家猫感染东方次睾吸虫形态学特征及分子鉴定

Morphological characteristics and molecular identification of Metorchis orientalis infection in domestic cats

目的了解家猫感染东方次睾吸虫的形态学及基因特征,为当地开展东方次睾吸虫病防控工作提供理论依据。方法采集浦城县感染有东方次睾吸虫囊蚴的麦穗鱼,剪碎囊蚴寄生处的鱼尾部肌肉,喂食2个月龄家猫。45 d后连续多天检测家猫粪便中的虫卵,直至发现虫卵后进一步深入检查。观察肝脏及胆囊变化和虫体寄生部位,捡出虫体置于生理盐水中,对虫体的形态学特征进行观察与鉴定描述。提取虫体基因组DNA,PCR扩增ITS基因序列,测序后在GenBank上进行BLAST比对,采用邻接法构建系统进化树。结果从胆囊和肝脏分离获得13条东方次睾吸虫。东方次睾吸虫为雌雄同体,虫体大小约为(1.95~2.86)mm×(0.61~0.93)mm;虫卵呈长椭圆形,似葵花籽状,虫卵大小约(28~32)μm×(16~18)μm。PCR结果显示,ITS基因序列扩增产物260 bp。BLAST比对结果显示,与国内已报道家鸭及黑天鹅感染的东方次睾吸虫(GenBank登入号分别为MT231323.1、MK482055.1、MK482052.1)的序列一致性均为99.62%。基于ITS基因的系统进化树显示,本研究分离获得的吸虫ITS序列属于次睾属,与东方次睾吸虫(GenBank登入号分别为HM347224、KX832894、MK482055、MT231323、KX857496、MK482052)属于同一个分支。结论从家猫分离的虫体经形态学及基因分析与文献报道的一致,鉴定为东方次睾吸虫。

This study sought to understand the morphological and genetic characteristics of domestic cats infected with Metorchis orientalis.The aim was to provide a theoretical basis for the prevention and control of M.orientalis in local areas.Pseudorasbora parva infected with metacercariae of M.orientalis were collected from Pucheng County.Fish tail muscles were cut,and areascontaining metacercariae were fed to 2-month-old domestic cats.After 45 days,the cats’feces were examined for several consecutive days until eggs were found and dissected.The changes in the liver and gallbladder,as well as the sites parasitized by worms,were observed.The worms were collected and placed in physiological saline.The morphological characteristics of the worms were observed and identified.The genomic DNA of the worms was extracted,and the ITS gene sequence was amplified by PCR and sequenced.Homology was analyzed with the BLAST tool in GenBank,and a phylogenetic tree was constructed through the neighbor-joining method.Thirteen M.orientalis were isolated from the gallbladder and liver.M.orientalis are hermaphrodites(1.95-2.86)mm×(0.61-0.93)mm in size.Their eggs are long and oval,similarly to sunflower seeds,and are approximately(28-32)μm×(16-18)μm in size.The ITS PCR amplification product was 260 bp in length.BLAST analysis indicated that the sequences were 99.62%consistent with those of M.orientalis(GenBank accession Nos.MT231323.1,MK482055.1,and MK482052.1)reported in domestic ducks and black swans.On the basis of the ITS gene phylogenetic tree,the ITS sequences of the trematodes isolated in this study belonged to Metorchis,and were on the same branch as[JP2]M.orientalis(GenBank accession Nos.HM347224,KX832894,[JP]MK482055,MT231323,KX857496,and MK482052).The morphology and genetic analysis findings of the worms isolated from the domestic cats were in good agreement with those reported in the literature,and the worms were identified as M.orientalis.