對產氣莢膜梭狀芽孢桿菌與空腸彎曲桿菌抗藥性以及海藻糖作為益生元潛力的研究

Abstract

家禽肉及其相關產品是亞洲以及全世界重要的蛋白質來源。然而，近年來，食品安全對於政府和整個社會來說變得越來越重要。食媒性病原體和抗生素抗藥性問題已經被指出對消費者健康造成傷害。隨著食品安全意識的抬頭，許多國家開始禁止抗生素作為生長促進劑使用。然而，部分疾病隨之重新浮現，例如產氣莢膜梭狀芽孢桿菌、空腸彎曲桿菌，對於家禽產業與人類健康帶來新的威脅。因此，本研究分別進行了三項研究，分別圍繞著產氣莢膜梭狀芽孢桿菌、空腸彎曲桿菌以及海藻糖在家禽健康管理中的作用。產氣莢膜梭狀芽孢桿菌感染以引起家禽群體中亞臨床和臨床性壞死性腸炎而惡名昭彰，全球每年的經濟損失估計為20億美元。第一項在台灣對五週齡、臨床健康上市前的肉雞進行的研究，旨在確定產氣莢膜梭狀芽孢桿菌的流行、毒力分型以及抗微生物藥物抗藥性的調查。該研究從2012年6月持續到2013年2月，總共從98個肉雞養殖場收集的435個雞迴腸內容物樣本。結果顯示產氣莢膜梭狀芽孢桿菌的檢出率為9.9%，有29.6%的肉雞養殖場為陽性。值得注意的是，所有分離出來的產氣莢膜梭狀芽孢桿菌都產生了A型毒素。該研究透過qPCR檢測雞隻腸內容物中的產氣莢膜梭狀芽孢桿菌數量平均為3.9×106 CFU/g，菌量範圍從6.85×102到1.61×107 CFU/g。在雞迴腸的病變分數與產氣莢膜梭狀芽孢桿菌數量之間有正相關（p<0.05）。對所有分離出來的產氣莢膜梭狀芽孢桿菌進行抗生素敏感性測試、量測各抗微生物藥物的MIC50值。大多數的產氣莢膜梭狀芽孢桿菌對amoxicillin、bacitracin及 enrofloxacin顯示出敏感，但對chlortetracycline、erythromycin及 lincomycin顯示出抗藥性。值得注意的是，從腸道有嚴重病變的雞體內分離出來的產氣莢膜梭狀芽孢桿菌的erythromycin及 lincomycin的MIC50值比腸道輕微病變的雞要高。在另一項研究中，2009年至2012年從國內水禽收集的95株空腸彎曲桿菌分離株進行了quinolone抗藥性分析。對nalidixic acid和ciprofloxacin具抗藥性的比率分別為61.2%和57.9%。此外，進一步篩查了這些分離物中gyrA基因的quinolone抗藥決定區（QRDRs）的突變情況。在gyrA突變分析中觀察到多種突變模式。高解析度熔解（HRM）分析中結果中顯示四種熔化溫度曲線模式，能夠區分突變型gyrA菌株和野生型菌株。試驗結果證明HRM可以在全面的quinolone抗藥性監測計劃中快速檢測gyrA突變。最後一項研究調查了海藻糖對肉雞生長性能和病原菌接種的影響。海藻糖是動物、植物和微生物中發現的一種雙醣，也是FDA和歐盟批准的食品添加劑。該研究包括兩個實驗—第一個旨在確定肉雞對海藻糖的耐受性。結果顯示，在飼養期間體重變化、每日體重增長、飼料攝取量或飼料轉換率方面都沒有顯著差異（p>0.05）。這個結果顯示飼糧中添加最高10%的海藻糖劑量也不會對肉雞養殖造成負面影響。第二個實驗評估了海藻糖對空腸彎曲桿菌和產氣莢膜梭狀芽孢桿菌的抗菌效果。試驗結果顯示，沒有觀察到肉雞生長性能或空腸彎曲桿菌和產氣莢膜梭狀芽孢桿菌在肉雞腸道和糞便中的菌數的顯著差異（p>0.05）。然而，乳酸桿菌的數量在添加3%和5%海藻糖的情況下顯著增加（p<0.05）。儘管海藻糖並未直接減少空腸彎曲桿菌和產氣莢膜梭狀芽孢桿菌的數量，但它似乎通過提高雞腸道中乳酸桿菌的數量來增強腸道健康，特別是在存在腸道病原菌的情況下。因此，雖然海藻糖並未直接影響肉雞的生長性能或空腸彎曲桿菌和產氣莢膜梭狀芽孢桿菌在肉雞腸道中的數量，但它可能通過提高乳酸桿菌的數量在肉雞飼養中作為一種益生元。總結來說，這些研究強調了台灣家禽中重要病原體如產氣莢膜梭狀芽孢桿菌和空腸彎曲桿菌的流行和抗藥性模式，並獲得海藻糖作為飼糧中添加劑提高家禽健康中的潛在作用。這些發現可能對制定未來的家禽疾病控制和管理策略提供參考，為更可持續的家禽養殖做出貢獻。

Poultry meat and related products are a vital source of protein not only in Asia but also in the global. However, recently, food safety has become increasingly important to governments, industry and consumers. Foodborne pathogens and antibiotic resistance have been reported to harm the health of consumers. With convenient transportation and increasing globalization, the food chain has become more and more complex, exacerbating these issues. The control and management of poultry diseases have significant implications for global food security, considering the potential economic losses in the industry. In this dissertation, there were three studies focusing on this challenge conducted in Taiwan: 1) revolving around Clostridium perfringens and 2) Campylobacter jejuni, as well as 3) the potential role of trehalose in managing poultry health. C. perfringens infection is notorious for causing subclinical and clinical necrotic enteritis in poultry flocks, with an estimated annual global economic toll of two billion US dollars. The first study on Taiwan's 5-week-old and clinically healthy broiler sought to determine C. perfringens prevalence, toxin types, and antimicrobial resistance levels. The study spanned from June 2012 to February 2013 and involved the collection of 435 samples of chicken ileum contents from 98 broiler farms. The results indicated a C. perfringens isolation rate of 9.9%, with 29.6% of the tested farms testing positive. It was noted that all isolated C. perfringens produced toxin type A. The study revealed a mean C. perfringens number of 3.9×106 CFU/g in the intestinal contents through qPCR. This number ranged from 6.85×102 to 1.61×107 CFU/g. There was a positive correlation (p<0.05) between the lesion score and C. perfringens number in the chickens' ilea. On conducting antimicrobial susceptibility tests on all C. perfringens isolates, the MIC50 values for several antimicrobial drugs were determined. Most C. perfringens isolates showed susceptibility to amoxicillin, bacitracin, and enrofloxacin but demonstrated resistance to chlortetracycline, erythromycin, and lincomycin. Notably, C. perfringens isolated from chickens with severe lesions had a higher MIC50 of erythromycin and lincomycin than chickens with mild lesions. In another study, 95 C. jejuni isolates collected from domestic waterfowl from 2009 to 2012 were subjected to quinolone resistance analysis. The resistance rates to nalidixic acid and ciprofloxacin were 61.2% and 57.9%, respectively. The isolates were further screened for mutations in the quinolone-resistant determining regions (QRDRs) of the gyrA genes. A variety of mutation patterns was observed in the gyrA mutation analysis. A high-resolution melting (HRM) analysis revealed four patterns of melting temperature curve that distinguished mutated-gyrA strains from wild-type strains. The HRM assay demonstrated its viability for rapidly detecting gyrA mutations in a comprehensive quinolone resistance monitoring program. The final study investigated the impact of trehalose, a disaccharide found in animals, plants, and microorganisms, and a legal food additive in the FDA and the European Union on broilers' growth performance and pathogenic bacteria inoculation. The research comprised two objectives—the first objective to determine broilers' tolerance to trehalose. The results showed no significant differences (p>0.05) on body weight changes, daily weight gain, feed intake, or feed conversion ratio during the feeding period. This result suggested that a trehalose dosage of up to 10% should not negatively affect broiler farming. The second objective evaluated the antibacterial effects of trehalose on C. jejuni and C. perfringens, respectively. No significant difference (p>0.05) was observed in broilers’ growth performance or bacterial counts of C. jejuni and C. perfringens in the intestine and feces of broilers during a 5-week feeding period. However, Lactobacillus counts significantly increased with 3% and 5% trehalose supplementation. While trehalose did not directly decrease C. jejuni and C. perfringens count, it seemed to enhance gut health by raising Lactobacillus counts in the chicken gut, especially in the presence of enteropathogenic bacteria. Thus, while trehalose does not directly impact the broiler’s growth performance or C. jejuni and C. perfringens counts in the broiler’s gut, it might serve as a prebiotic in broiler feeding by raising Lactobacillus counts. In conclusion, these studies highlight the prevalence and resistance patterns of critical poultry pathogens like C. jejuni and C. perfringens in Taiwan and underscore the potential role of dietary supplements like trehalose in enhancing poultry health. The findings could inform future strategies for controlling and managing poultry diseases, contributing to more sustainable poultry farming.