用于检测亚洲象出血性败血症抗体的内部间接酶联免疫吸附测定法的评估

Evaluation of an In-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants.

作者信息

Tankaew Pallop, Singh-La Thawatchai, Titaram Chatchote, Punyapornwittaya Veerasak, Vongchan Preeyanat, Sawada Takuo, Sthitmatee Nattawooti

机构信息

Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai 50100, Thailand; Center for Excellence in Elephant Research and Education, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai 50100, Thailand.

Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai 50100, Thailand.

出版信息

J Microbiol Methods. 2017 Mar;134:30-34. doi: 10.1016/j.mimet.2017.01.008. Epub 2017 Jan 12.

DOI:10.1016/j.mimet.2017.01.008

PMID:28088341

Abstract

Pasteurella multocida causes haemorrhagic septicemia in livestock and wild animals, including elephants. The disease has been reported in Asian elephants in India and Sri Lanka, but to date there have been no reported cases in Thailand. ELISA or indirect hemagglutination assays (IHA) have been demonstrated to be able to detect the antibody against the disease in cattle, but no data are available for elephants. The present study reports a novel in-house indirect ELISA for antibody detection of haemorrhagic septicemia in Asian elephants, and evaluates the sensitivity and specificity of the method using a Bayesian approach. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between these two diagnostic tests. The IHA was performed as recommended by the World Organization for Animal Health (OIE) manual for haemorrhagic septicemia. An in-house indirect ELISA was developed with a heat extract antigen of P. multocida strain M-1404 (serovar B:2) as a coating antigen and rabbit anti-immunoglobulin G conjugated with horseradish peroxidase (eIgG-HRP). The checkerboard titration method was done using elephant sera immunized with P. multocida bacterin and negative sera from colostrum-deprived elephant calves. The concentrations of heat extract antigen (160μg/ml), sample serum (1:100), and eIgG-HRP (1:1000) were optimal for the assay. The calculated cut-off value was 0.103. Of the elephant sera, 50.59% (43/85) were considered seropositive by ELISA. The sensitivity of the ELISA test was higher than that of the IHA test [median=86.5%, 95% posterior probability interval (PPI)=52.5-98.9%] while the specificity was lower (median=54.1%, PPI=43.6-64.7%). The median sensitivity and specificity of IHA were 80.5% (PPI=43.8-98.0%) and 78.4% (PPI=69.0-87.0%), respectively. These findings suggest that our in-house indirect ELISA can be used as a tool to detect the antibody against haemorrhagic septicemia in Asian elephants.

摘要

多杀性巴氏杆菌可导致家畜和野生动物（包括大象）患出血性败血症。在印度和斯里兰卡的亚洲象中曾有该病的报道，但迄今为止泰国尚未有病例报告。酶联免疫吸附测定（ELISA）或间接血凝试验（IHA）已被证明能够检测牛体内针对该疾病的抗体，但尚无大象的相关数据。本研究报告了一种用于检测亚洲象出血性败血症抗体的新型内部间接ELISA方法，并使用贝叶斯方法评估了该方法的敏感性和特异性。使用单总体贝叶斯模型分析ELISA和IHA的特征，该模型假设这两种诊断测试之间存在条件依赖性。IHA按照世界动物卫生组织（OIE）出血性败血症手册的建议进行。以多杀性巴氏杆菌M - 1404菌株（血清型B:2）的热提取物抗原作为包被抗原，辣根过氧化物酶偶联的兔抗免疫球蛋白G（eIgG - HRP），开发了一种内部间接ELISA。采用经多杀性巴氏杆菌菌苗免疫的大象血清和初乳缺乏的大象犊牛的阴性血清进行棋盘滴定法。热提取物抗原（160μg/ml）、样本血清（1:100）和eIgG - HRP（1:1000）的浓度对该检测最为适宜。计算得出的临界值为0.103。在大象血清中，ELISA检测显示50.59%（43/85）被认为血清呈阳性。ELISA检测的敏感性高于IHA检测[中位数 = 86.5%，95%后验概率区间（PPI）= 52.5 - 98.9%]，而特异性较低（中位数 = 54.1%，PPI = 43.6 - 64.7%）。IHA的敏感性和特异性中位数分别为80.5%（PPI = 43.8 - 98.0%）和78.4%（PPI = 69.0 - 87.0%）。这些发现表明，我们开发的内部间接ELISA可作为检测亚洲象出血性败血症抗体的工具。