Amplification and Analysis of ITS Sequence of Intestinal Parasites in Przewalski's Wild Horse (Equus ferus ssp. przewalskii)
LI Qiao1, ZHAO Jian-You3, DU Xiao-Hua2, LU Wei3, ZHANG Chun-Lin3, DONG Jian-Ying2, WEN Yong-Qiang1, ZHOU Ying-Jie3,*, LIU Xia1,*
1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 3 State Forest Administration Gansu Protection Centre of Threatened Species, Wuwei 733000, China
Abstract Przewalski's wild horse (Equus ferus ssp. przewalskii) is a subspecies of E. ferus and is considered as endangered animals. The healthy growth and continuous expansion in their population have special value for germplasm resource protection. The parasites in Przewalski's wild horse can produce different degrees of pathological damage to the organism. The present study aimed to identify the intestinal parasite species in Przewalski's wild horses which were taken from the State Forest Administration Gansu Protection Centre of Threatened Species. During sampling procedures, the worms were separated in the faeces of Przewalski's wild horses. Morphological characteristics were identified and ITS (internal transcribed spacer) sequences were compared using bioinformatic methods. The results showed that 2 kinds of worms named PE and CN were isolated from intestinal tracts of Przewalski's wild horses, and preliminarily identified as Parascaris equorum and Cylicocyclus sp. by morphology observation. The full length of PE ITS region was 865 bp (GenBank No. MT579850), and the length of ITS-1, 5.8S rDNA and ITS-2 was 421, 156 and 288 bp, respectively; and the homology with Parascaris equorum was 99.88%. The full length of CN ITS region was 843 bp (GenBank No. MT581488), and the length of ITS-1, 5.8S rDNA and ITS-2 was 370, 153 and 320 bp, respectively; and the homology with C. nassatus was 99.41%. The above results showed that PE sample collected from Przewalski's wild horses was Parascaris equorum and the CN sample was C. nassatus. The results of this study could provide a reference for healthy breeding of Przewalski's wild horses.
LI Qiao,ZHAO Jian-You,DU Xiao-Hua, et al. Amplification and Analysis of ITS Sequence of Intestinal Parasites in Przewalski's Wild Horse (Equus ferus ssp. przewalskii)[J]. 农业生物技术学报, 2020, 28(7): 1277-1286.
[1] 高远, 仇建华, 常巧呈等. 2017. 鼻状杯环线虫形态学与分子生物学鉴定[J]. 黑龙江八一农垦大学学报, 29(04): 45-48. (Gao Y, Qiu J H, Chang Q C, et al.2017. Identification of morphology and molecular biology of Cylicocyclusnassatus[J]. Journal of Heilongjiang Bayi Agricultural University, 29(04): 45-48.) [2] 韩亮. 2017. 白牦牛消化道线虫PCR-RFLP鉴定方法的建立及遗传变异研究[D]. 硕士学位论文, 甘肃农业大学, 导师: 孙晓林, pp. 24-26. (Han L.2012. Establishment of PCR-RFLP technique for identification and genetic variation studies of nematodes in digestive canal of yaks [D]. Thesis for M.S., Gansu Agricultural University, Supervisor: Sun X L, pp. 24-26.) [3] 何光志. 2009. 八种野生动物蛔虫的分子鉴定及两种蛔虫抗原基因的表达与免疫保护研究[D]. 博士学位论文, 四川农业大学, 导师: 杨光友, pp. 26-27. (He G Z.2009. Molecular identification of roundworms from eight wild animals, expression of antigen genes and immune protection from two Ascaris lumbricoides[D]. Thesis for Ph.D., Sichuan Agricultural University, Supervisor: Yang G Y, pp. 26-27.) [4] 何光志, 牛李丽, 杨光友, 等. 2008. 大熊猫等八种野生珍稀动物蛔虫ITS-2基因的序列分析[J]. 中国兽医科学, (11): 933-938. (He G Z, Niu L L, Yang G Y, et al. 2008. Sequence analysis of ITS-2 rDNA of roundworms from Ailuropoda melanoleuca# and seven rare wild animals[J]. Chinese Veterinary Science, (11): 933-938.) [5] 胡鹏辉. 2013. 斑马蛔虫ITS及5.8S rDNA的克隆及进化分析[J]. 中国动物检疫, 30(12): 62-65. (Hu P H.2013. Cloning and phylogenetic analysis of the ITS and 5.8S rDNA of Parascaris equorum from Equus burchelli[J]. China Animal Health Inspection, 30(12): 62-65.) [6] 卡丽比努尔·尔肯. 2015. 南疆驴常见寄生虫鉴定及其流行病学调查[D]. 硕士学位论文, 新疆农业大学, 导师: 巴音查汗, pp. 6-23. (Ka L B N E·E K.2015. The epidemiological research and the identification of common parasite donkey in southern of Xinjiang[D]. Thesis for M.S., Xinjiang Agricultural University, Supervisor: Ba Y C H, pp. 6-23.) [7] 罗晓平. 2012. 骆驼盘尾丝虫病流行病学及病原rDNA-ITS序列研究[D]. 硕士学位论文, 内蒙古农业大学, 导师: 杨晓野, pp. 18-22. (Luo X P.2012. Study on the epidemiology of onchocerciasis and the rDNA-ITS sequences of pathogen in camels[D]. Thesis for M. S., Inner Mongolia Agricultural University, Supervisor: Yang X Y, pp. 18-22.) [8] 卜艳珍. 2008. 河南省驴寄生圆线虫的研究[D]. 博士学位论文, 河北师范大学, 导师: 张路平, pp. 124-128. (Pu Y Z.2008. Studies on strongyloid nematoes of the donkeys in Henan province[D]. Thesis for Ph.D., Hebei Normal University, Supervisor: Zhang L P, pp. 124-128.) [9] 卜艳珍, 崔长海, 张路平. 2009. 河南省驴寄生圆线虫的种类记述—杯环属Cylicocyclus (Ⅱ)[J]. 河南农业科学, (12): 126-129. (Pu Y Z, Cui C H, Zhang L P.2009. Species accounts on strongyloid nematoes of the donkeys in Henan province-Cylicocyclus (Ⅱ)[J]. Journal of Henan Agricultural Sciences, (12): 126-129.) [10] 任娣, 陈武, 宋亚芬, 等. 2011. 斑鼻羚毛首线虫ITS序列的PCR扩增及分析[J]. 动物医学进展, 32(03): 47-50. (Ren D, Chen W, Sun Y F, et al.2011. PCR amplification and analysis of ITS sequence of Trichuris from Addax nasomaculatus[J]. Progress in Veterinary Medicine, 32(03): 47-50.) [11] 张赫凡, 李凯, 陈金良, 等. 2008. 普氏野马寄生虫的监测[J]. 新疆师范大学学报(自然科学版), (01): 94-96. (Zhang H F, Li K, Chen J L, et al.2008. Investigation of parasites of Przewalskii horse[J]. Journal of Xinjiang Normal University (Natural Sciences Edition), (01): 94-96.) [12] 张路平, 孔繁瑶. 2014. 中国动物志无脊椎动物第五十一卷线虫纲杆形目圆线亚目(二)[M]. 北京: 科学出版社, pp. 125-128. (Zhang L P, Kong F Y.2014. Fauna Sinica. Invertebrata. Vol. 51 Nematoda Rhabdida Strongyiata (Ⅱ)[M]. Science Press, Bejing, China, pp. 104-130.) [13] 郑小龙, 王群, 岳志芹, 等. 2013. 普氏野马物种的PCR鉴定[J]. 动物医学进展, 34(11): 81-84. (Zheng X L, Wang Q, Yue Z Q, et al.2013. PCR identification for Equus przewalsrii species[J]. Progress in Veterinary Medicine, 34(11): 81-84.) [14] 周杰. 2005. 中国畜禽寄生虫分类鉴定专家系统初步研究[D]. 硕士学位论文, 中国农业科学院, 导师: 黄兵, pp. 14-18. (Zhou J.2005. Development of parasites identifying expert system for livestock and poultry in China[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Huang B, pp. 14-18.) [15] Der S C.2015. Evolutionary genomics and conservation of the endangered Przewalski's Horse[J]. Current Biology, 25(19): 2577-2583. [16] George-Nascimento M, Gasser R, Zhu X, et al.2002. SSCP-based identification of members within the Pseudoterranova decipiens complex (Nematoda: Ascaridoidea: Anisakidae) using genetic markers in the internal transcribed spacers of ribosomal DNA[J]. Parasitology, 124(6): 615-621. [17] Gert H, Thomas F, Gerhard R, et al.2001. Identification of photobionts from the lichen family physciaceae using algal-specific ITS rDNA sequencin[J]. Lichenologist, 33(1): 73-86. [18] Hung G C, Chilton N B, Beveridge I, et al.1997. Molecular delineation of Cylicocyclus nassatus and C. ashworthi (Nematoda: Strongylidae)[J]. International Journal for Parasitology, 27(5): 601-605. [19] Hust J, Frydenberg J, Sauriau PG, et al.2004. Use of ITS rDNA for discriminating of larval stages of two microphallid (Digenea) species using Hydrobia ulvae (Pennant, 1777) and Corophium volutator (Pallas, 1766) as intermediate hosts[J]. Parasitol Research, 93(4): 304-310. [20] Lichtenfels J R, Kharchenko V A, Dvojnos G M.2008. Illustrated identification keys to strongylid parasites Strongyllidae nematoda of horses zebras and asses Equidae[J]. Veterinary Parasitology, 156(1-2): 117-120. [21] Lichtenfels J R, Kharchenko V A, Sommer C, et al.1997. Key characters for the microscopical identification of Cylicocyclus nassatus and Cylicocyclus ashworthi (Nematoda, Cyathostominae) of the horse, Equuscaballus[J]. Journal of Helminthological Society of Washington, 64(1): 120-127. [22] Nei M, Rooney A P.2005. Concerted and birth-and-death evolution of multigene families[J]. Annual Review of Genetics, 39(1): 121-152. [23] Prasad P K, Tandon V, Chatterjee A, et al.2007. PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA of giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899[J]. Parasitology Research, 101(6): 1581-1587. [24] Robert N, Walzer C, Ruegg S, et al.2005. Pathologic findings in reintroduced Przewalski's horses (Equus caballus przewalskii) in southwestern Mongolia[J]. Journal of Zoo and Wildlife Medicine, 36(2): 273-285. [25] Sim R R, Joyner P H, Padilla L R, et al.2017. Clinical disease associated with anaplasma phagocytophilum infection in captive Przewalski's horses (Equus ferus przewalskii)[J]. Journal of Zoo & Wildlife Medicine, 48(2): 492-497. [26] Tarav M, Tokunaga M, Kondo T, et al.2017. Problems in the protection of reintroduced Przewalski's horses (Equus ferus przewalskii) caused by piroplasmosis[J]. Journal of Wildlife Diseases, 53(4): 911-915. [27] Walzer C, Kaczensky P, Zimmermann W, et al.2012. Przewalski's horse reintroduction to Mongolia: Status and outlook[J]. WAZA Magazine, 13: 3-6.
