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目的了解大理地区肝片形吸虫(Fasciola hepatica)的中间宿主种类和虫体发育过程及特点。方法2012年7月~2013年7月,在大理地区收集牛源肝片形吸虫虫卵,置28℃水浴箱中孵化;并在大理农村无粪便污染的农田、沟渠内,捕捉尖膀胱螺(Physa acuta)、椭圆萝卜螺(Radix swinhoei)和小土蜗(Galba pervia),实验前证实无任何吸虫感染。毛蚴分别感染尖膀胱螺、椭圆萝卜螺和小土蜗,将螺放入泥盆中饲养并观察,每日捡出死螺,解剖观察幼虫在螺体内发育情况,直至尾蚴逸出结囊为止。最后收集10个囊蚴进行PCR,扩增其线粒体COX1基因部分序列(pCOX1)。结果前后8批次共感染1 146只椭圆萝卜螺、996只尖膀胱螺和3 307只小土蜗。2只椭圆萝卜螺感染后虽发现母雷蚴,但未见进一步发育到子雷蚴阶段,而尖膀胱螺则未能感染成功。仅小土蜗感染成功,感染率为27.2%(900/3 307)。在水温22℃时,毛蚴侵入小土蜗后,发育到胞蚴的时间为7~15 d、母雷蚴为11~20 d,子雷蚴为30~37 d,尾蚴成熟逸出,并结囊形成囊蚴的时间为42~55 d。囊蚴经PCR鉴定为肝片形吸虫,在约500 bp处出现明显的条带。结论大理地区采集的3种螺中,仅小土蜗成功感染肝片形吸虫。
Objective To understand the intermediate host species and developmental process of Fasciola hepatica and its characteristics in Dali area. Methods From July 2012 to July 2013, the eggs of S.alternatus were collected in Dali area and incubated in water bath at 28 ℃. In the ditch without farmland and dung pollution in Dali, Physa acuta, Radix swinhoei and Galba pervia, were confirmed as free from any trematode infection prior to the experiment. Mirabilis were infected with bladder cysts, oval radish snails and small earthworm, the snail into the mud in the feeding and observation, daily deadly snails were collected and dissected to observe the development of larvae in the spirochete, until the cercariae to escape from the cyst. Finally, 10 metacercariae were collected for PCR amplification of the mitochondrial COX1 gene partial sequence (pCOX1). Results A total of 1 146 eggs of Radix Elliottida, 996 Scirpus and 3 307 small earthworms were infected in 8 batches before and after the experiment. Two oval Radish snails although found after the mother mosquitoes, but no further development to the child oralei stage, while the sharp cyst is not successful infection. Only a small cochlear infection was successful, the infection rate was 27.2% (900/3 307). When the water temperature was 22 ℃, miracidia invaded the soil, the time to develop the larvae was 7-15 days, the mother larvae was 11-20 days, the sub-larvae were 30-37 days. The time of capsule formation of metacercaria was 42-55 days. The metacercariae were identified by PCR as Fasciola hepatica with obvious bands around 500 bp. Conclusion Only three soil snails were collected from Dali area, and only small soil snails were infected with Fasciola hepatica.