A型與D型Pasteurella multocida之分子鑑別及PMT毒素基因序列之比對

Molecular Characteristics and Genetic Analysis on PMT Gene of Pasteurella multocida Type A and Type D

廖志明(Chih-Ming Liao) ； 程明彥(Ming-Yann Chuieng) ； 周育賢(Yuh-Shyan Chou) ； 李維誠(Wei-Cheng Lee) ； 林正忠(Cheng-Chung Lin) ； 劉正義(Cheng-I Liu) ； 簡茂盛(Maw-Sheng Chien)

《臺灣獸醫學雜誌》 28卷4期 (2002/12) Pp. 281-293

https://doi.org/10.7009/TVJ.200212.0281

摘要

Pasteurella multocida為豬隻進行性萎縮性鼻炎（progressive atrophic rhinitis; PAR）之主要病原，其中D型P. multocida所分泌一分子量約為146kDa之毒素蛋白被稱為P. multocida toxin（PMT），並證實是PAR主要致病因子，但目前除D型外，A 型P. multocida產毒株亦被認為是PAR之致病原。為進一步探討A型與O型P. multocida在PMT毒素性狀與基因序列之異同性，本實驗首先證實若能導致vero cell產生特異性細胞病變的分離株中，不論A型或D型之分離株皆具有完整的PMT基因，但不會造成細胞病變之菌株則不具有PMT基因，也不會導致實驗BALB/c小鼠死亡及豬雙的臨床病徵。此外，在型別鑑定上以Hhal進行限制片段多樣性分析（restriction fragment length polymorphism; RFLP），以及應用隨機增幅多型性DNA聚合酵素連鎖反應（random amplified polymorphic DNA PCR; RAPD-PCR）分析後，可明顯區分A型與D型P. multocida，並可應用於快速鑑別菌株是否具有PMT基因。而當進一步比較A型及D型PMT之基因序列時，顯示國內兩血清型之產毒株其PMT核酸序列完全相同，並與Genbank登錄之D型PMT基因也僅有一胺基酸之改變，顯示PMT為一穩定而不易變異的毒素基因，而分析所萃取之兩型PMT毒素在細胞與動物實驗上的性狀也相當一致，且測試方法上以vero cell毒素測定最為敏感，可做為判定豬隻中和抗體力價之依據。

關鍵字

PMT毒素基因比對 ； 豬進行性萎縮性鼻炎 ； A型與D型P. multocida

並列摘要

Pasteurella multocida is characterized the primary causative agent of progressive atrophic rhinitis (PAR) in swine and the P. multocida toxin (PMT) with an approximate molecular weight of 146 kDa has been identified as the major virulent factor to induce nasal turbinate atrophy. In this study, both type D and type A of P. multocida, isolated from massive PAR-infected farm, could induce similar subcutaneous necrotic skin lesions in mice and rat and atrophic turbinate changes in experimental inoculated swine. The vero cell toxicity test and PMT gene identification were used to discriminate both type D and type A of toxigenic or non-toxigenic P. multocida. The results indicated PMT gene exist only in toxigenic P. multocida isolates. Moreover, both RFLP (restriction fragment length polymorphism) and RAPD (random amplification polymorphic DNA) PCR methods were established to identify the genetic variation between type A and type D, and successfully to recognize toxigenic or non-toxigenic of P. multocida isolates. In addition, southern blotting and DNA sequencing were utilized for identification the homology of PMT gene between type D and type A isolates. It revealed the DNA sequences of PMT gene were perfectly identical in our type D and type A isolates and showed only one amino acid change if in comparison with ATCC type D strains (NCTC 12178). The results suggested the PMT gene and its toxic activity in both type D and type A are highly conserved without serotype or geographic diversity among isolates.

並列關鍵字

PMT gene variation ； Swine progressive atrophic rhinitis ； PAR ； Type A and type D P. multocida