摘要
目的建立弓形虫快速鉴定方法,进行国内实验动物感染调查,为国家标准的修订提供参考依据。方法选择2012年12月至2016年12月期间全国60个不同厂家的12 394只实验动物(包括:猴290只、小型猪425只、犬579只、兔1 234只、地鼠372只、豚鼠1 363只、大鼠987只、小鼠7 144只)作为调查对象。采用ELISA方法检测动物血清中弓形虫抗体IgG和循环抗原CAg。利用PCR技术鉴定弓形虫p30基因。应用直接镜检实时动态显微视屏摄录技术识别弓形虫虫体。结果用ELISA从12 394份动物血清中检出4份弓形虫抗体IgG阳性。在这4份抗体IgG阳性样本中都发现了弓形虫虫体和DNA。通过PCR扩增p30基因的分子特性证明了样本中弓形虫病原体的存在。通过直接镜检实时动态显微视屏摄录技术,在这些样本中发现了弓形虫速殖子。2012年12月至2016年12月期间,对12 394只实验动物调查结果显示,弓形虫感染率为0.03%(4/12 394)。结论 ELISA、PCR、直接镜检实时动态显微视屏摄录技术相结合可完成实验动物的弓形虫检验工作。国内实验动物中存在弓形虫感染。因此,建议设计合理的筛查程序用于防止动物传播的弓形虫病。有必要开发新的诊断工具,特别是快检技术用于这一重要人兽共患病的未来研究。
Objective In order to establish a rapid identification method for Toxoplasma gondii(T.gondii), and acquire the prevalence data on T.gondii infestation in laboratory animals in China, and provide reference for the revision of national standard, a survey was conducted. Method A total of 12 394 laboratory animals(including 290 monkeys, 425 mini-pigs, 579 dogs, 1 234 rabbits, 372 hamsters, 1 363 guinea pigs, 987 rats, 7 144 mice) came from 60 different manufactures in China between December 2012 and December 2016 were chosen as respondents. Using enzyme-linked immunosorbent assay(ELISA), sera were tested for IgG antibody and CAg antigen of T. gondii. Polymerase chain reaction(PCR) was used to detect p30 gene of T. gondii. Direct microscopy real-time dynamic video recording technique was performed to identify the toxoplasma pathogen. Result Of 12 394 examined animals 4 were positive for T. gondii IgG antibody. Molecular characterization by PCR amplification of the p30 gene demonstrated the presence of T.gondii in these animals. Tachyzoites of T. gondii were detected in these animals by using direct microscopy real-time dynamic video recording technique. Among the total 12 394 laboratory animals investigated from December 2012 to December 2016, the prevalence of T. gondii infection was 0.03%(4/12 394). Conclusion ELISA and PCR combined with direct microscopy real-time dynamic video recording techniques can be used for the detection of T.gondii. T.gondii infection was present among laboratory animals in China. Thus, it is suggested to design reasonable screening programs for preventing animal-transmitted toxoplasmosis. New diagnostic tools, especially rapid detection technologies, which is necessary to be developed in the future studies on this important zoonotic disease.
引文
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