Eftekhari Mohsen, Mosavari Nader
Department of Microbiology, Saveh Branch of Islamic Azad University, Saveh, Iran.
Bovine Tuberculosis Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Tehran, Iran.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S222-S223. doi: 10.1016/j.ijmyco.2016.10.005. Epub 2016 Oct 27.
OBJECTIVE/BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is an etiological agent of Johne's disease in ruminant including cattle, sheep and goats. This disease is considered an economically important disease in cattle. Animals with paratuberculosis shed viable MAP, particularly in their milk and feces. MAP may be involved in the development of Crohn's disease in humans through the consumption of contaminated milk and dairy products. Common methods of pasteurization are not enough to kill all MAP present in the milk and the bacterium has been isolated from raw milk, pasteurized milk and cheese samples. The purpose of this study was to evaluate two different methods for detecting MAP in milk and milk products. We analyzed the commonly used methods such as culture and molecular biology for identification of MAP.
For this study, 50 milk samples from cows with suspected Johne's disease located in two dairy farms and 10 commercially available pasteurized milk and cheese samples from the market in Karaj city, Iran were selected. Following Ziehl-Neelsen staining of milk samples, direct microscopic detection of MAP was performed. All milk samples were centrifuged, and the concentrated samples were decontaminated using hexadecyl pyridinium chloride. The decontaminated milk suspensions were washed three times by centrifuging, and the collected filtrates were cultivated on Herrold's egg yolk medium enriched by Mycobactin J. Finally, identification and confirmation of isolates to MAP was performed using IS900-nested polymerase chain reaction (PCR).
According to the obtained results by culture and PCR methods, none of the pasteurized milk and cheese samples showed the presence of MAP. However, 10% of the tested raw milk samples collected from suspected cattle showed the presence of MAP by both culture and PCR methods.
Culture and PCR methods are reliable for identification of MAP from milk samples.
目的/背景：副结核分枝杆菌（MAP）是反刍动物（包括牛、绵羊和山羊）约翰氏病的病原体。这种疾病被认为是牛的一种经济上重要的疾病。患有副结核的动物会排出有活力的MAP，尤其是在它们的乳汁和粪便中。MAP可能通过食用受污染的牛奶和奶制品参与人类克罗恩病的发病过程。常见的巴氏杀菌方法不足以杀死牛奶中存在的所有MAP，并且该细菌已从生牛奶、巴氏杀菌牛奶和奶酪样品中分离出来。本研究的目的是评估两种检测牛奶和奶制品中MAP的不同方法。我们分析了用于鉴定MAP的常用方法，如培养法和分子生物学方法。
本研究选取了位于伊朗卡拉季市两个奶牛场的50份疑似患有约翰氏病奶牛的牛奶样本，以及10份市售的巴氏杀菌牛奶和奶酪样本。对牛奶样本进行齐-尼氏染色后，直接镜检检测MAP。所有牛奶样本均进行离心，浓缩样本用十六烷基氯化吡啶进行去污处理。将去污后的牛奶悬液通过离心洗涤3次，收集的滤液接种于添加了分枝杆菌素J的赫罗尔德蛋黄培养基上培养。最后，使用IS900巢式聚合酶链反应（PCR）对分离株进行MAP鉴定和确认。
根据培养法和PCR法获得的结果，所有巴氏杀菌牛奶和奶酪样本均未显示存在MAP。然而，从疑似患病牛采集的10%的测试生牛奶样本通过培养法和PCR法均显示存在MAP。
培养法和PCR法对于从牛奶样本中鉴定MAP是可靠的。
