The Research On Polymorphisms Of Ribosomal And Mitochondrial DNA Of Ascaris From Five Kinds Of Wild Animals

Adult worms of Ascaris are a kind of large nematode worms attach to the hosts’ small intestine which parasitise all major groups of vertebrates, and some of these ascaridoids are of major animal and/or human health hidden danger causing slow growth, malnutrition, sharp weight loss, anemia,bellyache and steatorrhea etc. The migration of larva results mechanical injury of hosts’ visceral organs causing visceral larva migrans (VLM), ocular larva migrans (OLM), even death if seriously. Ascariasis is a worldwide distributed disease having great ham to animals as well as human beings. Anciently, research on ascaridoid was mainly focus on the morphological description, life cycle and the ascariasis was on the epidemiology, diagnosis and comprehensive prevention. In recent years, the research of ascaridoid on molecule level is developing, but there are few reports on molecular classification and phylogenesis about ribosomal and mitochondrial gene sequences of ascaridoid from wild animals. Ribosomal DNA (rDNA) was generally existed in one or multi-chromosomes of living nature, and it has a great deal of features applying for as the molecular genetic maker DNA, for example in the different regions there is abundance of multicopy and different revolutionary rates; it’s suitable to differentiate populations between interspecies because its noncoding region of the rDNA has faster revolutionary rate; to contrive the universal primers in the coding region is much easier, in phylogenetic analysis, it is widely known and employed as the molecular genetic maker. There are total three ribosomal gene coding regions (18S,5.8S and28S) in the ribosomal DNA, two internal transcribed spacer regions (ITS-1and ITS-2). Because of maternal inheritance existed in mitochondrial DNA (mtDNA) one mitochondrial genome may stand for the whole variation. The ITS,28S and the cox1, nad1and nad4genes (derived from Equus burchelli, Ursus thibetanus, Panthera leo. Canis lupus and Sus scrofa) were amplified by PCR sequenced, then compared with other sequences, and constructed phylogeny trees of ascaris from5wild animals then determined the molecular phylogeny.Ascaris were isolated from the faeces of Equus burchelli, Ursus thibetanus, Panthera leo, Canis lupus and Sus scrofa after anthelminthic by morphology appraisal. Genomic DNA was extracted by the method of SDS-proteinase K. The each corresponding genes were amplified by PCR. The restriction fragment length polymorphism (RFLP) emthod was used to differentiate ascaris parasitize in Panthera leo and Canis lupus which were usually confused, and then, the5corresponding sequences were cloned into pMD18-T vector and sequenced, then compared with corresponding squences of other ascaris published on GenBankTM respectively, the phylogenic trees were generated by PAUP4.0, Modeltest3.7, MEGA and MrBayes3.1softwares. Phylogenetic relationships were reconstructed using the Maximum Likelihood methods (ML), the maximum parsimony method (MP) and Bayesian. The length of ITS-1+ITS-2of the ascaris from five kinds of wild animals was657bp、703bp、689bp、808bp and752bp, respectively. The similarity of the five samples with the corresponding sequence of Equus burchelli, Ursus thibetanus, Panthera leo, Canis lupus and Sus scrofa was98.3%、98.7%、96.1%、99.8%and99.9%. The ascaris from Equus burchelli, Ursus thibetanus, Panthera leo, Canis lupus and Sus scrofa were Parascaris equorum, Baylisacaris transfuga, Toxascaris leonina, Toxocara canis and Ascaris suum, respectively. The identification results of PCR-RFLP were the same with morphological identification results and successfully identified the Toxascaris leonina and Toxocara canis. Sequencing results showed that the evolutionary trees reconstructed by ITS, cox1, nad1and nad4were same. Parascaris equorum, Baylisacaris transfuga, Toxascaris leonina and Ascaris suum in relationship were near with each other while they were fay with Toxocara canis.This study identify the ascaris from five kinds of wild animals from zoo by molecular biology means and the results were same with that by Morphological methods. Identify the similar ascaris of Toxascaris leonina and Toxocara canis by PCR-RFLP method which is accurate efficient and saveing resources. The evolutionary analysis of the five kinds of wild animals were made by ML、MP and Bayesian based on sequences of ITS、 cox1、 nad1and nad4, the evolution and classification status were fixed accurately and lay the foundation for further research and prevention on ascaris from wild animals.