Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Min hang District, Shanghai, China.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China.
PLoS One. 2020 Jan 29;15(1):e0228184. doi: 10.1371/journal.pone.0228184. eCollection 2020.
The existing diagnostic techniques for detecting schistosomiasis turkestanica, such as aetiological assays, identify infection by parasitic worms via the incubation of miracidia from faeces or observing eggs under microscopy. However, they are limited in the diagnosis of low-grade and prepatent infections, which lead to a high misdetection rates. Therefore, a new method for parasite diagnosis with increased sensitivity is urgently needed.
Goats in Nimu County (Tibet, China) infected with Schistosoma turkestanicum in an epidemic area were selected according positivity for the infection by faecal examination. Adult worms were collected, eggs were extracted by the sodium hydroxide (NaOH) erosion method, and soluble worm antigen preparation (SWAP) and soluble egg antigen (SEA) were isolated. The best coating concentration of the antigens and the best degree of dilution for serum were determined by square array experiments, and the optimal blocking solution and serum diluents were selected. The specificity, sensitivity and crossover of the ELISA method were determined using 48 samples of goat sera positive for S. turkestanicum, 100 samples of goat sera negative for S. turkestanicum, and 54 samples of buffalo sera positive for S. japonicum. Serological assays were established with samples from goats naturally grazed in a rural area of Nimu County, Tibet Province, by using the indirect ELISA method for the diagnosis of schistosomiasis, and faeces were collected for miracidia hatching. The sensitivity of the two detection methods was compared.
Eggs of S. turkestanicum were distributed in the host duodenum and small intestine. Eggs in the host intestinal wall were extracted by the NaOH erosion method, which provided intact eggs with reduced impurities. The testing results obtained by isolating SEA were more stable than those obtained by using SWAP and less affected by the coating concentration and serum dilution. Additionally, the value of positive serum/negative (P/N) serum for SEA was much higher than that for SWAP. The optimal coating concentration of SEA was 0.5 μg/ml, and the optimal serum dilution was 1:100. The specificity and sensitivity of the indirect ELISA based on SEA (S. turkestanicum) were both 100%, and no cross-reactivity was found with schistosomiasis japonica. An epidemiological survey of goats in naturally infected areas showed that the prevalence rate of schistosomiasis turkestanica was 93%, and the infection rate increased with the ages of the goats.
We aimed to develop a sensitive method to utilize in the mass field screening of livestock. As a diagnostic antigen, SEA (S. turkestanicum) was more suitable for serological testing than SWAP (S. turkestanicum). The indirect ELISA using SEA (S. turkestanicum) exhibited good sensitivity, specificity and no cross-reactivity with schistosomiasis japonica. The degree of infectivity and prevalence of S. turkestanicum infection in endemic areas are serious and should be a focus of concern among local departments.
现有的检测间插血吸虫病的诊断技术，如病因学检测，通过孵化粪便中的尾蚴或在显微镜下观察虫卵来识别寄生虫感染。然而，这些方法在诊断低级别和潜伏性感染方面存在局限性，导致高误诊率。因此，迫切需要一种具有更高灵敏度的寄生虫诊断新方法。
选择来自西藏尼玛县流行区感染斯氏棘口吸虫的山羊，通过粪便检查阳性来确定感染。收集成虫，用氢氧化钠（NaOH）侵蚀法提取虫卵，分离可溶性虫体抗原制剂（SWAP）和可溶性虫卵抗原（SEA）。通过方阵实验确定抗原的最佳包被浓度和血清的最佳稀释度，选择最佳的封闭溶液和血清稀释液。用 48 份斯氏棘口吸虫阳性的山羊血清、100 份斯氏棘口吸虫阴性的山羊血清和 54 份日本血吸虫阳性的水牛血清来确定 ELISA 方法的特异性、敏感性和交叉反应性。利用间接 ELISA 方法对西藏尼玛县农村自然放牧的山羊进行血吸虫病血清学诊断，并采集粪便进行尾蚴孵化，建立血清学检测方法。比较两种检测方法的敏感性。
斯氏棘口吸虫的虫卵分布在宿主的十二指肠和小肠。用 NaOH 侵蚀法从宿主肠壁中提取虫卵，得到的虫卵完整，杂质减少。SEA 的分离结果更稳定，检测结果受包被浓度和血清稀释度的影响较小。此外，SEA 的阳性血清/阴性（P/N）血清值远高于 SWAP。SEA 的最佳包被浓度为 0.5μg/ml，最佳血清稀释度为 1:100。基于 SEA（斯氏棘口吸虫）的间接 ELISA 的特异性和敏感性均为 100%，与日本血吸虫无交叉反应。对自然感染地区山羊的流行病学调查显示，间插血吸虫病的流行率为 93%，感染率随山羊年龄的增长而增加。
我们旨在开发一种敏感的方法，用于家畜的大规模现场筛查。SEA（斯氏棘口吸虫）作为一种诊断抗原，比 SWAP（斯氏棘口吸虫）更适合血清学检测。间接 ELISA 用 SEA（斯氏棘口吸虫）检测具有良好的敏感性、特异性和与日本血吸虫无交叉反应性。流行地区斯氏棘口吸虫的感染程度和流行率严重，应引起当地部门的关注。
