Characterization Of Fasciola By SSR Markers And SCAR Markers

Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica.Fascioliasis in ruminants has a global geographical distribution and causes substantial economic losses,estimated at US$ 2 billion per annum worldwide.Importantly,human can also become infected with Fasciola.Fasciola hepatica and Fasciola gigantica are similar in their appearance,but different in antigenicity,immunogenicity,pathogenicity to host and sensibility to drug,so identifying exactly of them is important both for disease control and for epidemiological studies.For a long time,Fasciola were mainy characterized by morphology,growth cycle, epidemiology and so on,which played a very important role in the taxology.The use of traditional classification methods was limited because the wide variation of morphology in Fasciola hepatica and Fasciola gigantica.So other methods had to been sought.Along with the development of cytology and molecular biology,many cytological and molecular methods have been applied to the identification and genetic variation of Fasciola.Now,there are many classification methods including morphological,cytological and molecular ones,but they leaded different results.So we had to seek other methods.SSR marker and SCAR marker are new markers developed in recent years.SSR marker is very usefull for the identification and classification of parasite due to its abundance,uniform distribution,high polymorphism,codominance,rapid amplification by PCR. SCAR marker is convenient,shortcut,reliable,stable,high-repeatability and can detect numerous individium in high-speed.So we try to apply these two kinds of markers for the identification of Fasciola.In this study,the adult trematodes of Fasciola sampled from Chongqing,Sichuan and Guangxi area were conducted by SSR markers and SCAR markers.On one side,the DNA of Fasciola were extracted for amplification by 17 SSR primers from websid,and the PCR products of microsatellites were detected by polyacry lamide gel electrophoresis,then generic diversity analyses and cluster analysis were performed.Another.6 set of ISSR primer were used to amplificate in Fasciola and specific bands were cloned and sequenced,based on the sequences, SCAR pimers were designed to convert into SCAR markers which can identify Fasciola.The results were as follows:1.Webside http://bioinformatics.pbcbasc.latrobe.edu.au/cgi-bin/ssr provide EST-SSR sequences and primers of many species,from which 4 Fasciola sequnces,8133 Schistosoma mansoni sequences and 6183 Schistosoma japonicum sequences were abtained.Then 17 SSR primers including 4 SSR primers of Fasciola,6 SSR primers of Schistosoma japonicum and 7 SSR primers of Schistosoma manson,were chose to investigated the genetic variation of Fasciola.Out of 17 locus 13 could amplify and revealed 79 alleles,each locus had 6.2,and the effective number of allele varied from.4-10,which suggested that Fasciola had abundant genetic diversity.2.Genetic distance of 8 Fasciola individuals were computed by software NTSYS-pc.The result showed that the genetic distance between Fasciola hepatica and Fasciola gigantica was 0.425-0.5, the genetic distance in Fasciola hepatica was 0.2-0.4 and in Fasciola gigantica was 0.3625,which suggested variation between-group was greater than within-group which is also obvious.3.Clusteration was analyzed based on genetic distance by software NTSYS-pc.8 Fasciola individuals were clustered to two classes:the first class was Fasciola gigantica,including specimine in buffalo from Guangxi and specimine in buffalo from Chongqing,the second class was Fasciola hepatica,including specimines from Chongqing(except specimine in buffalo)and specimine from Sichuan.The second class were divided into two groups,specimine from Sichuan was in the first group;Specimines from Chongqing(except specimines in buffalo)were in the second group.The result indicated preliminarily that SSR markers not only could differentiate Fasciola but also could differentiate Fasciola from different Geographical location.4.6 ISSR primers were used to amplificate in Fasciola from Chongqing,Sichuan and Guangxi, and 2 ISSR primers(GACA)₄ and(AG)₈ could amplify 4 specific bands:(GACA)₄ could amplify 2 Ehepatica specific bands about 900 bp named ISSRF.h1 and ISSRF.h2 and 1 F.gigantica specific band about 900 bp named ISSRF.g,(AG)₈ could amplify 1 F.hepatica specific band about 300-400bp namely ISSRF.h3.These bands were cloned and sequenced,the result showed that the length of ISSRF.h1 was 919 bp,ISSRF.h2 was 806bp,ISSRF.h3 was 364bp and ISSRF.g was 845bp.5.The four set of SCAR primers containing ISSR primers were designed to convert into SCAR markers,in which 3 for F.hepatica named SCARF.h1,SCARF.h2 and SCARF.h3,and I for F.gigantica named SCARF.g.Verification showed that only F.hepatica could result the specialty PCR fragments with primers SCARF.h1,SCARFh2 and SCARF.h3,and only F.gigantica could result the specialty PCR fragment with primers SCARF.g,which indicated that the ISSR markers were successfully converted into SCAR markers.These 4 SCAR markers provide a new method to identify Fasciola..6.We find that there is one strain of Fasciola sp in buffalo from Chongqing with atypical sharpe,which was between F.hepatica or F.gigantica morphologically,and its body length was beyond 3 cm,length-width ratio was above 3,shoulder was not obvious,tail like "U",it was suspected "intermediate genotype" of Fasciola.It was identified as Fasciola gigantica by SSR analysis and SCAR analysis.