Objective To analyze the sequences of ribosomal second internal transcribed spacer（ITS2） and mitochondrial cytochrome c oxidase subunit Ⅰ (CO1) genes of Paragonimus metacercariae in 5 areas from Henan, Anhui, Fujian and Zhejiang Provinces of China, and identify the species of Paragonimus metacercariae. Methods From October 2018 to September 2019, freshwater crabs were collected from five survey sites, Lantian Town in Xiuning County of Anhui Province, Yongjia County in Zhejiang Province, Shaoyuan Town in Jiyuan City of Henan Province, as well as Zhenghe County and Zhangzhou City in Fujian Province. Genomic DNA was extracted from Paragonimus metacercariae from the crabs, and the ribosomal ITS2 and mitochondrial CO1 genes were amplified by PCR and sequenced. The amplified sequences were spliced and aligned by DNA Star software along with Blast alignment with the Paragonimus genes in GenBankTM. Based on the ITS2 and CO1 gene sequences, a phylogenetic tree was constructed with the neighbor-joining method using Fasciola hepatica as the outgroup. Results The metacercaria positive rates found in the crabs from the five survey sites in Lantian Town, Xiuning County (Anhui Province), Yongjia County (Zhejiang Province), Shaoyuan Town, Jiyuan City (Henan Province), Zhenghe County and Zhangzhou City (Fujian Province) were 82% (49/60), 41% (29/70), 55% (41/74), 65% (41/63), and 45% (32/71), respectively. The lengths of amplified ITS2 and CO1 gene sequences were approximately 500 bp and 450 bp, respectively. The ITS2 and CO1 gene sequences of Paragonimus metacercariae from Lantian Town of Xuning County(Anhui Province) and Yongjia County (Zhejiang Province) showed the highest similarity with those of P. westermani (KC417492.1 and AF219379.2), being 99%-100% and 96%-99%, respectively, and both clustered in the same branch with P. westermani. The ITS2 and CO1 genes of Paragonimus metacercariae from Shaoyuan Town of Jiyuan City(Henan Province) showed the highest similarity with those of P. skrjabini (KX129924.1 and MK568551.1), at 98%-100% and 95%-99%, respectively, both being clustered in the same branch with P. skrjabini. The ITS2 gene of Paragonimus metacercariae from Zhenghe County(Fujian Province) showed the highest similarity (98%-100%) with that of P. skrjabini (KX129924.1), forming a clade with P. skrjabini and P. miyazaki (the distance between the two species was not obvious). The CO1 gene from Zhenghe County showed the highest similarity (91%-94%) with that of P. miyazaki (AY618823.1 and AY618834.1), formed a clade with P. miyazaki but was distant from the clade of P. skrjabini. The ITS2 gene of Paragonimus metacercariae from Zhangzhou City (Fujian Province) showed the highest similarity (100%) with the gene of P. westermani (KC417492.1), and the CO1 gene sequence showed the highest similarity (97%-99%) with that of P. cenococopiosus (AF159595.1), being clustered in the with P. cenococopiosus branch. Conclusion The CO1 gene sequence amplified from the metacercariae of 5 areas in 4 Provinces was highly homologous to those of P. westermani, P. skrjabini, P. miyazaki and P. cenococopiosus. The CO1 gene of Paragonimus may be used as a potential molecular marker for species differentiation.
[1] David B.Paragonimiasis[J]. Adv Exp Med Biol, 2019, 1154: 105-138.
[2] Oh MY, Chu A, Park JH, et al. Simultaneous Paragonimus infection involving the breast and lung: a case report[J]. World J Clin Cases, 2019, 7(24): 4292-4298.
[3] Chen XT.Zoology of China (Platyhelminthes Trematoda Order Digenea)[M]. Beijing: Science Press, 1985. (in Chinese)
(陈心陶. 中国动物志(扁形动物门吸虫纲复殖目)[M]. 北京: 科学出版社, 1985.)
[4] World Health Organization.Investing to overcome the global impact of neglected tropical diseases: third WHO report on neglected tropical diseases[R]. Geneva: WHO, 2015.
[5] Yoshida A, Doanh PN, Maruyama H.Paragonimus and paragonimiasis in Asia: an update[J]. Acta Trop, 2019, 199: 105074.
[6] Zhu XP, Su C.Human parasitology[M]. Beijing: People’s Medical Publishing House, 2013. (in Chinese)
(诸欣平, 苏川. 人体寄生虫学[M]. 北京: 人民卫生出版社, 2013.)
[7] Kim JM, Hong ST.In memoriam: professor seung-yull Cho (1943-2019)[J]. J Korean Med Sci, 2019, 34(5): e43.
[8] Mei HY, Guan F, Niu AO.Genetic variation analysis of the ITS2 gene from Paragonimus in 10 districts of Hubei, Jiangxi and Zhejiang Provinces of China[J]. Chin J Zoonoses, 2009, 25(10): 935-939. (in Chinese)
(梅浩砚, 关飞, 牛安欧. 鄂赣浙3省10地并殖吸虫遗传变异的ITS2分析[J]. 中国人兽共患病学报, 2009, 25(10): 935-939.)
[9] Wu GL.Human Parasitology[M]. 3rd edition. Beijing: People’s Medical Publishing House, 2005. (in Chinese)
(吴观陵. 人体寄生虫学[M]. 3版. 北京: 人民卫生出版社, 2005.)
[10] Hong JH, Oh CS, Seo M, et al. Analysis of COI and ITS2 regions of DNA obtained from Paragonimus westermani eggs in ancient coprolites on Joseon dynasty mummies[J]. Mem Inst Oswaldo Cruz, 2019, 114: e180595.
[11] Hernández-Chea R, Jiménez-Rocha AE, Castro R, et al. Morphological and molecular characterization of the metacercaria of Paragonimus caliensis, as a separate species from P. mexicanus in Costa Rica[J]. Parasitol Int, 2017, 66(2): 126-133.
[12] Le TH, Blair D, McManus DP. Mitochondrial DNA sequences of human schistosomes: the current status[J]. Int J Parasitol, 2000, 30(3): 283-290.
[13] Velavan TP, Schulenburg H, Michiels NK.Detection of multiple infections by monocystis strains in a single earthworm host using ribosomal internal transcribed spacer sequence variation[J]. Parasitology, 2010, 137(1): 45-51.
[14] Shen YP, Zhang YJ.Preliminary observation on some aspects of biology and protein bands of metacercariae of Euparagonimus cenocopiosus[J]. Chin J Parasitol Parasit Dis, 1987, 5(4): 278-280. (in Chinese)
(沈一平, 张耀娟. 三平正并殖吸虫囊蚴的生物学与蛋白质区带初步观察[J]. 中国寄生虫学与寄生虫病杂志, 1987, 5(4): 278-280.)
[15] Technical specification for the quarantine of metacercariae in shellfish:SN/T 3504-2013[S], 2013. (in Chinese)
(甲壳类水产品中并殖吸虫囊蚴检疫技术规范:SN/T 3504-2013[S], 2013.)
[16] Sugiyama H, Morishima Y, Kameoka Y, et al. Polymerase chain reaction (PCR)-based molecular discrimination between Paragonimus westermani and P. miyazakii at the metacercarial stage[J]. Mol Cell Probes, 2002, 16(3): 231-236.
[17] Hong JL, Chen MG, Chang ZS, et al. Study on DNA sequence and karyotype of Paragonimus westermani in yongjia County, Zhejiang Province[J]. Chin J Parasit Dis Control, 2003(2): 42-45. (in Chinese)
(洪加林, 陈名刚, 常正山, 等. 浙江省永嘉县并殖吸虫DNA序列分析、形态及核型研究[J]. 中国寄生虫病防治杂志, 2003(2): 42-45.)
[18] Li YS, Hu Y.Contend of species of Paragonimus in China[M]. Fuzhou: Fujian Science & Technology Publishing House, 2015. (in Chinese)
(李友松, 胡野. 中国并殖吸虫虫种争鸣[M]. 福州: 福建科学技术出版社, 2015.)
[19] Lin BX, Wei HW, Li YS, et al. Investigation on epidemic foci of paragonimiasis in eastern part of Zhenghe County, Fujian Province[J]. Chin J Schisto Control, 2016, 28(4): 418-421. (in Chinese)
(林本翔, 魏焕旺, 李友松, 等. 福建省政和县东部并殖吸虫病疫源地调查[J]. 中国血吸虫病防治杂志, 2016, 28(4): 418-421.)
[20] Wang TP, Cao ZG.New trend of important parasite infection in Anhui Province and its influencing factors[J]. J Trop Dis Parasitol, 2017, 15(1): 1-6. (in Chinese)
(汪天平, 操治国. 安徽省重要寄生虫感染的新趋势及其影响因素[J]. 热带病与寄生虫学, 2017, 15(1): 1-6.)
[21] Chen WQ, Zhang YL, Deng Y, et al. Survey of knowledge and behavior of paragonimiasis in the primary and secondary school students in Jiyuan[J]. Henan J Prev Med, 2016, 27(10): 737-739. (in Chinese)
(陈伟奇, 张雅兰, 邓艳, 等. 济源市中小学生并殖吸虫病防治知识及行为调查[J]. 河南预防医学杂志, 2016, 27(10): 737-739.)
[22] Fang YM, Hu XS, Ling ZL, et al. Investigation on the second intermediate host(Potamon denticulate) of Paragonimus in Huangshan area[J]. J Trop Dis Parasitol, 2014, 12(4): 227-229. (in Chinese)
(方益民, 胡新三, 凌志利, 等. 黄山市肺吸虫第二中间宿主—溪蟹感染调查[J]. 热带病与寄生虫学, 2014, 12(4): 227-229.)
[23] Cheng YZ, Li LS, Lin GH, et al. Survey on the foci of Paragonimus in Youxi, Yongtai and Pinghe counties of Fujian Province[J]. Chin J Parasitol Parasit Dis, 2010, 28(6): 406-410. (in Chinese)
(程由注, 李莉莎, 林国华, 等. 福建省尤溪、永泰与平和县并殖吸虫病疫源地调查[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(6): 406-410.)
[24] Cai MR, Lin GH, Chen JZ, et al. Morphological observation of Sinopotamon pinghense and investigation of its infection with metacercariae of Euparagonimus cenocopiosus in Liudou Mountain Natural Reserve in Fujian Province[J]. Chin J Parasitol Parasit Dis, 2017, 35(5): 478-481, 494. (in Chinese)
(蔡茂荣, 林国华, 陈锦钟, 等. 福建省六斗山自然保护区平和华溪蟹感染三平正并殖吸虫囊蚴情况及其形态学研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 478-481, 494.)
[25] Liu DH, Guo JD, Wang TP, et al. Analysis of the results of paragonimiasis endemic in regular surveillance site in Anhui Province from 2013 to 2016[J]. J Trop Dis Parasitol, 2017, 15(3): 155-158. (in Chinese)
(刘道华, 郭见多, 汪天平, 等. 2013-2016年安徽省并殖吸虫病监测结果分析[J]. 热带病与寄生虫学, 2017, 15(3): 155-158.)
[26] Chen WQ, Deng Y, Zhang YL, et al. A case of group infections with Paraginimus species in Henan, central China[J]. Acta Trop, 2019: 105111.
[27] Ryu JS, Hwang UW, Min DY, et al. Molecular identification of Paragonimus ohirai and P. westermani from Anhui Province, China[J]. Parasite, 2000, 7(4): 305-309.
[28] Zhuo XH, Kong QM, Tong QB, et al. DNA detection of Paragonimus westermani: diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick[J]. Acta Trop, 2019, 200: 105185.
[29] Ohari Y, Suzuki Y, Shibahara T, et al. First report of Paragonimus skrjabini miyazakii metacercariae in Geothelphusa dehaani (Sawagani) occurring in Iwate Prefecture, Japan[J]. J Vet Med Sci, 2019, 81(8): 1109-1112.
[30] Gu MJ, Li YS, Dong HF, et al. Progress in taxonomy and genetic diversity of Paragonimus[J]. Chin J Zoonoses, 2019, 35(8): 744-750. (in Chinese)
(顾梦杰, 李友松, 董惠芬, 等. 并殖吸虫的分类和遗传多样性研究概述[J]. 中国人兽共患病学报, 2019, 35(8): 744-750.)
[31] Ma J, He JJ, Liu GH, et al. Complete mitochondrial genome of the giant liver fluke Fascioloides magna (Digenea ∶ Fasciolidae) and its comparison with selected trematodes[J]. Parasit Vectors, 2016, 9(1): 429.
