秦岭大熊猫槽盘吸虫的鉴定与遗传进化分析
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摘要
本研究旨在对采自大熊猫秦岭亚种(ailuropoda melanoleuca qinlingensis)小肠内的槽盘吸虫进行种类鉴定及遗传进化分析。通过形态学和分子生物学方法,对虫体样本分别进行染色显微镜观察和核糖体DNA内转录间隔区Ⅱ(ITS2)部分序列的PCR扩增和序列分析。经不同染色方法比较后,发现孔雀绿染色法处理后的虫体外部形态以及内部结构清晰,形态上符合印度槽盘吸虫(Ogmocotyle indica)的典型特征;序列及遗传进化分析表明,10个样本ITS2序列完全一致,其系统发生树显示10个样本与O.indica位于同一分枝。研究结果表明,分离的虫株从形态学和进化分析上均与印度槽盘吸虫具有高度一致性,提示该槽盘吸虫为印度槽盘吸虫。
The aim of the present study were to identify and genetically analyze Ogmocotyle sp. collected from the small intestine of ailuropoda melanoleuca qinlingensis. Adult trematodes were firstly identified based on morphological observation under microscopy after different staining methods,and then were analyzed their second internal transcribed spacer( ITS2) by PCR and sequencing. Comparison of different staining methods indicated that Malachite green worked clearly to show external and internal morphological features of parasites,and these characters were consistent with that of Ogmocotyle indica. Sequencing and genetic analysis showed that ITS2 sequences of ten samples were the same and they were located in the same clade with O. indica in phylogenetic tree based on ITS2 sequences. These results indicated that Ogmocotyle sp. in the present study had high identity to O. indica,suggesting that Ogmocotyle sp. collected from giant panda was O. indica.
The aim of the present study were to identify and genetically analyze Ogmocotyle sp. collected from the small intestine of ailuropoda melanoleuca qinlingensis. Adult trematodes were firstly identified based on morphological observation under microscopy after different staining methods,and then were analyzed their second internal transcribed spacer( ITS2) by PCR and sequencing. Comparison of different staining methods indicated that Malachite green worked clearly to show external and internal morphological features of parasites,and these characters were consistent with that of Ogmocotyle indica. Sequencing and genetic analysis showed that ITS2 sequences of ten samples were the same and they were located in the same clade with O. indica in phylogenetic tree based on ITS2 sequences. These results indicated that Ogmocotyle sp. in the present study had high identity to O. indica,suggesting that Ogmocotyle sp. collected from giant panda was O. indica.
引文
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[2]Peng J,Jiang Z,Hu J.Status and conservation of giant panda(ailuropoda melanoleuca):a review[J].Folia Zool,2001,50(2):81-88.
[3]张志和,魏辅文.大熊猫迁地保护理论与实践[M].北京:科学出版社,2006.
[4]中国濒危动物红皮书:兽类[M].北京:科学出版社,1998.
[5]冯文和,张安居.大熊猫繁殖与疾病研究[M].四川成都:四川科学技术出版社,1991.
[6]Zhang J S,Daszak P,Huang H L,et al.Parasite threat to panda conservation[J].Ecohealth J Consortium,2008,5(1):6-9.
[7]杨光友.大熊猫寄生虫与寄生虫病研究进展[J].中国兽医学报,1998(2):206-208.
[8]李德生,何燕,吴虹林,等.圈养大熊猫蠕虫病感染情况调查[J].经济动物学报,2014,18(4):214-216.
[9]杨光友,张志和.野生动物寄生虫病学[M].北京:科学出版社,2013.
[10]晏培松,苏明.七例大熊猫尸体解剖的病理学观察[C]∥大熊猫疾病治疗学术论文集.北京:中国林业出版社,1987:10-17.
[11]何承德,刘世修,陈兴汉.秦岭大熊猫寄生虫初报[C]∥大熊猫疾病治疗学术论文集.北京:中国林业出版社,1987:25-29.
[12]赖从龙,邱贤猛,罗秀芬,等.野外大熊猫内寄生虫病调查[J].中国兽医杂志,1993,19(5):10-11.
[13]潘广林.秦岭地区野生动物寄生虫种类的调查研究[D].陕西杨凌:西北农林科技大学,2012.
[14]Prasad P K,Tandon V,Biswal D K,et al.Molecular identification of the Indian liver fluke,Fasciola(Trematoda:Fasciolidae)based on the ribosomal internal transcribed spacer regions[J].Parasitol Res,2008,103(6):1247-1255.
[15]Amer S,Dar Y,Ichikawa M,et al.Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic(ITS1 and ITS2)and mitochondrial(NDI and COI)gene markers[J].Parasitol Int,2011,60(1):5-12.
[16]De Rojas M,U'beda J M,Cutillas C,et al.Utility of ITS1-5,8 S-ITS2 and 16S mitochondrial DNA sequences for species identification and phylogenetic inference within the Rhinonyssus coniventris species complex(Acari:Rhinonyssidae)[J].Parasitol Res,2007,100(5):1041-1046.
[17]Zhao G H,Li J,Mo X H,et al.The second transcribed spacer r DNA sequence:an effective genetic marker for inter-species phylogenetic analysis of trematodes in the order strigeata[J].Parasitol Res,2012,111(4):1467-1472.
[18]王运宏,薄新文,孙延鸣,等.绦虫成虫制片方法的改进[J].中国兽医寄生虫病,2008,15(6):17-20.
[19]奚琳琳,杨毅梅.带绦虫妊娠节片孔雀绿染色标本制作[J].中国热带医学,2008,8(3):474-474.
[20]危粹凡.羊体槽盘吸虫病(Ogmocotylosis)研究I病原形态分类[J].畜牧兽医学报,1965,8(3):205-212.
[21]Coil W H.Egg shell formation in the notocotylid trematode,Ogmocotyle indica(Bhalerao,1942)Ruiz,1946[J].Parasitol Res,1966,27(3):205-209.