豬腸毒性大腸桿菌線毛之主要黏附素之產製及試驗評估田間分離株於哺乳豬致病性及腸道趨向

Abstract

豬大腸桿菌病（swine colibacillosis）主要由腸毒性大腸桿菌（enterotoxigenic Escherichia coli ; ETEC）引起，於養豬產業中造成重大健康威脅，特別是在哺乳期豬隻造成早發型大腸桿菌下痢（neonatal diarrhea）及於離乳豬造成豬離乳後下痢（post-weaning diarrhea ; PWD）。早發型大腸桿菌下痢通常與母體移行抗體不足有關，豬離乳後下痢則發生於母乳轉換為固體飼料、移行抗體下降，加上免疫系統尚未成熟與環境壓力，使得仔豬特別容易受到腸道感染。ETEC菌株中，最常見的為表現出FaeG（F4 or K88）與FedF（F18）等菌毛黏附素的類型，兩者被認為與導致早發型（F4）及離乳後大腸桿菌（F4/F18）有關。菌株帶有這些貼附因子可附著於腸道上皮細胞，並分泌熱不穩定（heat-labile ; LT）與熱穩定（heat-stable ; STs）等腸毒素，導致體液平衡失調，進而引發分泌型下痢、脫水、生長遲滯甚至死亡。儘管已有包括抗生素與飼料添加劑等多項介入策略，但因抗生素抗藥性日益嚴重及歐美各國減少使用抗生素策略而更加棘手。近年來，針對F4與F18菌毛抗原所設計的疫苗，尤其是可誘導黏膜免疫的口服疫苗，被視為具潛力的防治策略。 雖然腸毒性大腸桿菌致病機轉及腸道貼附向性在離乳豬及田間豬隻已有多年之研究，然而帶有F4之腸毒性大腸桿菌對於新生仔豬動物模式和在不同腸道貼附向性研究有限，因此在第二章中，本研究首先以一株田間分離之 F4⁺ ETEC 菌株（PT4357），進行全基因定序，結果顯示，該菌株為 O8:H7 血清型，序列型別為 ST2521，且帶有多重毒力與抗藥性基因，包括 mcr-1。以ETEC PT4357對四日齡仔豬進行口服攻菌實驗，以評估其致病性及於不同腸道貼附向性，發現所有受試仔豬皆於接種後六小時內出現急性水樣下痢，這些豬隻於組織染色（H&E）檢查下無顯著組織病變，但可見細菌附著於十二指腸、空腸及迴腸全段小腸絨毛表面，且於Warthin-Starry與 Brown and Hopps染色下觀察更為顯著。腸道中也可重新分離出具有溶血特性的 ETEC，並經多重 PCR (multiplex PCR) 確認帶有與原接種菌株相同之F4、LT與STb基因。本研究不僅建立了一套可重現的新生仔豬 ETEC攻毒模型，了解早發型ETEC腸道組織向性，並提供該菌株於台灣流行之遺傳特徵與腸道定殖行為的重要資訊。 在第三章中，為進一步產製菌毛黏附素以做為未來疫苗候選，本研究進行ETEC F4的主要黏附亞基FaeG 與 F18的次要黏附亞基FedF的基因構築、表現與純化。經西方墨點法（Western blot）確認兩者皆有成功表現。此研究展示了表現與純化 FaeG 與 FedF 的可行性與挑戰，為未來開發診斷工具或免疫防治策略奠定基礎。 總結而言，本研究成功建立以田間分離 F4⁺ ETEC 菌株為基礎的新生仔豬攻毒模型，並研究其腸道向性為全段小腸。同時，本研究亦完成兩項關鍵黏附素 FaeG 與 FedF 的重組表現，可供未來驗證其作為次單位疫苗候選抗原之潛力。這些成果不僅提供評估 ETEC 致病性與防治策略的實驗平台，亦為未來針對豬隻新生仔豬下痢及斷奶後下痢之標靶疫苗與診斷工具開發奠定重要基礎。

Swine colibacillosis, primarily caused by enterotoxigenic Escherichia coli (ETEC), represents a significant health threat in pig farming, particularly leading to neonatal diarrhea in suckling piglets and post-weaning diarrhea (PWD) in weaned piglets. Neonatal diarrhea is typically associated with insufficient maternally derived antibodies, whereas PWD often occurs due to the transition from milk to solid feed, concurrent decline in passive immunity, immature immune function, and increased environmental stress, rendering piglets more susceptible to enteric infections. Among ETEC strains, those expressing fimbrial adhesins FaeG (F4 or K88) and/or FedF (F18) are most frequently identified and are considered major contributors to neonatal (F4) and post-weaning (F4/F18) colibacillosis. These adhesins facilitate bacterial adherence to the intestinal epithelium, followed by the secretion of enterotoxins, including heat-labile toxin (LT) and heat-stable toxins (STs), which disrupt fluid homeostasis, leading to secretory diarrhea, dehydration, growth retardation, and even death. Despite the use of antibiotics and feed additives as control measures, the growing concern over antimicrobial resistance and reduced antibiotic usage policies in Europe and other regions has made disease control increasingly challenging. Recently, vaccines targeting F4 and F18 fimbriae, particularly oral vaccines capable of inducing mucosal immunity, have been considered promising alternatives. Although the pathogenesis and intestinal tropism of ETEC have been extensively studied in weaned or field piglets, research focusing on F4⁺ ETEC in neonatal piglets remains limited. Therefore, in the Chapter II of this study, a field isolate of F4⁺ ETEC (PT4357) was characterized. Whole-genome sequencing revealed that the strain belongs to the O8:H7 serotype, sequence type ST2521, and harbors multiple virulence and antimicrobial resistance genes, including mcr-1. A challenge experiment was conducted by orally inoculating 4-day-old piglets with the ETEC PT4357 to evaluate its pathogenicity and intestinal adhesion pattern. All ETEC PT4357 inoculated piglets developed acute watery diarrhea within six hours post-infection. Although no significant histological lesions were observed by H&E staining, extensive adherence of bacteria to the villous surface across the duodenum, jejunum, and ileum was noted, which became more prominent under Warthin-Starry and Brown and Hopps staining. Hemolytic ETEC was successfully re-isolated from intestinal contents and confirmed by multiplex PCR to carry F4, LT, and STb genes. This study successfully established a reproducible neonatal piglet ETEC challenge model and provided insights into the intestinal tropism and genomic characteristics of a field-derived ETEC strain prevalent in Taiwan. In Chapter III, to facilitate the development of potential vaccine candidates, we cloned, expressed, and purified the major fimbrial adhesin FaeG (F4) and the minor adhesin FedF (F18). Western blotting confirmed successful expression of both recombinant proteins. The study demonstrated the feasibility and challenges of expressing and purifying FaeG and FedF, and laid a foundation for future development of diagnostic tools and immunoprophylactic strategies. In conclusion, this study established a neonatal piglet challenge model based on a field-isolated F4⁺ ETEC strain and confirmed its intestinal tropism encompassing the entire small intestine. Furthermore, two key recombinant fimbrial adhesins, FaeG and FedF, were successfully constructed and generated. Their potential as subunit vaccine candidates should be further investigated in the future. These findings provide not only an experimental platform for evaluating ETEC pathogenicity and intervention strategies but also a solid basis for the development of targeted vaccines and diagnostic tools against neonatal and post-weaning diarrhea in pigs.