中国实验动物中鼠管状线虫的分子鉴定和感染调查

Prevalence and molecular identification of Syphacia muris in laboratory animals in China

目的对鼠管状线虫进行分子鉴定和感染调查,为国家标准的修订提供参考依据。方法 923批5199只SPF动物(包括:1批5只猴,3批25只小型猪,28批55只兔,13批248只地鼠,37批198只豚鼠,93批459只大鼠,742批4179只小鼠,5批25只鸡和1批5只鸭)和145批1389只清洁动物(包括:1批3只兔,4批31只地鼠,16批157只豚鼠,32批268只大鼠和92批930只小鼠)来自全国50个不同的厂家。应用直接镜检实时动态显微视屏摄录技术结合形态学鉴定方法,进行鼠管状线虫感染筛查。应用多重PCR和测序技术,鉴定分离的鼠管状线虫ITS(内转录间隔区)、28S rRNA(28S核糖体RNA)、nad1(NADH脱氢酶亚单位1)和cox1(细胞色素C过氧化物酶亚基1)基因,从分子水平上确证鼠管状线虫感染。结果应用直接镜检实时动态显微视屏摄录技术,从动物中检出鼠管状线虫的虫卵、幼虫和成虫。根据鼠管状线虫的卵细胞、幼虫、雌雄成虫的大小和形态来鉴定虫种。应用多重PCR测序技术,能从分离的单个鼠管状线虫的虫卵、幼虫和成虫中鉴定出ITS、28S rRNA、nad1和cox1基因,与其他不同种属的寄生虫无交叉反应。应用直接镜检实时动态显微视屏摄录技术,从5199份SPF和1389份清洁动物样本中分别检出鼠管状线虫阳性样本285份和135份。应用多重PCR和测序技术,鉴定证明这些阳性样本中确实含有鼠管状线虫特异性的DNA。测序结果显示,不同动物分离的鼠管状线虫的ITS、28S rRNA、nad1和cox1部分基因序列核苷酸相似性达100%。SPF和清洁动物的鼠管状线虫感染率分别为5.5%(285/5199)和9.7%(135/1389)。结论应用直接镜检实时动态显微视屏摄录技术联合多重PCR测序技术能够快速精准检测鉴定出鼠管状线虫。鼠管状线虫的人兽共患本质可以视作为公共卫生的一个预警。良好的动物质量控制对保护人类身体健康和保障人民用药安全具有重要作用。本研究对中国SPF和清洁动物的鼠管状线虫进行了分子鉴定和感染调查。

Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals（ including one batch of 5monkeys,3 batches of 25 mini-pigs,28 batches of 55 rabbits,13 batches of 248 hamsters,37 batches of 198 guinea pigs,93 batches of 459 rats,742 batches of 4179 mice,5 batches of 25 chickens and one batch of 5 ducks） and 145 batches of1389 clean animals（ including one batch of 3 rabbits,4 batches of 31 hamsters,16 batches of 157 guinea pigs,32 batches of 268 rats and 92 batches of 930 mice） came from 50 different manufactures in China. Direct microscopy real-time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction（ multiple-PCR） testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer（ ITS）,28 S ribosomal RNA（ 28 S rRNA）,NADH dehydrogenase subunits 1（ nad1） and cytochrome c oxidase subunit 1（ cox1） genes,and the molecular sequencing of the multiple-PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs,larvae and adults were detected by using direct microscopy real-time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum,larvae,and female and male adult worms. Multiple-PCR and sequencing were performed to identify ITS,28 S rRNA,nad1 and cox1 genes of DNA extracted from the single egg,larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple-PCR amplification and sequencing of the ITS,28 S rRNA,nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple-PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real-time dynamic video recording technique in the study as containing Syphacia muris-specific DNA. Comparison of the partial sequences of the ITS,28 S rRNA,nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5. 5%（ 285 /5199） and 9. 7%（ 135 /1389）,respectively. Conclusions Direct microscopy realtime dynamic video recording technique,multiple-PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter,hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.