新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况及其分子鉴定

doi:10.12160/j.issn.1672-5190.2023.05.016

摘要/Abstract

摘要：

[目的]了解新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况和基因亚型分布特点。[方法]采集新疆维吾尔自治区某规模化绵羊养殖场4个品种1月龄以内腹泻羔羊新鲜粪便样本60份，使用饱和蔗糖溶液漂浮法检查隐孢子虫卵囊，进行种类初步鉴定；全部粪便样本提取基因组DNA后，基于隐孢子虫SSU rRNA基因位点和微小隐孢子虫gp60基因位点，对其进行PCR扩增、测序和序列分析，鉴定隐孢子虫种属和基因亚型，构建遗传进化树解析其分子遗传特征。[结果]经显微镜观察，发现38份样本呈隐孢子虫卵囊阳性，形态学初步鉴定为微小隐孢子虫；基于SSU rRNA基因位点，采用PCR方法检测出52份样本呈隐孢子虫阳性，感染率为86.67%（52/60），经序列比对分析，均为微小隐孢子虫；基于微小隐孢子虫gp60基因位点，PCR扩增后成功获得49条序列，经比对分析均为ⅡdA19G1基因亚型。[结论]该养殖场腹泻羔羊普遍感染微小隐孢子虫，其基因亚型均为ⅡdA19G1。调查结果为新疆南疆绵羊隐孢子虫种属鉴定与遗传进化研究提供了基础数据。

关键词: 隐孢子虫, 感染, 鉴定, 基因亚型, 羔羊

Abstract:

[Objective] This study was conducted to characterize the prevalence and the subgenotype distribution of Cryptosporidium in diarrheal lambs from a large-scale sheep farm in southern Xinjiang. [Method] A total of 60 fresh fecal samples were collected from the diarrheal lambs of four breeds less than 1 month old in a large-scale sheep farm in Xinjiang Uygur Autonomous Region, China. The species identification of Cryptosporidium oocysts was carried out by saturated sucrose flotation technique combined with microscopic examination. After the fecal genomic DNA was extracted, PCR amplifications and sequencing analyses targeting on SSU rRNA gene and gp60 gene were performed to confirm the species of Cryptosporidium and the subgenotypes of C. parvum, respectively. Furthermore, the phylogenetic tree based on the gp60 gene was constructed to reveal the molecular genetic characteristics of Cryptosporidium. [Result] Thirty-eight fecal samples were found to be positive for the presence of Cryptosporidium oocysts which were preliminarily identified as C. parvum by morphology observation. Based on the SSU rRNA gene sequence comparison, a total of 52 fecal samples were positive for C. parvum, with the infection rate of 86.67% （52/60）. Among them, 49 sequences of gp60 gene were obtained by PCR amplification, and the phylogenetic tree demonstrated that they all belonged to the subgenotype of ⅡdA19G1. [Conclusion] A high prevalence of C. parvum with the subgenotype of ⅡdA19G1 was observed in the diarrheal lambs in this farm. The results obtained in this study provides basic data for the species identification and genetic evolution analysis of Cryptosporidium in the sheep flocks in southern Xinjiang.

Key words: Cryptosporidium, infection, identification, subg-enotype, lamb

中图分类号:

S858.262.723

张博文, 司俊飞, 张振杰, 郭嘉栋, 陈荣, 赫永强, 余复昌, 齐萌. 新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况及其分子鉴定[J]. 畜牧与饲料科学, 2023, 44(5): 115-120.

ZHANG Bowen, SI Junfei, ZHANG Zhenjie, GUO Jiadong, CHEN Rong, HE Yongqiang, YU Fuchang, QI Meng. Prevalence and Molecular Identification of Cryptosporidium in Diarrheal Lambs from a Large-scale Sheep Farm in Southern Xinjiang[J]. Animal Husbandry and Feed Science, 2023, 44(5): 115-120.

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参考文献 20

[1]	RYAN U, ZAHEDI A, PAPARINI A. Cryptosporidium in humans and animals-A one health approach to prophylaxis[J]. Parasite Immunology, 2016, 38(9): 535-547. doi: 10.1111/pim.2016.38.issue-9
[2]	YANG X, GONG Q, ZHAO B, et al. Prevalence of Cryptosporidium infection in sheep and goat flocks in China during 2010—2019:A systematic review and meta-analysis[J]. Vector-Borne and Zoonotic Diseases, 2021, 21(9): 692-706. doi: 10.1089/vbz.2020.2713
[3]	RYAN U M, FENG Y, FAYER R, et al. Taxonomy and molecular epidemiology of Cryptosporidium and Giardia-A 50 year perspective (1971—2021)[J]. International Journal for Parasitology, 2021, 51(13/14): 1099-1119. doi: 10.1016/j.ijpara.2021.08.007
[4]	MI R, WANG X, HUANG Y, et al. Sheep as a potential source of zoonotic Cryptosporidiosis in China[J]. Applied and Environmental Microbiology, 2018, 84(18): e00868-18.
[5]	杨佳冰, 米荣升, 张烨华, 等. 宁夏吴忠市羊隐孢子虫感染情况调查及虫种鉴定[J]. 中国动物传染病学报, 2019, 27(6): 72-78.
[6]	XIAO L, FENG Y. Molecular epidemiologic tools for waterborne pathogens Cryptosporidium spp. and Giardia duodenalis[J]. Food and Waterborne Parasitology, 2017(8/9): 14-32.
[7]	李文超, 汪凯, 唐莉, 等. 安徽及周边省份绵羊和山羊隐孢子虫分子特性分析[J]. 中国血吸虫病防治杂志, 2019, 31(5): 474-478.
[8]	QI M, ZHANG Z, ZHAO A, et al. Distribution and molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi amongst grazing adult sheep in Xinjiang, China[J]. Parasitology International, 2019, 71:80-86. doi: 10.1016/j.parint.2019.04.006
[9]	YE J, XIAO L, WANG Y, et al. Periparturient transmission of Cryptosporidium xiaoi from ewes to lambs[J]. Veterinary Parasitology, 2013, 197(3/4): 627-633. doi: 10.1016/j.vetpar.2013.07.021
[10]	李治国, 徐春艳, 侯旻昱, 等. 新疆部分地区牧马肠道寄生虫感染情况调查[J]. 畜牧与兽医, 2019, 51(9): 87-90.
[11]	RYAN U, XIAO L, READ C, et al. Identification of novel Cryptosporidium genotypes from the Czech Republic[J]. Applied and Environmental Microbiology, 2003, 69(7): 4302-4307. doi: 10.1128/AEM.69.7.4302-4307.2003
[12]	ALVES M, XIAO L, SULAIMAN I, et al. Subgenotype analysis of Cryptosporidium isolates from humans,cattle,and zoo ruminants in Portugal[J]. Journal of Clinical Microbiology, 2003, 41(6): 2744-2747. doi: 10.1128/JCM.41.6.2744-2747.2003
[13]	CONNELLY L, CRAIG B H, JONES B, et al. Genetic diversity of Cryptosporidium spp. within a remote population of soay sheep on St. Kilda Islands, Scotland[J]. Applied and Environmental Microbiology, 2013, 79(7): 2240-2246. doi: 10.1128/AEM.02823-12
[14]	CHEN Y, QIN H, HUANG J, et al. The global prevalence of Cryptosporidium in sheep:A systematic review and meta-analysis[J]. Parasitology, 2022, 149(12): 1652-1665. doi: 10.1017/S0031182022001196
[15]	TAKO S, FLEIDEROVITZ L, MARKOVICH M P, et al. Cryptosporidium parvum gp60 subtypes in diarrheic lambs and goat kids from Israel[J]. Parasitology Research, 2023, 122(9): 2237-2241. doi: 10.1007/s00436-023-07925-0
[16]	IMRE K, LUCA C, COSTACHE M, et al. Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries)[J]. Veterinary Parasitology, 2013, 191(1/2): 119-122. doi: 10.1016/j.vetpar.2012.08.020
[17]	DÍAZ P, NAVARRO E, PRIETO A, et al. Cryptosporidium species in post-weaned and adult sheep and goats from N.W. Spain: Public and animal health significance[J]. Veterinary Parasitology, 2018, 254:1-5. doi: 10.1016/j.vetpar.2018.02.040
[18]	PAPANIKOLOPOULOU V, BAROUDI D, GUO Y, et al. Genotypes and subtypes of Cryptosporidium spp. in diarrheic lambs and goat kids in northern Greece[J]. Parasitology International, 2018, 67(4): 472-475. doi: 10.1016/j.parint.2018.04.007
[19]	KAUPKE A, MICHALSKI M M, RZEZUTKA A. Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland[J]. Parasitology Research, 2017, 116(3): 871-879. doi: 10.1007/s00436-016-5360-3
[20]	PAZ E SILVA F M, LOPES R S, BRESCIANI K D, et al. High occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil[J]. Acta Parasitologica, 2014, 59(1): 193-196.