Teymouri Hedyeh, Mosavari Nader, Poor Taghi Hadi
Department of Microbiology, School of Science, Karaj Azad University, Alborz, Iran.
Bovine Tuberculosis Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S220-S221. doi: 10.1016/j.ijmyco.2016.10.006. Epub 2016 Oct 27.
OBJECTIVE/BACKGROUND: Mycobacterium avium subsp. paratuberculosis is a slow growing, Gram-positive, and acid-fast bacillus. It is the causative agent of the chronic enteritis disease of ruminants called Johne's disease. Most of the infected animals are young. However, most clinical cases belong to adult cattle aged 3-5years. Due to a prolonged incubation period, identification of subclinically infected animals is one of the most crucial problems in Johne's disease. The aim of this study was to detect M. avium subsp. paratuberculosis in cattle using indirect absorbed enzyme-linked immunosorbent assay (ELISA) and culture for positive samples in Alborz Province, Iran.
Briefly, 384 blood samples were taken from cattle of five areas of Alborz Province. Blood samples were tested by indirect absorbed ELISA (Razi paratuberculosis kit, Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran). Soluble antigens of Mycobacterium phlei were used to remove nonspecific antibodies in the bovine serum. Before transferring serum samples of cattle to the plate that coated with (MAP) antigens, serum samples of cattle were diluted and pre-incubated with a dilution buffer containing M. phlei antigens. During this project, we cultured fecal samples of cattle who displayed positive results for ELISA test. Anti-ruminant immunoglobulin G and Horse Radish Peroxidase (HRP) were added to all microwells. After washing, the substrate solution tetra-methyl benzidine (TMB) was added to eliminate the excess conjugate. The microplate was read using a spectrophotometer (ELISA reader, Bio Rad-Model 620) at 450nm in the Razi Vaccine and Serum Research Institute (Karaj, Iran). All ELISA-positive samples were cultured in media tubes (3 tubes of Herrold's egg with Mycobactin and 1 tube of Herrold's egg without Mycobactin for every sample).
In total, 3.19% of cattle serum samples showed significant antibodies titer to infection with M. avium subsp. paratuberculosis in all the areas of sample source, whereas 96.81% serum samples were negative. Of the 12 ELISA-positive samples, six samples showed growth in the media.
Because there is no treatment or cure for Johne's disease, detection of infected cattle and subsequent culling is very important for preventing infection in other cattle. Fecal culture is a standard method for the identification of the disease. However, in Johne's disease, due to prolonged incubation and shedding of the disease, the probability of isolating the responsible agent is very low. To identify the infection, the indirect absorbed ELISA method is used for eradication. This technique is considered as one of the most reliable for identification of the disease worldwide due to its ease of use and low cost. However, for confirmation of ELISA-positive results, culture method has been recommended.
目的/背景：副结核分枝杆菌是一种生长缓慢、革兰氏阳性、抗酸的杆菌。它是反刍动物慢性肠炎疾病——约内氏病的病原体。大多数受感染动物为幼龄动物。然而，大多数临床病例属于3至5岁的成年牛。由于潜伏期较长，亚临床感染动物的识别是约内氏病最关键的问题之一。本研究的目的是在伊朗阿尔伯兹省使用间接吸收酶联免疫吸附测定（ELISA）检测牛体内的副结核分枝杆菌，并对阳性样本进行培养。
简要来说，从阿尔伯兹省五个地区的牛身上采集了384份血液样本。血液样本通过间接吸收ELISA（伊朗阿尔伯兹卡拉季拉齐疫苗和血清研究所的拉齐副结核试剂盒）进行检测。用草分枝杆菌的可溶性抗原去除牛血清中的非特异性抗体。在将牛血清样本转移到包被有副结核分枝杆菌（MAP）抗原的平板之前，将牛血清样本稀释并用含有草分枝杆菌抗原的稀释缓冲液进行预孵育。在这个项目中，我们对ELISA检测呈阳性结果的牛的粪便样本进行了培养。向所有微孔中加入抗反刍动物免疫球蛋白G和辣根过氧化物酶（HRP）。洗涤后，加入底物溶液四甲基联苯胺（TMB）以去除过量的结合物。在伊朗卡拉季的拉齐疫苗和血清研究所使用分光光度计（ELISA读数仪，伯乐620型）在450nm处读取微孔板。所有ELISA阳性样本均在培养基管中培养（每个样本3管含分枝杆菌素的赫罗尔德卵培养基和1管不含分枝杆菌素的赫罗尔德卵培养基）。
在所有样本来源地区，总共3.19%的牛血清样本显示出针对副结核分枝杆菌感染的显著抗体效价，而96.81%的血清样本为阴性。在12个ELISA阳性样本中，有6个样本在培养基中生长。
由于约内氏病没有治疗方法，检测感染牛并随后进行扑杀对于防止其他牛感染非常重要。粪便培养是诊断该疾病的标准方法。然而，在约内氏病中，由于潜伏期长且疾病传播，分离病原体的概率非常低。为了识别感染，间接吸收ELISA方法用于根除。由于其易于使用和成本低，该技术被认为是全球范围内诊断该疾病最可靠的方法之一。然而，为了确认ELISA阳性结果，建议采用培养方法。
