The Preliminary Investigation Of Paragonimus Bangkokensis And The Morphological Observation And Genetic Markers Analysis Of Its Metacercaria In Yunnan Province

[Objective]This study aimed to investigate the geographical distribution of Paragonimus bangkokensis from Yunnan,the kind of natural second intermediate host and appropriate definitive host of P. bangkokensis, furthermore, to describe the kind of intermediate host, the infection status of P.bangkokensis and the morphology of metacercaria.Based on morphological classification and combined with DNA sequence analysis, to further study of the independence and taxonomic status of P. bangkokensis from Yunnan.The Genetic evolutionary tree was censtructed to confirm the homology and phylogenetic relationship with the P. bangkokensis of different areas or other species.[Methods] Crabs were collected from eight citys of Hekou,Maguan,Cangyuan, Baoshan, longchuan,Puer,Jinghong,Mengla and so on in southern and southwest of Yunnan Province.Metacercarias were separated,after measured and identified,part of them were fed to experimental cats.The DNA of Metacercarias was extracted by Kit.The ITS2and COI gene were amplified through PCR,which were compared in GenBank with Blast software and the Genetic evolutionary tree was constructed by MEGA5.0.[Result]Thirty one metacercarias of P.bangkokensis were obtained from859crabs collected from seventeen Streams of eight citys in southern and southwest of Yunnan Province.but the metacercaria of P. bangkokensis was found only in Potamon chinghungense in Jinghong city and Mengla county, while it was not found in other six citys. The natural infection rate of metacercarias from Jinghong and Mengla is2.33%and11.1%separately,the infection degree is1.2and1.25separately. The crab was identified with Potamon chinghungene that is the second intermediate host of P.bankokensis.The metacercaria is roundness or approximate roundness,which has two layer cyst walls, the outer wall is thin,crisp and easy to be broken,while the inner wall is tenacious.There is a big space between the outer and inner wall. The average size of eight metacercarias with outer wall is (551.25±29.40) μm (512.66±33.74)μm, The average thickness of its’outer wall is (5.67±1.61) μm; The average size of seventeen metacercarias which took off outer wall is (465.64±23.38) μm×(438.84±23.50) μm, the average thickness of its’inner wall is (9.77±2.80) μm. The larval body crimps as the shape of "U"style which is the most significant characteristic of metacercaria of P.bankokensis. There is a big space between larval body and inner wall when the excretory bladder arrives at intestinal bifurcation,the activity of larval body is free in the cyst. any adult worm was not found in two cats through artificial infection in this experiment.According to comparation of ITS2and COI gene of metacercaria of P. bangkokensis obtained from Jinghong city and Mengla county,it shows that the homology of ITS2and COI gene between the P.bangkokensis of this study from Yunnan Province and the P.bangkokensis from Vietanm is100%and99%, and the P. bangkokensis from Thailand is100%and96%, and the Paragonimus harinasutai from Thailand is99%and95%, the P. harinasutai from Vietnamis98%and94%, the Paragonimus microrchis from Yunnan is98%and93%. The Genetic evolutionary tree of ITS2gene shows that the P.bangkokensis of this study and the P. bankokensis from Vietanm, Thailand and Yunnan,China,and the P.harinasutai from Thailand,Vietanm and the P. microrchis from Yunnan clusted into same clade a branch at degree of confidence as100%and separate apart from other species. the Genetic evolutionary tree of COI gene shows that the P.bangkokensis of this study and the P. bankokensis from Vietanm and Yunnan,China clusted into same clade two branches at degree of confidence as65%, these two branches clusted into same clade a branch at degree of confidence as99%,then the branch and the P. bankokensis from Thailand clusted into same clade a branch at degree of confidenc as93%.[conclusion] The morphology of meta.cercaria of P.bankokensis of this study is the same as P.bankokensis from Wanyafang(2011) and other ares,which have benn researched, confirming that Jinghong city also is the distribution area of P. bangkokensis for the first time,but the infection rates of the second intermediate host is much less than Mengla county, the Potamon chinghungense is the second intermediate host of P. bangkokensis. Cat’s susceptibility is poor,it is Maybe not appropriate experimental definitive host. The analysis of DNA shows that there is the highest homology between the P. bangkokensis of this study with the P. bangkokensis from Yunnan,Thailand and Vietanm of having been reported,which are the same species.lt also shows that the homology is more close between the P.bankokensis of this study and the P.harinasutai from Vietanm,thailand and the Paragonimus microrchis from Yunnan are affinis species.