藉乳汁傳播細菌性疾病之分子檢測

Molecular Detection of Milk borne Bacterial Disease

邱泓錫(Hung-Shi Chiou)

指導教授 ： 龐飛

共同指導教授 ： 鄭謙仁(Chian-Ren Jeng)

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02368

摘要

乳汁內包含許多豐富的蛋白質及維生素，對人類及動物是一個很重要的營養來源，但同時也提供一個很好的環境供微生物生長，尤其是細菌性病原。在人類的食物媒介性人畜共通傳染性細菌性疾病中，主要是由沙門氏桿菌、李斯特菌、金黃色葡萄球菌、彎曲桿菌及大腸桿菌O157:H7所引起。藉由乳汁傳播引起疾病的病原也包含了牛型分枝桿菌及副結核分枝桿菌，且未經巴斯德滅菌的乳汁是一個重要的傳播媒介。由於巴斯德滅菌法無法將細菌所產生的毒素完全消滅，故市售鮮乳有其一定的風險性存在。鑒於上述，本研究目的主要為建立分子生物學的檢測方法，評估藉乳傳播細菌性疾病之病原菌及其毒素經由生乳及熟乳傳播的風險性。針對Mycobacterium spp.、Mycobacterium bovis、Mycobacterium paratuberculosis、Salmonella enterica subspecies enterica serovar Typhimurium、Listeria monocytogenes、Campylobacter jejuni、Staphylococcus aureus及Escherichia coli O157的細菌或其毒素基因序列，依序包含HSP65、TbD1、IS900、InvA、PlcA、MapA、Nuc及Stx，設計特異性引子對及建立其聚合酶鏈鎖反應檢測是否有上述細菌存在乳汁中之風險。樣本的收集包含從2個牧場收集到12個皮內結核菌素陽性反應乳牛的乳汁樣本(群組I)、以及自5個牧場收集到461個皮內結核菌素陰性反應乳牛及山羊的乳汁樣本(群組II)。另外，也自5個廠牌共收集80個市售商品化的鮮乳樣本 (群組III)。檢測結果發現，所有群組中主要的檢出細菌族群為分枝桿菌群及金黃色葡萄球菌，HSP65在各群組的陽性率為66.8%、14.1、及40.0%； ITT陽性牛隻生乳樣本的HSP65檢出率顯著高於其他群組，且市售鮮乳樣本的檢出率亦顯著高於ITT陰性牛隻的生乳樣本；另外，Nuc基因的陽性率在各群組為50.0%、16.5%及13.8%，且ITT陽性牛隻生乳樣本檢出率顯著高於ITT陰性牛隻生乳樣本及市售鮮乳樣本。除上述兩個主要細菌族群外，M. bovis可在一頭ITT陽性牛隻生乳樣本中檢測到，且S. Typhimurium則在一頭ITT陰性牛隻生乳樣本中被檢測到。由於HSP65及Nuc基因可在生乳及市售鮮乳中檢出，顯示乳汁受到細菌及(或)其毒素汙染的風險的確是存在的；然而活菌的存在與否仍需藉由傳統的細菌培養的方法以驗證之。實驗結果顯示M. bovis可在生乳樣本中檢測到，牧場工作者須重視初乳滅菌的重要性，以降低細菌藉由生乳傳播給小牛的風險性。本實驗可藉由分子生物學方法檢測到市售鮮乳樣本中有細菌核酸的存在，進一步推論現行的巴斯德滅菌法可能有滅菌不全的風險存在或在操作過程中有人為疏失；因此政府及企業應重視此問題並予以檢討並加強管理以確保消費者的安全。

關鍵字

牛乳汁 ； 滅菌乳汁 ； 藉乳傳播之細菌 ； 核酸檢測 ； 聚合酶鏈鎖反應

並列摘要

Milk is a nutritious food for humans and animals because it contains several important nutrients, including proteins and vitamins, but it also serves as a good medium for the growth of many microorganisms, especially bacterial pathogens. The major food-borne pathogens in humans include Salmonella enterica subspecies enterica serovar Typhimurium, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, and Escherichia coli O157:H7. Milk consumption is also considered as an important transmission pathway for various diseases to animals and humans. Non-pasteurized milk is by far the most probable vehicle for the transmission of pathogenic mycobacteria, including Mycobacterium bovis and M. avium subsp. Paratuberculosis. Potential hazard exists in the commercial milk products since bacterial endotoxin could not completely be destroyed by current pasteurization methods. In order to evaluate the potential of milk-borne bacterial diseases via raw and pasteurized milk, molecular detecting methods were developed for potential milk-borne bacterial pathogens. Polymerase chain reaction with specific primer sets was used to detect specific genes of Mycobacterium spp., M. bovis, M. paratuberculosis, S. Typhimurium, L. monocytogenes, C. jejuni, S. aureus, and E. coli O157, and in milk. There were a total of 12 and 461 raw milk samples obtained from 12 tuberculin skin test-positive dairy cows in 2 dairy farms (group I) and 461 tuberculin skin test-negative dairy cows and goats from 5 dairy farms (group II). In addition, 80 commercialized pasteurized milk products were also collected from 5 brands (group III) during the surveyed period. Mycobacterium spp. and Staphylococcus aureus were the most predominant bacteria and/or bacterial toxin detected in all sample groups. The positive rates of HSP65 were 66.8%, 14.1%, and 40.0% in group I, II, and III, respectively; with a significantly higher positive rate in ITT-positive than in ITT-negative raw milk samples and in pasteurized milk than in ITT-negative raw milk samples. The positive rates of Nuc gene were 50.0%, 16.5, and 13.8 in groups I, II, and III, respectively it was significantly higher in ITT-positive raw milk samples than in ITT-negative raw milk samples and pasteurized milk samples, but there was no difference between ITT-negative raw milk samples and pasteurized milk samples. One M. bovis-positive raw milk sample was detected in group I and one S. Typhimurium-positive raw milk sample was detected in group II. Owing to the presence of detectable HSP65 and Nuc in both raw milk samples and commercial pasteurized milk products, the risk of bacterial contamination is present; however, a definitive confirmation of the presence of live bacteria in the milk should rely on bacterial culture. Since M. bovis could be detected in the raw milk samples, the dairy workers should follow the procedure of pasteurization of the colostrum restrictively prior to feeding it to calves to eliminate the possible transmission of bacterial pathogens through milk to calves. It is speculated that there might be failure in pasteurization or negligence in the procedure; therefore, the management of enterprise, and efforts from the government and the entire productive chain are required to attain consumer’s safety.

並列關鍵字

cow raw milk ； pasteurized milk ； milk borne bacteria ； nuclear acid detection ； PCR

參考文獻

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