Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats
Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats
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来自捻转血矛线虫的粘附调节分子：诊断山羊早期感染的潜在抗原
DOI：
10.3390/pathogens9010034
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发表时间：
2020-01-01
期刊：
影响因子：
3.7
通讯作者：
Yan, Ruofeng
中科院分区：
文献类型：
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作者：
Aimulajiang, Kalibixiati;Naqvi, Muhammad Ali-ul-Husnain;Yan, Ruofeng
捻转血矛线虫是反刍动物的一种吸血线虫，在世界范围内造成巨大的经济损失。感染的诊断主要依赖于临床体征和粪便检查的评估。然而，这对于早期或轻度感染的诊断具有局限性，其中血清学诊断似乎更准确和可靠。本研究中，重组H.表达并纯化了捻转肌粘附调节分子蛋白（rHCADRM），并对其诊断价值进行了评价。实验感染H.在感染后0天（感染前，阴性对照）、7天、14天、21天、35天、49天、63天、85天和103天（DPI）收集扭体（n = 5）。用Western blot（WB）分析rHcADRM与山羊血清的反应。结果表明，rHcADRM在感染后14 d即能在血清中被识别，且抗体的维持时间可达89 d以上。未发现rHcADRM与旋毛虫、肝片吸虫和弓形虫抗体发生反应。以rHcADRM为基础，建立了间接酶联免疫吸附试验（ELISA）。通过棋盘滴定法确定最佳包被抗原（279 ng rHcADRM/孔）和血清稀释度（1：50）。共检测了64份血清标本，其中32份来自H.感染捻转虫的山羊和32只无蠕虫的山羊，用于确定ELISA的阳性（0.362）和阴性（0.306）临界值。结果表明，该方法具有较高的敏感性（90.6%）和特异性（93.75%）。批内和批间变异系数小于11%。为将该方法应用于现场检测，随机采集了51份山羊血清样品进行检测。结果表明，19.6%（10/51）的山羊感染H.扭转与尸检结果符合率为100%，高于粪检（15.7%，8/51）。这些结果提示rHcADRM可能是诊断H.山羊的捻转感染。
Haemonchus contortus, a blood-sucking nematode of ruminants, causes large economic losses worldwide. Diagnosis of infection mainly depends on the evaluation of clinical signs and fecal examination. However, this has limitations for the diagnosis of early or light infections, where serological diagnosis seems to be more accurate and reliable. In this study, the recombinant H. contortus adhesion-regulating molecule protein (rHCADRM) was expressed and purified, and its diagnostic potential was evaluated. Serum samples from goats experimentally infected with H. contortus (n = 5) were collected at 0 (before infection, negative control), 7, 14, 21, 35, 49, 63, 85, and 103 days post-infection (DPI). The reactions between rHcADRM and goat serum were tested using Western blot (WB) analysis. The results show that rHcADRM can be recognized in the serum as early as 14 DPI, and the antibody against rHcADRM in infected goat could be maintained for over 89 days. No reaction was found between rHcADRM and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immune sorbent assay (ELISA) was developed based on rHcADRM. The optimal coating antigen (279 ng of rHcADRM/well) and serum dilutions (1:50) were determined by checkerboard titration. A total of 64 serum samples, including 32 from H. contortus infection goats and 32 from helminth-free goats, were used to determine the positive (0.362) and negative (0.306) cut-off values for the ELISA. The results show this serological diagnosis method is highly sensitive (90.6%) and specific (93.75%). The coefficient of variation within run and between runs was less than 11%. To apply this indirect ELISA during field examination, 51 serum samples were randomly collected from goat farms and tested using this method. The result showed that 19.6% (10/51) of goats were infected with H. contortus, which was 100% consistent with the necropsy result, higher than that of fecal examination (15.7%, 8/51). These results indicate that rHcADRM could be a potential antigen for diagnosis of H. contortus infection in goats.