Bhide Mangesh, Chakurkar Eaknath, Tkacikova Ludmila, Barbuddhe Sukhadeo, Novak Michal, Mikula Ivan
Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine, Komenskeho-73, Kosice, Slovakia.
Vet Microbiol. 2006 Jan 10;112(1):33-41. doi: 10.1016/j.vetmic.2005.10.004. Epub 2005 Nov 21.
Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.
副结核病是反刍动物经济损失的主要原因之一，也是发展中国家和发达国家的主要健康危害。到目前为止，已经尝试了许多微生物学、血清学和分子方法来检测副结核分枝杆菌。在本研究中，我们尝试基于PCR检测牛（n = 262）和羊（n = 78）血沉棕黄层中副结核分枝杆菌特有的插入序列IS900，并通过单链构象多态性（SSCP）进行直接基因分型。共有30头（11.45%）牛和1只羊（1.28%）的副结核分枝杆菌IS900呈阳性。这种基于IS900的PCR检测具有高度特异性，尤其是在对其他非副结核分枝杆菌菌株进行检测时。SSCP分析根据不同的条带模式将副结核分枝杆菌IS900分为四个不同的簇。在本研究中，注意到从绵羊（GenBank登录号 ）和牛（GenBank登录号 -）检测到的副结核分枝杆菌之间存在核苷酸序列变异性。虽然近年来基于IS900-PCR从白细胞中检测副结核分枝杆菌的方法已应用于人类，但在动物中的应用仍然有限。我们的工作不仅支持其在动物中的应用，还建议进一步基于IS900-SSCP的副结核分枝杆菌基因分型，并结合DNA测序，作为一种快速有效的副结核病监测的有前景的工具。
