Hulten K, Karttunen T J, El-Zimaity H M, Naser S A, Almashhrawi A, Graham D Y, El-Zaatari F A
Department of Medicine, Baylor College of Medicine, Houston, TX, USA.
Vet Microbiol. 2000 Dec 20;77(3-4):513-8. doi: 10.1016/s0378-1135(00)00336-9.
Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.
分枝杆菌的细胞壁缺陷型可能在克罗恩病和结节病的发病机制中起重要作用。然而，尚无方法可在组织切片中定位这类微生物。我们开发了一种原位杂交方法，用于在石蜡包埋的组织切片中显示副结核分枝杆菌原生质球（细胞壁缺陷型）。
副结核分枝杆菌原生质球通过用甘氨酸和溶菌酶处理制备。将制备好的原生质球注射到牛肉块中。样品用缓冲福尔马林固定并石蜡包埋。使用源自IS900基因的副结核分枝杆菌特异性探针。通过使用无关探针以及将切片与来自其他细菌的原生质球杂交来控制特异性。注射了副结核分枝杆菌原生质球的牛肉样品是唯一与探针杂交的样品。含有耻垢分枝杆菌和结核分枝杆菌的抗酸或原生质球形式以及副结核分枝杆菌抗酸形式的牛肉样品未与探针杂交。无关细菌对照，即幽门螺杆菌和大肠杆菌在该检测中也呈阴性。用IS900探针进行原位杂交为在组织切片中定位副结核分枝杆菌原生质球提供了一种特异性方法，可能有助于研究副结核分枝杆菌与克罗恩病和结节病之间的联系。该检测对于研究患有约内氏病的动物也可能有价值。
