Gene Identification And Genetic Relationship Analysis Of Plerocercoid And T.callipaeda

Introduction With the development of molecular biology,Molecular Parasitology has also made great progress,and has become the most important branch of modern parasitology and the frontier of research.By using molecular biology techniques to study parasites,species identification and classification can be conformed at the genetic level,parasitic phenomena can be recognized at the molecular level,molecular mechanism of parasite invasion into the host can be explored,molecular targets of anti-parasitic drugs can be found,and specific antigen molecules and vaccine candidate antigen molecules for diagnosis can be isolated and identified to solve the problems of diagnosis,treatment and prevention of parasitic diseases.At present,there are many sequences of genes used to identify and classify parasite species,including nuclear DNA,mitochondrial DNA,kinetoplast DNA and plastid DNA.Mitochondrial DNA(cytb1,cox1,cox3,nad1,nad4,etc.)and ribosomal DNA(ITS1,ITS2,rrnS,etc.)are widely used to analyze the genetic structure and relationship of parasites.In particular,mitochondrial DNA has the characteristics of maternal inheritance and rapid evolution,which is easier to reflect species,strains and even interspecific differences than nuclear DNA.Mitochondrial cytochrome C oxidase I subunit gene(cox1)is recognized as a genetic marker and DNA barcode for species identification because of its small variation within species.At present,it has been used for the identification of various human and animal parasites.It has been confirmed that there are two kinds of spargania that can infect humans: S.erinaceieuropaei and S.decipiens.Both kinds have been reported in Korea.So far,only S.erinaceieuropaei has been reported in China.Especially,the reports of sparganosis in Liaoning and Hubei are limited to human case reports or animal infection rate detection.There is no basic genetic analysis and accurate species identification,and it is unclear whether they belong to S.erinaceieuropaei or S.decipiens,and it is unknown if the genotypes of isolates from both provices are similar to Korean isolates.At present,three kinds of Thelazia have been confirmed causing human infection,concluded T.callipaeda,T.californiensis and T.gulosa.There are differences not only morphological character but also geographical distribution among different Thelazia.China is the country with the largest number of reported cases diagnosed mainly on morphological characteristics or clinical symptoms,the genetic and evolutionary relationships among different species caused by long-term environmental factors such as natural geography can not be distinguished.So,we can identify the parasite species,subspecies and genetic variation using DNA sequence analysis.In this study,cox1 gene was used as a molecular marker to identify the genes of two rare parasites: plerocercoid and T.callipaeda.The phylogenetic relationship between the two parasites was analyzed to clarify the taxonomic status of S.erinaceieuropaei and T.callipaeda in China.The data obtained are of great significance to the epidemiological investigation of two rare parasitic diseases,and lay a foundation for further research on the differential diagnosis,prevention and control of diseases.Methods Morphological identification 1.The worms were placed in a dish containing physiological saline,and the general morphology of the worms was observed.2.Microstructure of the specimens observed under a microscope and then the worms were stored at 4° C in a 75% ethanol solution.PCR amplification and Sequencing of target genes 1.The worms were removed from the preservative and shredded by surgical scissors.Then,the genomic DNA was extracted by conventional phenol chloroform method after digested by SDS/protein kinase K,finally,added TE buffer solution and preserved at-20° C.2.The first set of PCR primers was p1 f and p1 r,which amplified a 440 bp product corresponding to the positions 707-1,146 bp of the cox1 gene.The second set of PCR primers,used for sequencing,was p1f1 and p1r1,which yielded a 390 bp product corresponding to positions 732-1,122 bp of the cox1 gene.These were designed from S.erinaceieuropaei(KJ599680)and S.decipiens(KJ599679);a pair of PCR primers was NTF and NTR,which amplified a 689 bp product of cox1 gene of T.callipaeda.3.The amplified products were sent to Shanghai biotech Co.,Ltd.for sequencing.Sequence alignment and analysis of genetic structure 1.The Contig Express software was used to stitch the obtained sequences,and then the Bio Edit software was used for manual correction.The corrected sequences were identified by BLAST searches and compared with the homologous sequences published in GenBank.2.The cox1 gene sequences of other tapeworms were obtained from GenBank.The DNA Star software was used to alignment the obtained sequences.Phylogenetic tree analysis of cox1 gene The phylogenetic analysis was performed with MEGA version 7.0 by Maximum Likelihood(ML),Maximum Parsimony(MP),and Neighbor Joining(NJ)methods.Clades were assessed by bootstrap resampling(1,000 replicates).Result Morphological identification 1.The size of the spargana observed in this study was 9.8~15.1 cm in length and 0.3~0.4 cm in width.And all the samples had histopathological characteristics of the larvae: body folds,dense body hair,Calcareous corpuscle and muscle bundle.2.Two isolates of T.callipaeda with a typical hexagonal trapezoidal mouth on the front supported its morphological diagnosis.NEST PCR amplification and Sequencing of target genes 1.The cox1 gene fragments(390 bp)obtained by NEST PCR amplification showed97%~100% similarity to the reference sequence of S.erinaceieuropaei(KJ599680.1)and 88%~89% similarity to the reference sequence of S.decipiens(KJ599679.1).There were differences in the bases of the cox1 gene amplified between these worms(0~12),but no obvious genetic variation(0~3.3%).There was little difference of cox1 gene between the human and frog(1%~3%).2.Both of the isolates of T.callipaeda showed 99% similarity to the reference sequence.The similarity of two samples was 98.3%.There were 13 base mutation sites(G/A at 89,149,206,221,236,467,473,608;C/T at 39,47,225,401,539).Phylogenetic tree analysis of cox1 gene 1.The phylogenetic trees constructed by MP method,NJ method and ML method were basically the same,only the bootstrap values were different.Liaoning isolates(LNZH1,LNDD1,LNJZ1 and LNJZ2)and Hubei isolates(HBXG1,HBHH1 and HBXN1)located in the same branch with the isolate of Korean cat(KJ599680.1)were highly homologous and far apart from other branches of the genus tapeworm.2.The isolate from Liaoning located in the same branch with the isolate of Italian dog(AM042555.1)were homologous to European strains,and the similarity of two specimens is 99.1%.Six base mutation sites(G/T at 8;G/A at 311,468;C/T at 39,47,401);the isolate from Shandong located in the same branch with the isolate of Romanian dog(KP087796.1),Slovak dog(KY476400.1),Hungarian cat(KX372681.1)and Serbian cat(KJ433983.1)belonged to the same branch and was also homologous to European strains.The similarity was 99.6%.There were three base mutation sites(G/T is 8;G/A is 221,257).Conclusions 1.Isolates from Liaoning and Hubei provinces could be confirmed as S.erinaceieuropaei by morphology and genotypes.2.Isolates from Liaoning and Shandong provinces could be confirmed as T.callipaeda by morphology and genotypes.3.The phylogenetic trees constructed by three different methods have consistency,and all of them shows that the isolates of Liaoning and Hubei are in the same large branch of Korean cat(KJ599680.1).It was the first time to confirm the type of the genus Spirometra of human sparganosis in both provinces.4.The human isolates from Liaoning and Shandong were homologous to European strains,which was consistent with the previous research results of our group.5.There was a small variation within species from different regions and hosts,which proved that cox1 gene could be used as molecular diagnostic marker for parasites.