青海藏羊片形吸虫生物学特性研究

发布时间：2018-04-21 17:46

本文选题：椎实螺 + 片形吸虫　；参考：《青海大学》2016年硕士论文

【摘要】：本研究以青海藏羊体内片形吸虫和片形吸虫中间宿主椎实螺为研究对象,结合形态学和分子分类学方法进行了螺体内蚴虫以及成虫的虫种分类、鉴定。采用压片镜检的方法对青海省部分地区中间宿主螺、螺体内蚴虫感染率进行了调查,同时,用大蒜素和苦参碱两种植物提取物对椎实螺进行了急性毒性试验,以在实验室条件下,筛选出杀死中间宿主螺的有效药物,结果如下:1.在椎实螺中收集到雷蚴、尾蚴并在培养青海萝卜螺的水中收集到囊蚴。经形态学初步鉴定三种蚴虫均为肝片吸虫蚴虫,进一步提取寄生于蚴虫基因组DNA,对18S r RNA基因片段进行克隆与测序,对收集到的蚴虫进行分子分类学鉴定,所测片段长度为1805bp,用DNAMAN进行序列比对分析,显示与Gen Bank中的大片吸虫(Fasciola gigantica)18S r RNA基因序列相似性为92.03%,与肝片吸虫(Fasciola hepatica)的相似性为99.12%。综合同源进化树图谱可以进一步确定所收集的蚴虫为肝片吸虫的蚴虫。2.传统的形态学鉴定方法无法对肝片吸虫及大片吸虫的中间种类进行分类鉴定,本试验对其成虫采用18S r RNA基因克隆及序列分析的方法,测出目的片段大小为1834bp。并将测得序列用DNAMAN软件与Gen Bank中已发表的大片吸虫的18S r RNA基因序列比对,其相似性为92.92%,与肝片吸虫的18S r RNA基因序列相似性为99.78%,结合同源进化树图谱可以确定所采成虫样品为肝片吸虫。3.试验中发现所调查地区的椎实螺主要有三个种,分别为青海萝卜螺(Radix cucnnorica)、椭圆萝卜螺(Radix swinhoei)和狭萝卜螺(Radix lagotis)。通过对螺体进行压片镜检及在实验室条件下培养椎实螺,结果发现:椭圆萝卜螺体内未发现肝片吸虫蚴虫寄生,狭萝卜螺体内发现雷蚴寄生,感染率较低,10%。青海萝卜螺体内发现有雷蚴和尾蚴寄生,感染率较高,达41.33%,随后在培养青海萝卜螺的器皿中成功收集到囊蚴,而在狭萝卜螺和椭圆螺的培养过程中并未收集到囊蚴。表明青海藏羊肝片吸虫的主要中间宿主螺为青海萝卜螺。4.利用大蒜素和苦参碱两种植物提取物对椎实螺在实验室条件下浸杀灭螺效果进行了试验,研究发现,大蒜素经48h,浓度为8mg/L的条件下,死亡率为70%;苦参碱经96h,浓度为24mg/L的条件下,死亡率为100%。这两种提取物的48h的半数致死浓度(LC50)分别为6.02mg/L和45.55mg/L。表明大蒜提取物对椎实螺的灭杀效果较苦参提取物的灭杀效果好,即椎实螺对大蒜提取物的抵抗能力比对苦参提取物的抵抗能力弱。
[Abstract]:In this study, the parasites of cercariae and adults in Qinghai Tibetan sheep were classified and identified by means of morphology and molecular taxonomy. The infection rate of the intermediate host snail and the parasitic parasite in some parts of Qinghai Province was investigated by the method of pressing microscopic examination. At the same time, the acute toxicity test was carried out with two kinds of plant extracts, allicin and matrine. The effective drugs to kill intermediate host snails were screened under laboratory conditions. The results are as follows: 1. The cercariae and cercariae were collected from the vertebrae and the cysticercaria was collected in the water of the cultured turnip. The three species of cercariae were identified by morphology as paragonimiasis hepatica. The genomic DNA of the three species were further extracted, the 18s r RNA gene fragment was cloned and sequenced, and the collected cercariae were identified by molecular taxonomy. The length of the fragment was 1805bp.The sequence alignment analysis with DNAMAN showed that the similarity with the sequence of Fasciola gigantica)18S r RNA gene in Gen Bank was 92.03 and 99.12% with that of Fasciola hepatica. According to the phylogenetic tree map, we can further determine that the collected metacercaria is a metacercaria of clonorchiasis liver. The traditional morphological identification method could not classify and identify the middle species of paragonimiasis and paragonimiasis. In this experiment, 18s r RNA gene cloning and sequence analysis were used to detect the target fragment size of 1834bp. The sequence was compared with the 18s r RNA gene sequence of the trematode published in Gen Bank by DNAMAN software. The similarity was 92.92%, and the similarity of 18s r RNA gene was 99.78 with that of paragonimus hepatica. The adult samples were determined to be hepatic paragonimiasis by homologous phylogenetic tree map. It was found that there are three main species in the investigated area, Radix cucnnorica, Radix swinhoeii and Radix lagotis. The results showed that there was no parasitism of paragonimiasis in oval radish snail, while that of Rayleidae in radish snail was lower than that in radish snail, and the infection rate was lower than that in radish snail. The parasitic larvae and cercariae were found in the body of turnip snails in Qinghai province, and the infection rate reached 41.33. The cysticercaria was collected successfully in the vessel of cultivating turnip snail, but no cysticercaria was collected in the culture process of radish snail and elliptical snail. The results showed that the main intermediate host snail of the Tibetan sheep liver clonorchiasis in Qinghai was the turnip snail. 4. Two plant extracts, allicin and matrine, were used to study the effect of imbibition on molluscicidal effect in laboratory. The results showed that the mortality rate of allicin was 70 under the condition of 48 h and concentration of 8mg/L, and the concentration of matrine was 24mg/L for 96 h. The mortality rate is 100. The LC50 for 48h of the two extracts was 6.02mg/L and 45.55 mg / L, respectively. The results showed that garlic extract was more effective than Sophora flavescens extract in killing vermicelli, that is to say, the resistance ability of garlic extract was weaker than that of Sophora flavescens extract.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.7

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