PMT toxoid, competent cells, and expression products of recombinant PMT gene clone Tox4, Tox5, Tox7 were used as immunogen. The piglets were vaccinated twice at 5 and 7 weeks of age followed by challenging with either 0.5 mg or 20 mg PMT at 11 weeks of age. All vaccination groups other than competent cells and nonvaccination control groups could mount good protective immunity 1 week after challenge, and the neutralization antibody titer increased up to 1,024 x with vero cell toxicity test, and in the other two groups, piglets showed turbinate atrophy and poor protective immunity during experimental period. In evaluation of mucosal immunity of recombinant PMT vaccine, piglets were vaccinated intranasally with expression products of Tox4+Tonx5, and challenged with 5 mg (5 LD100) PMT. The result showed that the neutralization ntibody titer markedly increased up to 128 x with vero cell toxicity test. All these results demonstrated that the recombinant PMT vaccines could provide good protection and mount highly specific immunity against AR in piglets and sows.
URI: http://hdl.handle.net/11455/14156
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