Naser S A, Shafran I, Schwartz D, El-Zaatari F, Biggerstaff J
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando 32816, USA.
Mol Cell Probes. 2002 Feb;16(1):41-8. doi: 10.1006/mcpr.2001.0395.
The diversity in the methodology employed to investigate Crohn's disease (CD) etiology has added significantly to the controversy of the mycobacterial role in this chronic inflammatory bowel disease. Mycobacterium avium subsp paratuberculosis (MAP), a proposed and suspected agent in many CD patients, is a fastidious and very slow grower bacillus, which causes Johne's disease (JD) in cattle. The methodology that has been widely and successfully used for isolation and identification of MAP from and in JD animals is not reliable and has proven to be unsuccessful in achieving the same objectives for CD diagnosis. In this study, a Confocal Scanning Laser Microscopy (CSLM) system has been employed in an attempt to detect MAP in CD patient. In situ hybridization was performed on full thickness tissue using rabbit anti-MAP polyclonal antibody that was adsorbed with E. coli protein extracts. Consequently, MAP was detected in the microvilli region in tissue specimens from CD patient and not in the controls. In the same CD tissue specimen, MAP was not detected when isotype normal rabbit sera was employed. The polyclonal antibody marker may be replaced with monoclonal antibodies, if available, or with MAP-specific-DNA or RNA probes. This technique adds an additional approach to investigate MAP role in CD etiology especially when the culture approach is long and inconsistent.
用于研究克罗恩病（CD）病因的方法学多样性，显著加剧了分枝杆菌在这种慢性炎症性肠病中作用的争议。副结核分枝杆菌（MAP），在许多CD患者中被认为是可疑病原体，是一种苛求且生长非常缓慢的杆菌，可导致牛的副结核病（JD）。在JD动物中广泛且成功用于从体内分离和鉴定MAP的方法不可靠，并且已证明在实现CD诊断的相同目标方面不成功。在本研究中，采用共聚焦扫描激光显微镜（CSLM）系统试图检测CD患者体内的MAP。使用与大肠杆菌蛋白提取物吸附的兔抗MAP多克隆抗体对全层组织进行原位杂交。结果，在CD患者的组织标本微绒毛区域检测到MAP，而在对照中未检测到。在相同的CD组织标本中，使用同型正常兔血清时未检测到MAP。如果有可用的单克隆抗体，或者用MAP特异性DNA或RNA探针，可以用其替代多克隆抗体标记物。该技术为研究MAP在CD病因中的作用增加了一种额外的方法，特别是当培养方法耗时且结果不一致时。
