基于高通量测序的家犬粪便寄生虫病原调查

doi:10.12140/j.issn.1000-7423.2023.03.010

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (3): 325-330.doi: 10.12140/j.issn.1000-7423.2023.03.010

基于高通量测序的家犬粪便寄生虫病原调查

谢宜¹(), 王莹¹, 王旭¹, 施丹丹¹, 付梅花², 李春阳¹, 伍卫平¹, 丹巴泽里³, 廖沙⁴, 张凯歌¹, 邓雪莹¹, 官亚宜¹^,^*()

1 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，世界卫生组织热带病合作中心，国家级热带病国际联合研究中心，上海 200025
2 上海市疾病预防控制中心，上海 200023
3 甘孜藏族自治州疾病预防控制中心，四川甘孜 626000
4 四川省疾病预防控制中心，成都 610041

收稿日期:2022-12-31 修回日期:2023-02-13 出版日期:2023-06-30 发布日期:2023-06-20
通讯作者: *官亚宜（1963-），女，博士，研究员，从事寄生虫病学、全球卫生研究。E-mail：guan_ml@163.com
作者简介:谢宜（1996-）女，硕士研究生，从事寄生虫病流行病学研究。E-mail：xie08112021@163.com

Investigation of fecal parasite pathogens in domestic dogs based on high-throughput sequencing

XIE Yi¹(), WANG Ying¹, WANG Xu¹, SHI Dandan¹, FU Meihua², LI Chunyang¹, WU Weiping¹, DAN Bazeli³, LIAO Sa⁴, ZHANG Kaige¹, DENG Xueying¹, GUAN Yayi¹^,^*()

1 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; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, China
2 Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200023, China
3 Garzê Tibetan Autonomous Prefecture Center for Disease Control and Prevention, Garzê 626000, Sichuan, China
4 Sichuan Province Center for Disease Prevention and Control, Chengdu 610041, China

Received:2022-12-31 Revised:2023-02-13 Online:2023-06-30 Published:2023-06-20
Contact: *E-mail: guan_ml@163.com

摘要/Abstract

摘要：

目的调查大兴安岭地区家犬寄生虫感染情况，为当地人兽共患寄生虫病的防控提供参考。方法于2020年9月在大兴安岭地区加北村、白桦村、东山村、图强镇、幸福村和古源镇等6个自然村的养犬户院内收集新鲜家犬粪便，提取粪样总DNA，使用顶复门（Apicomplexa）、阿米巴属（Amoeba）、双滴虫目（Diplomonadida）、动质体目（Kinetoplastida）、毛滴虫属（Parabasalia）、线形动物门（Nematoda）、扁形动物门（Platyhelminthes）和小孢子虫目（Microsporidia）等8个分类学群的13对寄生虫通用引物进行PCR扩增，扩增产物经高通量测序后获得目的基因片段，产物序列在NCBI数据库中进行比对分析以鉴定虫种，统计家犬粪便的寄生虫检出率。结果 202份家犬粪便中有37份检出寄生虫DNA，寄生虫总检出率为18.32%。共计检出7个门15个科的19种寄生虫，不同虫种检出率差异具有统计学意义（χ² = 69.488，P < 0.05）。检出原虫13种，蠕虫6种。其中，以原虫眼虫门动质体纲Parabodonida科的Parabodo caudatus检出率最高，为6.44%（13/202）。在37份检出寄生虫DNA的样品中，混合检出阳性占比为32.43%（12/37），其中检出1种寄生虫的占72.97%（27/37），同时检出2种、3种、4种寄生虫的分别占27.03%（10/37）、0.27%（1/37）和0.27%（1/37）。家犬粪便寄生虫DNA检出率以加北村最高，为37.70%（23/61），其次为图强镇，检出率为36.36%（20/55），白桦村未检出（0/13）。不同采样点的检出率差异具有统计学意义（χ² = 19.717，P < 0.05）。结论大兴安岭地区家犬体内存在多种寄生虫感染，且部分地区检出率较高，当地居民存在一定的犬源性寄生虫感染风险。

关键词: 寄生虫, 家犬, 高通量测序, 人兽共患寄生虫病

Abstract:

Objective To investigate the parasitic infection in domestic dogs in Da Hinggan Ling area, and provide reference for the prevention and control of local zoonotic parasitic diseases. Methods In September 2020, fresh domestic dog feces were collected from dog owner’s household yard of 6 natural villages including Jiabei Village, Baihua Village, Dongshan Village, Tuqiang Town, Xingfu Village and Guyuan Town in the Da Hinggan Ling area, and total DNA was extracted from the fecal samples. PCR amplification was conducted using 13 pairs of parasite universal primers targeting 8 taxonomy groups including Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Parabasalia, Nematoda, Platyhelminthes and Microsporidia, and the target gene fragments were obtained after high-throughput sequencing of the amplified products. The product sequences were compared and analyzed in the NCBI database to identify the parasite species, and the detection rate of parasites in domestic dog feces was calculated. Results Parasite DNA were detected in 37 of 202 domestic dog feces, and the total detection rate was 18.31%. A total of 19 species of parasites which belongs to 15 families from 7 phyla were detected, and the detection rate of different species were significantly different (χ² = 69.488, P < 0.05). Thirteen protozoa species and 6 helminth species were detected. The detection rate of Parabodo caudatus in the family Parabodonidae was the highest at 6.44% (13/202). Among the 37 samples in which parasite DNA was detected, the mixed positive ratio was 32.43% (12/37), of which 72.97% (27/37) detected 1 type of parasite, and 2 types, 3 kinds and 4 kinds accounted for 27.03% (10/37), 0.27% (1/37) and 0.27% (1/37) respectively. Jiabei Village had the highest detection rate of parasite DNA in domestic dog feces, which was 37.70% (23/61), followed by Tuqiang Town, with a detection rate of 36.36% (20/55), and Baihua Village, which was not detected (0/13). There was a statistically significant difference in the detection rate of different sampling sites (χ² = 19.717, P < 0.05). Conclusion In Da Hinggan Ling area, domestic dogs were found infected with multiple parasites, showing considerably higher positive rate detected in some parts of the area, thus, there exists risk of infection caninee-source parasites to local residents.

Key words: Parasites, Domestic dogs, High-throughput sequencing, Zoonotic parasitic disease

中图分类号:

谢宜, 王莹, 王旭, 施丹丹, 付梅花, 李春阳, 伍卫平, 丹巴泽里, 廖沙, 张凯歌, 邓雪莹, 官亚宜. 基于高通量测序的家犬粪便寄生虫病原调查[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 325-330.

XIE Yi, WANG Ying, WANG Xu, SHI Dandan, FU Meihua, LI Chunyang, WU Weiping, DAN Bazeli, LIAO Sa, ZHANG Kaige, DENG Xueying, GUAN Yayi. Investigation of fecal parasite pathogens in domestic dogs based on high-throughput sequencing[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(3): 325-330.

图/表 3

表1

表2

大兴安岭地区202份犬粪寄生虫DNA检测结果

寄生虫 Parasite	门 Phylum	纲 Class	目 Order	科 Family	虫种 Species	阳性份数 No. positive	阳性检出率/% Positive rate/%	95%置信区间 95% CI
原虫 Protozoa	原生动物门 Fornicata	动鞭毛亚纲 Zoomastigina	双滴虫目 Diplomonadida	六鞭毛刚科 Hexamitidae	蓝氏贾第鞭毛虫 Giardia lamblia	1	0.50^a	0.03~3.16
	毛滴虫门 Parabasalia	—	毛滴虫目 Trichomonadida	毛滴虫科 Trichomonadidae	人五毛滴虫 Pentatrichomonas hominis	8	3.96^b	1.85~7.93
					鸽源禽毛滴虫 Tetratrichomonas gallinarum	4	1.98^a	0.64~5.32
	眼虫门 Euglenozoa	动质体纲 Kinetoplastida	Parabodonida	—	Parabodo caudatus	13	6.44^b	3.62~11.00
			Neobodonida	—	博多弯毛虫 Neobodo curvifilus	2	1.00^a	0.17~3.91
				Rhynchomonadidae	桑顿锥虫	1	0.50^a	0.03~3.16
				Dimastigella trypaniformis
	顶复门 Apicomplexa	Aconoidasida	梨形虫目 Piroplasmida	巴贝虫科 Babesiidae	驽巴贝虫 Babesia caballi	1	0.50^a	0.03~3.16
		Conoidasida	Eucoccidiorida	Sarcocystidae	犬等孢子虫 Cystoisospora canis	1	0.50^a	0.03~3.16
	丝足虫门 Cercozoa	—	Cercomonadida	Heteromitidae	球形丝足虫 Heteromita globosa	1	0.50^a	0.03~3.16
				Cercomonadidae	海绵体单胞丝足虫 Cavernomonas mira	1	0.50^a	0.03~3.16
					丝足虫 Cavernomonas stercoris	1	0.50^a	0.03~3.16
	纤毛门 Ciliophora	Spirotrichea	Sporadotrichida	Gonostomatidae	纤毛虫1 Metagonostomum gonostomoida	1	0.50^a	0.03~3.16
		Colpodea	Cyrtolophosidida	Platyophryidae	纤毛虫2 Platyophrya spumacola	1	0.50^a	0.03~3.16
蠕虫 Worms	线虫门 Nematode	线虫纲 Chromadorea	杆形目 Strongylida	钩口科 Ancylostomatidae	狭首钩刺线虫 Uncinaria stenocephala	7	3.47^b	1.53~7.31
				毛圆科 Molineidae	摩利诺斯线虫 Molineus patens	1	0.50^a	0.03~3.16
				Chabertiidae	食道口线虫 Oesophagostomum muntiacum	1	0.50^a	0.03~3.16
			小杆（线虫）目 Rhabditida	弓首科 Toxocaridae	犬弓首蛔虫 Toxocara canis	4	1.98^a	0.64~5.32
				小杆（线虫）总科 Rhabditidae	纳氏隐杆线虫 Rhabditoides inermiformis	3	1.49^a	0.39~4.64
				Thelaziidae	螺旋体线虫 Spirocerca vulpis	1	0.50^a	0.03~3.16
合计 Total						37^c	18.32

表2

图1

参考文献 19

[1]	Chen YD, Zhou CH, Zhou XN, et al. National survey on the current status of important human parasitic diseases in China in 2015[J]. Chin J Parasitiol Parasit Dis, 2020, 38(1): 5-16. (in Chinese)
	(陈颖丹, 周长海, 周晓农, 等. 2015年全国人体重点寄生虫病现状调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 5-16.)
[2]	Tang L, Xing ZF, Ge T, et al. Current status of key parasitic infections of humans in Heilongjiang Province in 2015[J]. Chin J Parasitol Parasit Dis, 2018, 36(4): 388-391, 398. (in Chinese)
	(唐磊, 邢智锋, 葛涛, 等. 2015年黑龙江省人体重点寄生虫感染现状调查[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 388-391, 398.)
[3]	Lyons MA, Malhotra R, Thompson CW. Investigating the free-roaming dog population and gastrointestinal parasite diversity in Tulúm, México[J]. PLoS One, 2022, 17(10): e0276880. doi: 10.1371/journal.pone.0276880
[4]	Wang Y, Sun BB, Yu SK. Investigation on the species and infective status of enteroparasites of scattered dogs in a town of Huxian[J]. Prog Vet Med, 2015, 36(3): 130-132. (in Chinese)
	(王银, 孙彬彬, 于三科. 户县某镇散养犬肠道寄生虫种类及感染情况的调查[J]. 动物医学进展, 2015, 36(3): 130-132.)
[5]	Ma DY, Lang P, Xin LY, et al. Investigation on intestinal parasite infection in dogs in Emin County[J]. Vet Orientat, 2021(16): 224. (in Chinese)
	(马丁允, 郎平, 信璐瑶, 等. 额敏县犬肠道寄生虫感染情况调查[J]. 兽医导刊, 2021(16): 224.)
[6]	Xu ZJ. General situation of human parasitic diseases in Heilongjiang Province[C]. Compile Abstracts 8th National Symposium Parasitology Zoological Society of China. Chinese Society of Parasitology, 2001: 2. (in Chinese)
	(徐之杰. 黑龙江省人体寄生虫病概况[C]. 中国动物学会第八次全国寄生虫学学术讨论会论文摘要汇编. 中国动物学会寄生虫学专业委员会, 2001: 2.)
[7]	Cannon MV, Bogale H, Rutt L, et al. A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples[J]. Microbiome, 2018, 6(1): 195. doi: 10.1186/s40168-018-0581-6 pmid: 30373673
[8]	Zhang ZP, Hu GW, Zhao QB, et al. Discovery of Lepidoptera stenocellata in the three rivers source area[J]. Chin J Animal Husb Vet Med, 2019(11): 29-30. (in Chinese)
	(张志平, 胡广卫, 赵全邦, 等. 三江源地区发现狭头钩刺线虫[J]. 畜牧兽医科技信息, 2019(11): 29-30.)
[9]	Miller TA. Hookworm infection in man[J]. Adv Parasitol, 1979, 17: 315-384.
[10]	Li CY, Wang X, Chen QL, et al. Investigation of helminth infection in wild canines in Shiqu County, Sichuan Province based on molecular biology technology[J]. J Pathog Biol, 2022, 17(6): 675-681. (in Chinese)
	(李春阳, 王旭, 陈齐鲁, 等. 基于分子生物学技术对四川省石渠县野外犬科动物蠕虫感染情况调查[J]. 中国病原生物学杂志, 2022, 17(6): 675-681.)
[11]	Wu TK, Bowman DD. Toxocara canis[J]. Trends Parasit, 2022, 38(8): 709-710. doi: 10.1016/j.pt.2022.01.002
[12]	Zhang YL, Zhang SZ, Gu XB, et al. Morphological observations of Toxocara canis egg development in vitro[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 486-489. (in Chinese)
	(张艺琳, 张森棹, 古小彬, 等. 犬弓首蛔虫卵体外发育的形态观察[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 486-489.)
[13]	Mahittikorn A, Udonsom R, Koompapong K, et al. Molecular identification of Pentatrichomonas hominis in animals in central and western Thailand[J]. BMC Vet Res, 2021, 17(1): 1-7. doi: 10.1186/s12917-020-02730-8
[14]	Mei RF, Lin XY, Sun TB, et al. Investigation on Pentatrichomonas hominis infection of wild zoo animal in Hebei Province[J]. Chin J Vete Med, 2022, 58(6): 123-126.
	(梅汝蕃, 林小雨, 孙通宝, 等. 河北省某野生动物园园养动物人五毛滴虫感染调查[J]. 中国兽医杂志, 2022, 58(6): 123-126.)
[15]	Li WC, Gong PT, Ying M, et al. Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China[J]. Parasitol Res, 2014, 113(5): 1795-1801. doi: 10.1007/s00436-014-3825-9
[16]	Zhang N, Zhang HB, Yu YH, et al. High prevalence of Pentatrichomonas hominis infection in gastrointestinal cancer patients[J]. Parasit Vectors, 2019, 12(1): 1-9. doi: 10.1186/s13071-018-3256-z
[17]	Vandersea MW, Birkenheuer AJ, Litaker RW, et al. Identification of Parabodo caudatus (class Kinetoplastea) in urine voided from a dog with hematuria[J]. J VET Diagn Invest, 2015, 27(1): 117-120. doi: 10.1177/1040638714562827 pmid: 25525146
[18]	Li YF. Classification and biological characteristics of Trichomonas avium isolates from pigeon in some cities of Shandong Province[D]. Taian: Shandong Agric Univ, 2022: 31. (in Chinese)
	(李俞锋. 山东省部分地市鸽源禽毛滴虫分离虫株分型及生物学特性分析[D]. 泰安: 山东农业大学, 2022: 31.)
[19]	Jiang XH, Sun JJ, Xiao YH, et al. Investigation and genetic evolution analysis of T. gallinae infection from pigeons in some areas of Shandong Province[C]. Proceedings 7th Congress 12th Symposium Animal Parasitology Branch Chinese Association Animal Husbandry Veterinary Medicine. Chinese Society of Animal Husbandry and Veterinary Medicine, 2013: 261-262. (in Chinese)
	(姜夕月, 孙静静, 肖一红, 等. 山东省部分地区鸽源禽毛滴虫 (T. gallinae) 感染情况调查及遗传进化分析[C]. 中国畜牧兽医学会家畜寄生虫学分会第七次代表大会暨第十二次学术研讨会论文集. 2013: 261-262.)

物种 Species	引物名称 Primer name	引物序列 (5'→3') Primer sequence (5'→3')	产物长度/bp Product length/bp	退火温度/℃ Annealing temperature/℃
阿米巴属 Amoeba	AmoF AmoR	GAATTGACGGAAGGGCACAC GCCCYRTCTAAGGGCATCAC	333~420	57.8
顶复门A Apicomplexa A	ApiAF ApiAR	GACCTATCAGCTTTCGACGG CCCTCCAATTGWTACTCTGGR	244~248	55.0
顶复门B Apicomplexa B	ApiBF ApiBR	TGYGTTTGAATACTAYAGCATGG TCTGATCGTCTTCACTCCCTT	228~244	53.7
顶复门C Apicomplexa C	ApiCF ApiCR	TTGGMCTACCGTGGCARTGA TCAAGGCAAHWGCCTGCTT	420~470	57.5
芽囊原虫属 Blastocystis	BlaF BlaR	TGGTCGCAAGGCTGAAACTT TTGCCTCCAGCTTCCCTACA	303~307	58.1
双滴虫目 Diplomonadida	DipF DipR	RGGGACRGGTGAAATAGGATG CAAATTGAGCCGCAGACTCC	277~285	56.5
动质体门 Kinetoplastida	KinF KinR	AAATTAAACCGCACGCTCCA GCAAACGATGACACCCATGA	250~300	55.4
小孢子虫门 Microsporidia	MicF MicR	BCAGGTTGATTCTGCCTGACR ACCAGWCTTGCCCTCCARTT	370~440	57.9
副基体纲 Parabasalia	ParF ParR	TAGGCTATCACGGGTAACGG GCGTCCTGATTTGTTCACAG	326~364	55.3
扁形动物门 Platyhelminthes	PlaF PlaR	CAATTGGAGGGCAAGTCTGG TGCTTTCGCWKTAGTTTGTCTG	350~550	55.4
线性动物门A Nematode A	NemAF NemAR	CACCCGTGAGGATTGACAG CGATCACGGAGGATTTTCAA	320~335	54.1
线性动物门B Nematode B	NemBF NemBR	CGTCATTGCTGCGGTTAAAA CCGTCCTTCGAACCTCTGAC	380~410	56.0
线性动物门C Nematode C	NemCF NemCR	AGTGGAGCATGCGGCTTAAT TGCAATTCCCTRTCCCAGTC	380~440	56.6

基于高通量测序的家犬粪便寄生虫病原调查

Investigation of fecal parasite pathogens in domestic dogs based on high-throughput sequencing

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