Sonawane Ganesh G, Tripathi Bhupendra N
Animal Health Division, ICAR-Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India.
ICAR-National Research Centre on Equines, Hisar, Haryana, India.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S88-S89. doi: 10.1016/j.ijmyco.2016.09.005. Epub 2016 Oct 21.
Paratuberculosis or Johne's disease is a chronic infectious granulomatous enteritis, mainly of cattle, sheep, goats, and other domestic and wild animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). Currently, MAP has been recognized as an important animal pathogen with significant zoonotic and public health concerns. The early detection of infected animals using suitable diagnostic methods helps in developing control and preventive strategies for the herd. Therefore, the present study was aimed to determine the comparative efficacy of certain diagnostic methods used in the identification and confirmation of MAP in the ovine tissues with distinct pathology of paratuberculosis.
The ileum and mesenteric lymph node (MLN) tissues were collected from 38 sheep infected with paratuberculosis from organized farms of Rajasthan. These animals were further classified as paucibacillary (PB; n=15) or multibacillary (MB; n=23) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN tissues of these animals were subjected to IS900 and 251 gene polymerase chain reaction (PCR) and bacterial culture. The tissue sections from MB, PB, and uninfected control sheep groups were stained using indirect immunoperoxidase technique (IPT) and Ziehl-Neelsen (ZN) method.
On bacterial culture examination of the ileum and MLN tissues using Herrold's egg yolk medium, MAP was isolated in 14 (60.9%) MB and 5 (33.3%) PB sheep. Of 38 sheep, IS900 PCR detected 21 (55.2%) positive for MAP, of which 19 (82.6%) were MB and 2 (13.3%) were PB sheep. Similarly, 251 gene PCR detected 25 (65.7%) sheep positive for MAP infection, of which 21 (91.3%) were MB and 4 (27%) were PB sheep. Thus, 251 gene PCR was found superior to IS900 PCR in the detection of MAP from the tissues. In PB sheep, IPT and ZN tests were positive in 87.5% and 50% of ileum sections and 70% and 37.5% in MLN sections, respectively. In MB sheep, IPT and ZN tests detected all animals as positive for MAP organisms or antigen and had equal sensitivity in the detection of MAP. The overall sensitivity of IPT was found superior (95%) to ZN staining (80%) in the demonstration of acid-fast bacteria or its antigen in the tissues.
The sensitivity of all the tests in the detection of MAP was lower in PB sheep than in MB sheep. Bacterial culture detected MAP in only 50% of sheep and was found less sensitive than other tests used in the present study. Comparing the overall sensitivity of both the PCR assays, 251 gene PCR was found superior to IS900 gene PCR. The sensitivity of IPT was found superior (95%) to the ZN staining (80%) in the demonstration of acid-fast bacteria in the tissues. In the present study, IPT was found superior in the detection of MAP in PB and MB form of ovine paratuberculosis. This test can be used in the confirmation of post mortem diagnosis and research of paratuberculosis along with histopathology. However, 251 gene PCR assay was found easier to perform than IPT and could be used as paratuberculosis screening test in endemic sheep farms for blood and fecal samples.
副结核病或约翰氏病是一种慢性传染性肉芽肿性肠炎，主要发生于牛、绵羊、山羊以及其他家养和野生动物，由副结核分枝杆菌（MAP）引起。目前，MAP已被公认为一种重要的动物病原体，对人畜共患病和公共卫生具有重大影响。采用合适的诊断方法对感染动物进行早期检测，有助于制定畜群的控制和预防策略。因此，本研究旨在确定某些诊断方法在鉴定和确认具有副结核病明显病理特征的绵羊组织中MAP的比较效果。
从拉贾斯坦邦有组织养殖场的38只感染副结核病的绵羊中采集回肠和肠系膜淋巴结（MLN）组织。根据组织病理学结果和分枝杆菌载量，将这些动物进一步分为少菌型（PB；n = 15）或多菌型（MB；n = 23）。对这些动物的回肠和MLN组织进行IS900和251基因聚合酶链反应（PCR）以及细菌培养。对MB、PB和未感染对照绵羊组的组织切片采用间接免疫过氧化物酶技术（IPT）和齐-尼氏（ZN）法进行染色。
使用赫罗尔德蛋黄培养基对回肠和MLN组织进行细菌培养检查时，在14只（60.9%）MB绵羊和5只（33.3%）PB绵羊中分离出MAP。在38只绵羊中，IS900 PCR检测出21只（55.2%）MAP阳性，其中19只（82.6%）为MB绵羊，2只（13.3%）为PB绵羊。同样，251基因PCR检测出25只（65.7%）绵羊MAP感染阳性，其中21只（91.3%）为MB绵羊，4只（27%）为PB绵羊。因此，在从组织中检测MAP方面，发现251基因PCR优于IS900 PCR。在PB绵羊中，IPT和ZN试验在回肠切片中的阳性率分别为87.5%和50%，在MLN切片中的阳性率分别为7%和37.5%。在MB绵羊中，IPT和ZN试验检测到所有动物的MAP生物体或抗原均为阳性，在检测MAP方面具有相同的敏感性。在组织中显示抗酸菌或其抗原方面，发现IPT的总体敏感性（95%）优于ZN染色（80%）。
在检测MAP方面，所有试验在PB绵羊中的敏感性均低于MB绵羊。细菌培养仅在50%的绵羊中检测到MAP，并发现其敏感性低于本研究中使用的其他试验。比较两种PCR检测方法的总体敏感性，发现251基因PCR优于IS900基因PCR。在组织中显示抗酸菌方面，发现IPT的敏感性（95%）优于ZN染色（80%）。在本研究中，发现IPT在检测绵羊副结核病的PB和MB形式的MAP方面更具优势。该试验可与组织病理学一起用于死后诊断的确认和副结核病的研究。然而，发现251基因PCR检测比IPT更容易操作，可作为地方病绵羊场血液和粪便样本的副结核病筛查试验。
