黑龙江省林蛙肺线虫的鉴定及感染情况调查

Identification and Infection Investigation of Wood Frog Lung Nematodes in Heilongjiang Province

为了解黑龙江省林蛙肺部寄生线虫的种类和感染情况,2019年-2020年在黑龙江省采集黑龙江林蛙778只和东北林蛙174只,从其肺脏内检获线虫进行形态学鉴定后,提取虫体DNA,用通用引物对其核糖体DNA的内转录间隔区(ITS)序列进行PCR扩增,并进行克隆和测序,经Blast进行同源性分析,并应用MEGA 6.0软件构建系统发生树;同时分析林蛙肺线虫的感染情况。结果表明,黑龙江林蛙和东北林蛙检出的肺线虫形态一致,初步鉴定为蟾蜍棒线虫(Rhabdias bufonis)。获得的ITS序列长度为711 bp,与GenBank中已公布的蟾蜍棒线虫(KF999593)序列的相似性为99.43%,与蟾蜍棒线虫(KF999593)处于同一分支中。依据形态学与分子生物学鉴定结果该虫体为蟾蜍棒线虫。2种林蛙肺脏线虫总感染率为90.97%,平均感染强度为13.45条。东北林蛙肺线虫的感染率和平均感染强度分别为89.66%和18.19条;黑龙江林蛙感染率和平均感染强度分别为91.26%和12.41条,2种林蛙的感染率和感染强度差异均不显著(P>0.05)。研究结果可为黑龙江省林蛙肺线虫病的防控提供科学依据。

In order to understand the species and infection of parasitic nematodes in the lungs of wood frogs in Heilongjiang province,778 Rana amurensis and 174 Rana dybowskii were collected in Heilongjiang province in 2019-2020.After morphological preliminary identification of nematodes detected in their lungs,worm DNA was extracted.The internal transcribed spacer sequences(ITS)of its ribosomal DNA was amplified by PCR with universal primers,cloned and sequenced,and homology was analyzed by Blast.The phylogenetic tree was constructed by MEGA 6.0.At the same time,the infection of wood frog lung nematodes were analyzed.The results showed that the morphology of lung nematodes detected in R.amurensis and R.dybowskii was the same,and it was preliminarily identified as Rhabdias bufonis according to the literature.The length of its sequence was 711 bp,and the similarity with the published sequence of R.bufonis(KF999593)in GenBank was 99.43%;phylogenetic analysis showed that it was in the same branch with R.bufonis(KF999593).According to the results of morphology and molecular biology,the worms were identified R.bufonis.The total infection rate of lung nematodes of the two wood frog species was 90.97%,and the average infection intensity was 13.45.The infection rate and average infection intensity of lung nematodes in R.dybowskii were 89.66%and 18.19,respectively;The infection rate and average infection intensity of lung nematodes in R.amurensis were 91.26%and 12.41,respectively.There was no significant difference in infection rate and intensity between the two species of wood frogs in different locations(P>0.05).This study can provide a scientific basis for the prevention and control of parasitic nematode disease of cultured wood frogs in Heilongjiang province.