入境黄鳝颚口线虫检疫及虫种鉴定

Detection and Identification for Gnathostoma sp. in Imported Monopterus albus

目的检查进口黄鳝（Monopterus albus）体内的颚口线虫Ⅲ期幼虫,并鉴定虫种。方法对2010年1月至2011年3月间从上海口岸入境的10批黄鳝进行颚口线虫寄生情况检疫。采样52尾,3-10尾/批,分别来自菲律宾（25尾）、印度尼西亚（24尾）和孟加拉国（3尾）,分尾解剖、切碎、蛋白酶消化后,悬液用10目铜筛过滤,取滤液沉淀。体视镜下挑出完整虫体,进行形态学鉴定,并计算感染率和感染度。提取颚口线虫基因组DNA,PCR扩增核糖体DNA第二内转录间隔区（internal transcribed spacer region 2,ITS2）和线粒体细胞色素C氧化酶亚基Ⅰ（cytochromec oxidase subunit 1,cox1）基因,对产物进行电泳和测序。测序结果与GenBank相应序列进行多重序列比对。结果从菲律宾和印度尼西亚进口的黄鳝中均检出有颚口线虫Ⅲ期幼虫寄生,阳性率分别为36.0%（9/25）和50.0%（12/24）,平均感染度分别为7.8（70/9）和2.8（34/12）。孟加拉国进口的黄鳝采样中未检出虫体。镜下显示,检获的虫体有头球,头球上有4环小钩,体表有横纹和小棘,体前部棘明显大而密,体后部棘渐小而疏。有1对颈乳突和4个颈囊。形态学特征与棘颚口线虫（Gnathostoma spinigerum）Ⅲ期幼虫相似。PCR结果显示,ITS-2和cox1扩增产物的长度分别为647 bp和441 bp,与预期大小一致。经测序后比对分析结果显示,2个扩增产物分别与棘颚口线虫ITS-2（GenBank登录号为AB181155和Z97175）和cox1（GenBank登录号为AY501388、AB180099和AB551552）基因片段序列一致性为99%~100%。结论从菲律宾和印度尼西亚进口黄鳝中检出的颚口线虫均为棘颚口线虫。

Objective To inspect the third stage larvae of Gnathostoma in imported Monopterus albus,and identify its species.Methods Ten batches of M.albus imported to Shanghai were detected for nematode Gnathostoma from January 2010 to March 2011.Fifty-two M.albus imported from the Philippines（25）,Indonesia（24） and Bangladesh（3） were sampled（3-10/batch）,which were dissected,minced,and digested.The suspension was filtered with 10 mesh screen to take the disposit.The complete parasites were picked out under stereoscope followed by morphological identification.The rate and intensity of infection were calculated.Genomic DNA of Gnathostoma was extracted to amplify internal transcribed spacer region 2（ITS-2） and cytochrome C oxidase subunit 1（cox1） by PCR,the product of which was analyzed by electrophoresis and sequencing.The sequences were aligned with corresponding sequences in GenBank.Results The third stage larvae of Gnathostoma were detected in M.albus from Indonesia and Philippines with infection rate of 36.0%（9/25） and 50.0%（12/24） and average infectiosity of 7.8（70/9） and 2.8（34/12）,respectively.No Gnathostoma was found in M.albus imported from Bangladesh.Under microscope,the larvae showed one cephalic bulb with 4 rings of hooklets on it,cross striations and small spines on the body surface.The front body spines were bigger and denser,while the rear spines were smaller and sparser.It had 1 cervical papilla and 4 cervical capsules.Morphological characteristics were similar to the third stage larvae of G.spinigerum.PCR results showed that the length of the ITS-2 and cox1 PCR products was 647 bp and 441 bp,respectively.Sequence alignment analysis showed that the two PCR products had 99%-100% consistency with G.spinigerum ITS-2（GenBank Accession No.AB181155 and Z97175） and cox1（GenBank Accession No.AY501388,AB180099,and AB551552）.Conclusion All the larvae detected in M.albus imported from the Philippines and Indonesia have been identified as G.spinigerum.