孔雀源异刺线虫的形态学特征及分子鉴定

doi:10.12140/j.issn.1000-7423.2021.06.013

中国寄生虫学与寄生虫病杂志 ›› 2021, Vol. 39 ›› Issue (6): 816-820.doi: 10.12140/j.issn.1000-7423.2021.06.013

孔雀源异刺线虫的形态学特征及分子鉴定

刘路瑶¹(), 杨聪山¹, 章翔¹, 程蓉蓉¹, 洪宇航¹, 吴成龙¹, 孙萌¹, 陈木新², 艾琳², 徐前明¹^,^*()

1 安徽农业大学动物科技学院,合肥 230036
2 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室（中国疾病预防控制中心寄生虫病预防控制所）,上海 200025

收稿日期:2021-03-20 修回日期:2021-05-31 出版日期:2021-12-30 发布日期:2021-12-17
通讯作者: 徐前明
作者简介:刘路瑶（1998-）,女,硕士研究生,从事动物寄生虫与寄生虫病研究。E-mail： 2010798534@qq.com

Morphological characteristics and molecular identification of Heterakis sp. peacock origin

LIU Lu-yao¹(), YANG Cong-shan¹, ZHANG Xiang¹, CHENG Rong-rong¹, HONG Yu-hang¹, WU Cheng-long¹, SUN Meng¹, CHEN Mu-xin², AI Lin², XU Qian-ming¹^,^*()

1 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology (National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention), Shanghai 200025, China

Received:2021-03-20 Revised:2021-05-31 Online:2021-12-30 Published:2021-12-17
Contact: XU Qian-ming

摘要/Abstract

摘要：

目的用形态学和分子鉴定方法分析采自合肥市野生动物园孔雀的异刺线虫的特征。方法从孔雀盲肠内分离虫体并鉴定虫体形态学特征。提取虫体基因组DNA,PCR扩增28S rRNA D2-D3基因和第二内转录间隔区（ITS2）,测序后在GenBank上进行BLAST比对,用邻接法构建系统进化树。结果共分离获得48条线虫。雌虫体长7.30~10.28 mm,雄虫体长5.34~7.38 mm,雌虫较雄虫长2~5 mm；雄虫交合刺长80.00~322.73 μm,雌虫阴门靠近虫体的尾端；雄虫与雌虫头部均含有3片唇,口腔短,食道前部圆柱状,末端膨大为食道球；雄虫尾端含有两根不等长的交合刺,雌虫尾部尖细光滑；虫卵呈椭圆形,灰褐色,含两层卵壳,卵壳较厚,壳面光滑,内含有单个胚细胞。PCR结果显示,28S rRNA D2-D3基因和ITS-2扩增产物分别为750 bp和1 053 bp。BLAST比对结果显示,其28S rRNA D2-D3基因序列与圆线虫（GenBank登录号LC133189.1、LC133188.1）和异刺线虫（GenBank登录号LC185992.1、LC185989.1、LC186015.1、LC186006.1）的序列一致性分别为84.59%和84.46%；其ITS-2序列与贝拉异刺线虫（GenBank登录号KU529974、LC592732、LC592731）、鸡异刺线虫（GenBank登录号LC59279、LC5928003、LC592805、LC592797)和印度异刺线虫（GenBank登录号LC592808、LC592809、LC592807）的序列一致性分别为85.29%、80.32%和79.85%。基于28S rRNA D2-D3基因的系统进化树显示,本研究分离的线虫与圆线虫和异刺线虫较近,但不在同一分支上；基于ITS-2基因系统进化树显示,其与贝拉异刺线虫、鸡异刺线虫和印度异刺线虫较为接近,但不在同一分支上。结论从孔雀分离的异刺线虫与已报道的异刺线虫在形态学上高度一致,初步判断其为异刺属线虫,但系统进化树分析结果显示其与已报道的异刺线虫存在一定的差异。

关键词: 异刺线虫, 形态学鉴定, 分子鉴定, 系统进化树, 孔雀

Abstract:

Objective To analyze taxonomic characteristics of Heterakis sp. from peacocks collected from Hefei Wildlife Zoo using morphological and molecular identification methods. Methods The worms were isolated from the cecum of peacocks and were indentified according to the morphological characteristics. The genomic DNA of the worms was extracted, then 28S rRNA D2-D3 gene and second internal transcribed spacer (ITS2) were amplified by PCR, and sequenced, the homology were analyzed by BLAST tool in GenBank, the phylogenetic tree was constructed by using the neighbour-joining method. Results A total of 48 nematodes were isolated. Male worm was 5.34-7.38 mm in length, while female 7.30-10.28 mm, which was about 2-5 mm longer than male. The copulatory spines in male was 80.00-322.73 μm in length, the pubic valve in female located near the caudal end of the worm. Both males and females contain three lamellae on the head, with a short oral cavity and a esophagus with cylindrical anterior and expanded posterior esophageal bulb. The male contains two unequal copulatory spines at the caudal end, while the female has a thin and smooth caudal tip. The egg is oval-shaped and gray-brown in color, covered by two layers of egg shell, which is fairly thick and smooth-surfaced, containing a single embryonic cell inside. PCR results showed that the 28S rRNA D2-D3 and ITS-2 amplification products were 750 bp and 1 053 bp in length respectively. BLAST showed that the 28S rRNA D2-D3 sequence was 84.59% homologous to S. calotis (GenBank Acession No. LC133189.1, LC133188.1) and 84.46% to H. gallinae (GenBank Acession No. LC185992.1, LC185989.1, LC186015.1, LC186006.1); the ITS-2 sequences was 85.29% homologous to H. beramporia (GenBank Acession No. KU529974, LC592732, LC592731), 80.32% to H. gallinarum (GenBank Acession No. LC59279, LC5928003, LC592805, LC592797) and 79.85% to H. indica (GenBank Acession No. LC592808, LC592809, LC592809). Based on the 28S rRNA D2-D3 gene phylogenetic tree, the Heterakis isolated in this study was genetically closer to S. calotis and the H. gallinae, but not on the same branch; based on the ITS-2 gene phylogenetic tree, it was genetically closer to the H. beramporia, H. gallinarum and H. indica, but not on the same branch. Conclusion The Heterakis worm isolated from peacock is morphologically highly consistent with the reported finding, thus, is preliminarily cassified as the Heterakis sp., but the phylogenetic trees based on the marker 28S rRNA D2-D3 and ITS-2 present certain variance with the reported.

Key words: Heterakis, Morphological identification, Molecular identification, Phylogenetic tree, Peacock

中图分类号:

R383.19

刘路瑶, 杨聪山, 章翔, 程蓉蓉, 洪宇航, 吴成龙, 孙萌, 陈木新, 艾琳, 徐前明. 孔雀源异刺线虫的形态学特征及分子鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(6): 816-820.

LIU Lu-yao, YANG Cong-shan, ZHANG Xiang, CHENG Rong-rong, HONG Yu-hang, WU Cheng-long, SUN Meng, CHEN Mu-xin, AI Lin, XU Qian-ming. Morphological characteristics and molecular identification of Heterakis sp. peacock origin[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(6): 816-820.

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参考文献 17

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特征Characteristics	雄虫 Male	雌虫 Female
体长/mm Body length/mm	5.34~7.38	7.30~10.28
最大体宽/μm Body width of maximum/μm	280.31~361.24	322.45~378.59
食道长度/μm Length of esophagus/μm	680.00~920.33	810.63~1 085.47
排泄孔到头端距离/μm Distance from drain hole to tip/μm	310.08~362.53	418.92~540.00
尾长/μm Caudal length/μm	254.30~350.12	703.78~56.22
交合刺长度/μm Copulatory spine length/μm	80.00~322.73
阴门到尾端距离/mm Distance from vulva to caudal end/mm		3.76~5.41

孔雀源异刺线虫的形态学特征及分子鉴定

Morphological characteristics and molecular identification of Heterakis sp. peacock origin

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