丹顶鹤感染前殖吸虫的种类鉴定及其ITS序列分析

doi:10.12140/j.issn.1000-7423.2025.01.015

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (1): 97-102.doi: 10.12140/j.issn.1000-7423.2025.01.015

丹顶鹤感染前殖吸虫的种类鉴定及其ITS序列分析

曹禹¹^,²^,^*(), 李烨¹, 金振华¹, 王丽坤¹, 高忠燕³, 张显光³, 江波涛¹^,²

1 黑龙江省农业科学院畜牧兽医分院，黑龙江齐齐哈尔 161005
2 黑龙江省兽用药物重点实验室，黑龙江齐齐哈尔 161005
3 黑龙江省扎龙国家级自然保护区管理局，黑龙江齐齐哈尔 161002

收稿日期:2024-07-31 修回日期:2024-12-17 出版日期:2025-02-28 发布日期:2025-03-26
作者简介:曹禹（ORCID：0000-0001-7387-8349），男，博士，助理研究员，从事动物分子寄生虫学与寄生虫病防治研究。E-mail：yu_cao2021@163.com
基金资助:
黑龙江省农业科技创新跨越工程农业科技基础创新项目优秀青年项目(CX23BS06);黑龙江省博士后面上资助项目(LBH-Z24275)

Species identification and ITS sequence analysis of Prosthogonimus in Grus japonensis

CAO Yu¹^,²^,^*(), LI Ye¹, JIN Zhenhua¹, WANG Likun¹, GAO Zhongyan³, ZHANG Xianguang³, JIANG Botao¹^,²

1 Branch of Animal Husbandry and Veterinary Branch of Heilongjiang Academy of Agricultural Sciences, Qiqihar 161005, Heilongjiang, China
2 Heilongjiang Province Key Laboratory of Veterinary Drugs, Qiqihar 161005, Heilongjiang, China
3 Heilongjiang Zhalong National Natural Reserve Administration, Qiqihar 161002, Heilongjiang, China

Received:2024-07-31 Revised:2024-12-17 Online:2025-02-28 Published:2025-03-26
Contact: *E-mail: yu_cao2021@163.com
Supported by:
Heilongjiang Province Agricultural Science and Technology Innovation Leapfrog Project Agricultural Science and Technology Basic Innovation Project (Excellent Young Scholars)(CX23BS06);Doctoral Postdoctoral Funding Project in Heilongjiang Province(LBH-Z24275)

摘要/Abstract

摘要：

目的鉴定丹顶鹤感染前殖吸虫的种类并分析其核糖体DNA内转录间隔区（ITS）。方法从黑龙江省扎龙自然保护区丹顶鹤的泄殖腔采集前殖吸虫，PCR扩增前殖吸虫ITS序列并测序，测序结果在NCBI进行BLAST比对。采用基因重复序列计算器查找重复序列，MegAlign软件进行序列相似性分析，MEGA 7.0、Clustal X和Paup软件进行序列对齐，采用最大简约法构建系统进化树。结果在丹顶鹤体内共采集到5条前殖吸虫，PCR扩增共获得5条ITS序列，其中3条长1 240 bp（GenBank登录号：PQ634967，PQ634968，PQ634969），与楔形前殖吸虫（Prosthogonimus cuneatus）（GenBank登录号：OQ344776）的序列相似性为98.2%，鉴定为楔形前殖吸虫；2条长为1 183 bp（GenBank登录号：PP956934，PP956935），与透明前殖吸虫（P. pellucidus）（GenBank登录号：KP192732）的序列相似性为97.7%，鉴定为透明前殖吸虫。楔形前殖吸虫和透明前殖吸虫的5.8S和ITS2无重复序列，ITS1则分别有16、17个重复序列。系统进化树结果显示，本研究中的楔形前殖吸虫先与寄生于乌鸫、绿头鸭的楔形前殖吸虫（捷克）聚为一个小分支，再与本研究中的透明前殖吸虫聚为一个分支。结论扎龙自然保护区丹顶鹤体感染的前殖吸虫为楔形前殖吸虫和透明前殖吸虫，其ITS1分别有16、17个重复序列。楔形前殖吸虫和透明前殖吸虫与来自捷克的楔形前殖吸虫的亲缘关系较近。

关键词: 楔形前殖吸虫, 透明前殖吸虫, 内转录间隔区序列, 丹顶鹤

Abstract:

Objective To identify the species of Prosthogonimus in Grus japonensis and analyze the ribosomal DNA internal transcribed spacer (ITS) gene sequence. Methods Prosthogonimus samples were collected from the G. japonensis cloaca in Zhalong National Nature Reserve, Heilongjiang Province. The ITS gene of Prosthogonimus was amplified using PCR assay, and the amplified product was sequenced. The sequencing results were aligned with sequences recorded in the NCBI database using the BLAST tool. Repetitive sequences were identified using the Repeat Masker tool, and sequence similarity analysis was performed using the MegAlign software. Sequence alignments were conducted using the MEGA 7.0, Clustal X, and Paup software, and a phylogenetic tree was created using the maximum parsimony method. Results A total of 5 Prosthogonimus specimens were collected from G. japonensis, and five ITS gene sequences were obtained. Three sequences (GenBank accession numbers: PQ634967, PQ634968 and PQ634969) were 1 240 bp in size and showed 98.2% sequence similarity with P. cuneatus (GenBank accession number: OQ344776), which were identified as P. cuneatus. The other two sequences (GenBank accession numbers: PP956934 and PP956935) were 1 183 bp in size and showed 97.7% sequence similarity with P. pellucidus (GenBank accession number: KP192732), which were identified as P. pellucidus. There were no repetitive sequences in 5.8S or ITS2 genes of P. cuneatus and P. pellucidus, and there were 16 and 17 repetitive sequences the ITS1 gene of P. cuneatus and P. pellucidus, respectively. Phylogenetic analysis revealed that the P. cuneatus from this study was firstly clustered with P. cuneatus from Turdusmerula and Anasplatyrhynchos (Czech Republic isolate) into a subclade, and then clustered with P. pellucidus from this study into a clade. Conclusion The Prosthogonimus species infecting G. japonensis in the Zhalong National Nature Reserve are P. cuneatus and P. pellucidus, with 16 and 17 repetitive sequences in the P. cuneatus and P. pellucidus ITS1, respectively. P. cuneatus and P. pellucidus share a closer phylogenetic relationship with the Czech Republic isolate of P. cuneatus.

Key words: Prosthogonimus cuneatus, Prosthogonimus pellucidus, ITS sequences, Grus japonensis

中图分类号:

R383.2

曹禹, 李烨, 金振华, 王丽坤, 高忠燕, 张显光, 江波涛. 丹顶鹤感染前殖吸虫的种类鉴定及其ITS序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(1): 97-102.

CAO Yu, LI Ye, JIN Zhenhua, WANG Likun, GAO Zhongyan, ZHANG Xianguang, JIANG Botao. Species identification and ITS sequence analysis of Prosthogonimus in Grus japonensis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(1): 97-102.

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重复序列（3'→5'） Repetitive sequence (3'→5')	长度/bp Length/bp	位置/bp Position/bp	重复次数Repetition
GGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTCGTCTGCCTACGG-TAGAGCGCCTCAGTTCTATCAAAC	73	1，93	2
GGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTCGTCTGCCTACGG-TAGAGCGCCTCAGTTCTATC	69	1，93，140	3
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTGT	49	220，318	2
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTG	48	220，269，318	3
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATCAAACACTGCAGCCTC	47	83，130	2
GTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAACTCTG	47	220，270，319	3
TCTGCAGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATC	43	215，362	2
AGTCTTGCCTGCCTACGGTAGAGCGCCTCAGTTCTATC	38	269，318，367	3
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATCAAAC	36	38，83，130	3
CTGCCTACGGTAGAGCGCCTCAGTTCTATCGGAAA	35	179，228，277，326	4
GTCTGCCTACGGTAGAGCGCCTCAGTTCTATC	32	38，83，130，178	4
TCTGCCTACGGTAGAGCGCCTCAGTTCTATC	31	39，84，131，178	4
CTGCCTACGGTAGAGCGCCTCAGTTCTATC	30	40，85，132，179，228，277，326，375	8
GGTAGAGCGCCTCAGTTCTATCAAAC	26	1，48，93，140,	4
GGTAGAGCGCCTCAGTTCTATC	22	1，48，93，140，187，236，285，334，383	9
GTTGTCAGTC	10	525，590	2

重复序列（3'→5'）Repetitive sequence (3'→5')	长度/bp Length/bp	位置/bp Position/bp	重复次数 Repetition
CTGCCTACGGTAGAGCGCCTCAGTTCTATC	30	224，371	2
CGGTAGAGCGCCTCAGTTCTATCGGAAA	28	231，280	2
AGTCTTGCCTGCCTACGGTAGAGCGC	26	314，363	2
CGGTAGAGCGCCTCAGTTCTATC	23	231，280，378	3
TCAGTTCTATCGGAAACTCTG	21	292，341	2
TCTGCAGTCTTGCCTGCCT	19	260，358	2
CTGCCTACGGTAGAGCGC	18	224，322，371	3
GAAAATCTGCAGTCTTG	17	206，255	2
TCAGTTCTATCGGAAA	16	243，341	2
TGCCTACGGTAGAG	14	83，225，323，372	44
AGTCTTGCCTGCCT	14	265，314，363	3
TCTGCAGTCTTG	12	211，260，358	3
CGGTAGAGCGC	11	231，280，329，378	4
AGAGCGCGTCA	11	186，333	2
TCAGTTCTATC	11	243，292，341，390	4
GTTGTCAGTC	10	521，586	2
GGTAGAGAGC	10	1，90	2

丹顶鹤感染前殖吸虫的种类鉴定及其ITS序列分析

Species identification and ITS sequence analysis of Prosthogonimus in Grus japonensis

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