基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查

doi:10.11843/j.issn.0366-6964.2021.012.021

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (12): 3546-3556.doi: 10.11843/j.issn.0366-6964.2021.012.021

基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查

尚婧晔¹, 张光葭¹, 丹巴泽里², 王奇¹, 喻文杰¹, 何伟¹, 廖沙¹, 沈芸舟³, 黄燕^1*, 王谦¹, 钟波¹, 刘阳^1*

1. 四川省疾病预防控制中心, 成都 610041;
2. 甘孜藏族自治州州疾病预防控制中心, 康定 626099;
3. 凉山彝族自治州疾病预防控制中心, 西昌 615000

收稿日期:2021-03-17 出版日期:2021-12-25 发布日期:2021-12-22
通讯作者: 黄燕,主要从事寄生虫病防控与研究,Tel:028-85589532,E-mail:huangyancdc@163.com;刘阳,主要从事寄生虫病防控与研究,Tel:028-85589501,E-mail:45481283@qq.com
作者简介:尚婧晔(1987-),女,四川成都人,硕士生,主要从事寄生虫病防控与研究,E-mail:jy_shang@126.com;张光葭(1991-),女,四川成都人,本科,主要从事寄生虫病防控与研究,E-mail:274125589@qq.com。尚婧晔和张光葭为同等贡献作者
基金资助:
四川省卫健委科研课题（17PJ440）

Identification of Wild Canidae Species and Investigation of Echinococcus Infection Based on Fecal Samples

SHANG Jingye¹, ZHANG Guangjia¹, DANBA Zeli², WANG Qi¹, YU Wenjie¹, HE Wei¹, LIAO Sha¹, SHEN Yunzhou³, HUANG Yan^1*, WANG Qian¹, ZHONG Bo¹, LIU Yang^1*

1. Sichuan Center for Disease Control and Prevention, Chengdu 610041, China;
2. Ganzi Tibetan Autonomous Prefecture Center for Disease Control and Prevention, Kangding 626099, China;
3. Liangshan Yi Autonomous Prefecture Center for Disease Control and Prevention, Xichang 615000, China

Received:2021-03-17 Online:2021-12-25 Published:2021-12-22

摘要/Abstract

摘要： 旨在探索粪便样本分析对犬科动物物种鉴别的方法及其可行性，同时对野外犬科动物棘球绦虫感染情况进行调查。采用哺乳动物线粒体DNA控制区通用引物对101份采集自野外环境的犬科动物粪便DNA进行PCR扩增和测序，通过核苷酸位点变异分析、BLAST相似性比对、遗传距离分析和系统进化树构建，对粪样来源物种进行分子鉴别。同时，采用粪-抗原ELISA检测，对粪样棘球绦虫感染情况进行调查。结果表明：粪样来源物种鉴别的检测成功率为73.27%，获得的74条序列共定义单倍型20个，所有单倍型序列均可在GenBank数据库中匹配到单一物种，序列相似度为98.52%~100%。单倍型根据序列差异可分为4个单倍型集，各单倍型集间平均碱基差异为17.83~70.10，各单倍型集内单倍型碱基差异在1~10个之间。各集合间遗传距离为0.068~0.342，远大于各集合内单倍型间遗传距离0.009~0.022。系统进化分析结果显示，同一集合的单倍型以高自展值聚集成一支。综合各项分析结果，可推测所检测粪样宿主来源分别为犬、灰狼、赤狐和红狐。粪-抗原ELISA检测发现阳性样本11份，样本棘球绦虫抗原阳性率为10.89%。综上表明：线粒体DNA控制区序列分析可用于犬科动物的分子鉴别，可结合核基因等其他分子标记进一步提升检测准确率。研究区域野外犬科动物棘球绦虫粪抗原阳性率较高。

关键词: 犬科动物, 棘球绦虫, 终末宿主, 线粒体基因, 物种鉴定

Abstract: This study was conducted to explore the methods and its feasibility of identifying Canidae species and to investigate the infection of Echinococcus in wild Canidae based on non-invasive fecal sample analysis. A total of 101 DNA samples extracted from feces of wild Canidae were amplified and sequenced with a pair of universal primers designed for the mitochondrial DNA control region of mammalian species. The species origins of the fecal samples were molecularly identified by the analysis of nucleotide variation, the comparison of sequence similarity, the analysis of genetic distance and the construction of phylogenetic tree. Meanwhile, copro-antigen ELISA was used to investigate the infection of Echinococcus. The test success rate for the species identification of the samples was 73.27%. A total of 20 haplotypes were inferred from the 74 sequences obtained in the study. Each of the haplotype sequences was found to match with one single species in the GenBank database with 98.52%-100% sequence similarity. All the haplotypes can be divided into 4 haplotype sets according to sequence differences. The average number of nucleotide differences among the 4 haplotype sets is 17.83-70.10, and the number of nucleotide differences among the haplotypes within each set is 1-10. The genetic distance among the 4 haplotype sets is 0.068-0.342. The value is much larger than that of the genetic distance among the haplotypes within each set, which is 0.009-0.022. The phylogenetic analysis showed that haplotypes in the same sets clustered into same clades with high bootstrap values. Based on all the analysis results, it can be inferred that the sources of the fecal samples were Canis lupus familiaris, Canis lupus, Vulpes vulpes and Vulpes ferrilata respectively. A total of 11 positive samples were found in the copro-antigen ELISA test. The positive rate of Echinococcus antigen was 10.89%. The analysis of mitochondrial DNA control region sequence can be used for the molecular identification of the Canidae. The detection accuracy rate would be further improved if other molecular markers such as nuclear genes were aslo used. The positive rate of Echinococcus antigen is high in feces of wild Canidae in the study area.

Key words: Canidae, Echinococcus, definitive host, mtDNA, species identification

中图分类号:

尚婧晔, 张光葭, 丹巴泽里, 王奇, 喻文杰, 何伟, 廖沙, 沈芸舟, 黄燕, 王谦, 钟波, 刘阳. 基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查[J]. 畜牧兽医学报, 2021, 52(12): 3546-3556.

SHANG Jingye, ZHANG Guangjia, DANBA Zeli, WANG Qi, YU Wenjie, HE Wei, LIAO Sha, SHEN Yunzhou, HUANG Yan, WANG Qian, ZHONG Bo, LIU Yang. Identification of Wild Canidae Species and Investigation of Echinococcus Infection Based on Fecal Samples[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3546-3556.

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基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查

Identification of Wild Canidae Species and Investigation of Echinococcus Infection Based on Fecal Samples

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