Observation By Scanning Electron Microscopy And Molecular Biological Identification On Fasciola In Dali,Yunnan

[Objective]To provide some basic information about the biological characteristics and genetic differentiation of Fasciola in Dali,Yunnan.Some typical structures of cephalic cone of Fasciola(oral sucker、ventral sucker、genital pore、spines)in Dali,Yunnan were observed by Scanning Electron Microscopy,meanwhile,Using molecular biology method to identify the species of Fasciola in this area.[Methods]1.Partial structures of Fasciola in Dali,Yunnan were observed by Scanning Electron Microscopy:Obtained 10 article Fasciolas from four natural infected cattle liver bile duct in Dali market,Rinse with saline to the blood,then each divided into four bottles filled with saline,marking by 1、2、3、4.Select two complete worms marking byA、B from bottle 4;Select two complete worms marking byC.Dfrom bottle 3.collect four cephalic cone of Fasciola,After the cold overnight treatment,According to the steps of making the sample of scanning electron microscope:Drawn,fixed,dehydrated,embedded,sliced,stained,after these steps,then observed by Scanning Electron Microscopy.2.Extract the target genomic DNA from mature Fasciola:Select six complete worms marking by 1、2、3、4、5、6.Select two complete worms marking by 7、8,Take 1/3 front part of these parasites,inclouding cephalic cone,After rinsing with saline,then washed with ultra-pure water 4 to 5 times,at last assembled by disinfected 1.5ml centrifuge tube.pound these parasites to pieces with hand-held tissue grinding machine,extract the target genomic DNA according to the method on TaKaRa MiniBEST Universal Genomic DNA Extraction KitVer.5.0 kit instruction,the DNA concentration of the eight samples was then examined using a nucleic acid quantifier.3.PCR amplification and sequencing:The ribosomal ITS-2 gene fragment,mitochondrial cox l partial gene fragment and mitochondrial nad5 gene fragment of Fasciola were amplified,then the PCR product was sented to Dalian Bao biological company and sequenced.4.Sequence analysis:The resulting sequence was compared with the previously reported standard sequence using DNAStar7.0 software.[Results]1.Except that one of the them its genital pore was not obviously observed anthoer three Fasciolas both have the typical structure of Fasciola:oral sucker、ventral sucker.genital pore and the spines spread all over the worms,the diameter of oral sucker range from 310μm to540μm,the diameter of ventral sucker range from 45μm-600μm.After partial enlargement of these oral sucker and ventral sucker,there are two forms of papillae:dome-shaped papillae and ciliate papillae,part of the spines were under high magnification using Scanning electron microscopy,The results show that it grows in the surface of tegument of worm,with a wider substrate and the end is a narrow arc-shaped structure,the end of the growth has a zigzag structure.2.Eight Fasciolas were identified by molecular biology showing that:Two of them were amplified by PCR and agarose gel electrophoresis showed the target band of F.gigantica.these samples are identified as F.gigantica;Another six samples were amplified by PCR and agarose gel electrophoresis showed the target band of F.hepatica.these samples are identified as F.hepatica.3.The mitochondrial partial coxl gene of Fasciola in Yunnan was PCR amplificated,then sequenced by DNAStar software,its intaraspecific variation is 0.9%-2.3%,its interaspecific variation is 3.0%-10.0%;the mitochondrial partial nad5 gene of Fasciola in Yunnan,its intaraspecific variation is 0.9%-1.2%,its interaspecific variation is 5.0%-13.0%,so the intaraspecific variation of Fasciola was low,whereas the interaspecific variation f Fasciolai was signifiacnt.[Conclusions]1.Scanning Electron Microscopy of Partial Structure of Fasciola in Yunnan shows that three of them are F.hepatica,one of them are F.gigantica.except that one of the them its genital pore was not obviously observed,another three Fasciolas have its typical structure(oral sucker、ventral sucker、genital pore),the tegument was armed with dense spines and the distribution of the spines around oral sucker are intensive than ventral sucker.2.Based on the study of ribosomal ITS-2 gene as a classification of genetic markers,there are two kinds of Fasciola in Dali,Yunnan.3.The ribosomal ITS-2 gene and the mitochondrial coxl and nad5 gene sequences were sequenced and analyzed by DNAstar software and its result shows that the intaraspecific variation of Fasciola was low,whereas the interaspecific variation f Fasciolai was signifiacnt.