- 學位論文
豬致病性大腸桿菌分離、致病力評估及類毒素產製
Isolation, Pathogenicity Evaluation, and Toxoid Generation of Pathogenic Escherichia coli in Pigs
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摘要
大腸桿菌（Escherichia coli，E. coli）引起豬隻下痢是全球豬業面臨的重要問題，導致豬場經濟困難。現今，具病原性的大腸桿菌主要依據其毒力因子進行分類，包括黏附素、毒素、脂多醣和莢膜多醣等。在病原性大腸桿菌中，腸毒性大腸桿菌（Enterotoxigenic E. coli，ETEC）、志賀毒素大腸桿菌（Shiga toxin-producing E. coli，STEC）和腸致病性大腸桿菌（Enteropathogenic E. coli，EPEC）對新生豬和離乳後仔豬侵襲性非常高。在新生與離乳仔豬中，ETEC和EPEC與水樣下痢有關。ETEC通常產生耐熱毒素（ST）和不耐熱毒素（LT），而EPEC中則檢測到Intimin。另一方面，帶有志賀毒素2e型（Shiga toxin type 2e，Stx2e）的STEC會破壞離乳豬的全身性內皮細胞造成水腫性疾病。 在第二章中，使用傳統多重PCR分析2017年至2023年間在台灣收集到的病原性大腸桿菌的毒力因子。在十株溶血性大腸桿菌中，有五株（50％）被鑑定為ETEC，一株為STEC，一株為EPEC。有趣的是，其中有三株（30％）大腸桿菌菌株同時攜帶ST、LT和Stx2e基因並認為是混合型STEC/ETEC菌株。為了評估混合型STEC/ETEC的致病性，將一株野外分離菌株口服接種到離乳豬上，在研究期間，控制組的所有豬隻糞便評分正常，並保持健康，而STEC/ETEC接種組中的大部分豬隻出現水樣性腹瀉，但沒有水腫病相關症狀。與ETEC的發現相似，接種STEC/ETEC的豬隻沒有明顯的組織學病變。鑒於觀察到的混合型STEC/ETEC的高盛行率和致病性，應將其視為新興的病原體。 為了控制病原性大腸桿菌，目前常用的方法是在新生豬和離乳豬中使用黏菌素（colistin）和其他抗菌劑。然而，許多國家目前禁止將抗生素用作生長促進劑、或在豬使用氧化鋅治療。此外，目前商業化的ETEC疫苗只對預防新生仔豬下痢有效，對離乳豬的下痢無效。因此替代性抗菌劑及對混合型STEC/ETEC和ETEC都有效的高效疫苗的研發有其迫切需求。在第三章中，我們構築及合成包括野生型STaLTB及LTASTb及減毒突變型STaN11SLTB和LTAR192G/L211ASTb的ST和LT合成毒素。帶有毒素蛋白基因的重組質體被轉染到BL21（DE3）細胞中，並在大腸桿菌表達系統中成功表現毒素。 本研究中分離病原性大腸桿菌分離株和合成類毒素可作為抗病原性大腸桿菌的死毒和次單位蛋白疫苗候選物。建立的混合型STEC/ETEC豬攻毒模式將可用於未來評估對病原性大腸桿菌感染的保護確效指標。
並列摘要
Porcine diarrhea caused by Escherichia coli (E. coli) has been a major problem in the global swine industry, leading to the financial issues of swine farms. Nowadays, pathogenic E. coli is usually classified based on their virulence factors, including adhesins, toxins, lipopolysaccharides, and polysaccharide capsules. Among pathogenic E. coli, the enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) attacking neonatal and post-weaning pigs are important. In neonatal and post-weaning piglets, ETEC and EPEC are associated with watery diarrhea. The heat-stable toxin (ST) and heat-labile toxin (LT) are usually found in ETEC, while intimin is detected in EPEC. The STEC, on the other hand, carrying Shiga toxin type 2e (Stx2e) causes systemic endothelial cell destruction and edema illness in weaned pigs. In the chapter II, conventional multiplex PCR were used to analyze the virulence factors of pathogenic E. coli collected in Taiwan from 2017 to 2023. While five of the ten (50%) hemolytic E. coli were ETEC, one isolate was STEC, and one was EPEC, interestingly, concurrently carrying ST, LT, and Stx2e genes, namely the hybrid STEC/ETEC strains, were noted in three of the ten (30%) E. coli isolates. In order to evaluate pathogenicity of hybrid STEC/ETEC, a field isolated strain was orally inoculated in post-weaning pigs. While all pigs in the mock-inoculated group had normal fecal consistency score and were healthy during the study, the majority of piglets in the STEC/ETEC-inoculated group had watery diarrhea without signs of edema disease. Similar to ETEC, histological examination of STEC/ETEC-inoculated pigs reveals no apparent lesion. Due to the high prevalence and pathogenicity of hybrid STEC/ETEC observed, hybrid STEC/ETEC should be considered a new emerging pathogen. For controlling pathogenic E coli, colistin and other antimicrobials were commonly used in neonatal and post-weaning pigs. However, many countries currently prohibit the usage of antibiotics as growth promoters or therapeutic usage of zinc oxide in pigs. Furthermore, the current commercial ETEC vaccination is only useful in preventing neonatal diarrhea not for post-weaning diarrhea. These findings suggest that alternative antimicrobials are urgently needed and an efficient vaccine against both hybrid STEC/ETEC and ETEC is required. In the Chapter III, chimeric toxoids, including wild typed STaLTB and LTASTb and attenuation mutants of STaN11SLTB and LTAR192G/L211ASTb, were generated. All of recombinant plasmids were transformed into BL21 (DE3) competent cells and successfully expressed in the E. coli expression system. The pathogenic E. coli isolates and chimeric toxoids generated in the present study could be potential inactivate and toxoid subunit vaccine candidates against pathogenic E. coli, respectively. The established hybrid STEC/ETEC porcine challenge model could be used to evaluate the vaccine efficacy and protectivity against pathogenic colibacteriosis.
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