| Objective Paragonimus, commonly known as lung flukes, some species of which in the human body can cause paragonimiasis. Paragonimus belongs to the general community, animal kingdom, phylum Platyhelminthes, class Trematodes, order Digenea. Many disputes were related with traditional classification of Paragonimus. They mainly distribute in eastern Asia, southeast Asia, Africa and Latin Aamerica. In China, they distribute in Yangtze River basin, southwest and northwest areas. It has been already 100 years for researching Paragonimus since 1879 when Ringer first reported Paragonimus parasitized in the lung of a sailor in Taiwan. Scholars have different perspectives about the characteristics, independent status, geographical distribution and pathogenesis of Paragonimus. There are some limitations about the traditional classification, which based primarily on the morphology of adult Paragonimus and life history, such as differences in morphology of adult from different regions, different hosts, different developmental stages and individual divergence of the specimens. At present, the life history was clarified, only in a small number of Paragonimus. The first and second intermediate hosts of mang Paragonimus had not yet been confirmed. Since 1990, molecular biological methods have been popularly used in studies on genetic variation and classification. In this research,12 strains of Paragonimus were collected from 10 districts of 3 provinces in China. The technique of amplification by PCR, and sequencing was used to analyze the genetic variations and to investigate the phylogenetic relationship among these strains.Materials and methods 12 strains of Paragonimus were collected from 10 areas of 3 provinces in China:hot springs area of Hubei-Xianning-Wenquan, Jiangxi-Wuning, Zhejiang-Shaoxing-Lanting, Hubei-Chibi-Zhaoliqiao, Hubei-Shiyan, Hubei-Wufeng, Hubei-Hefeng-Wuli, Hubei-Hefeng Zouma, Hubei-Suizhou and Hubei-Shennongjia. The samples collected from hot springs area of Hubei-Wenquan, Jiangxi-Wuning, Zhejiang-Lanting and Hubei-Shiyan were adults. The samples collected from hot springs area of Hubei-Suizhou were larva. The rest were metacercariae. The populations were named after the names of collecting sites temporarily. We observed the exterior morphological features of forth adults, one larva and five metacercariae by naked eyes or stereoscope and then recorded them and extracted the genomic DNA. The ITS2 gene fragment was amplified with 3s'and BD2 using PCR. Then, the genetic distances and sequence similarity of Paragonimus from various strains were calculated by Bioedit, Clustalx and MEGA4.0. The phylogenetic tree was thereby mapped.Results 1. Exterior morphological features Adult:the shape of Hubei-Shiyan strains is long-fusiform and another three is oval.; Metacercaria:the shape of all metacercariae is round and there are two layers of capsule wall. The inner layer of Hubei-Chibi strains is thicker than that of Wufeng(a), Wufeng(b), Zouma(a), Zouma(b) and Wuli strains. The exterior morphological features of Shennongjia strains cannot be clearly observed because of damages, but it can be confirmed as metacercaria of Paragonimus. Larva:the shape of Hubei-Suizhou strains is long-fusiform 2. DNA extraction:The A260/A280 value of DNA solution ranged from 1.60-2.00, the purity is qualified.3. PCR amplification and sequencing:The genetic distances from Hubei-xianning, Jiangxi-Wuning, Zhejiang-Lanting and Hubei-Chibi strains ranged from 0.01897 to 0.11278, while the genetic distances from the 7 populations of Paragonimus ranged from 0.00004 to 0.08401. The genetic distances between Shennongjia strains and P. westermani ranged from 0.01897 to 0.11278, while the genetic distances between Shennongjia strains and P. skrjabini ranged from 0.01355 to 0.12195. The dendrogram shows that 12 strains of Paragonimus can be divided into three groups:â‘ Jiangxi-Wuning, Zhejiang-Lanting, Hubei-xianning and Hubei-Chibi strains, which were Paragonimus westermani;â‘¡Hubei-Zouma(a), Hubei-Zouma(b), Hubei-Wufeng(a), Hubei-Wufeng(b), Hubei-Shiyan and Hubei-Wuli strains, which were P. skrjabini;â‘¢Shennongjia strains, which has been unkown. There are two Paragonimus epidemic areas: southeast of Hubei Province where P. westimani is mainly prevalent and west of Hubei Province where P. skrjabini is mainly prevalent.Conclusion 1. According to sequence analysis,12 strains of Paragonimus involved in this study can be divided into three groups:â‘ Jiangxi-Wuning, Zhejiang-Lanting, Hubei-Wenquan and Hubei-Chibi strains, which were Paragonimus westermani;â‘¡Hubei-Zouma(a), Hubei-Zouma(b), Hubei-Wufeng (a), Hubei-Wufeng (b), Hubei-Shiyan and Hubei-Wuli strains, which were P. skrjabini;â‘¢Shennongjia strains, which has not been reported before.2. There are two Paragonimus epidemic areas:southeast of Hubei Province where P. westimani is mainly prevalent and west of Hubei Province where P. skrjabini is mainly prevalent.3. Genomic DNA extracted from different stage of Paragonimus doesn't affect the results of PCR and sequencing. |
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