深圳某引种奶牛场奶牛感染隐孢子虫的分子特征

doi:10.12140/j.issn.1000-7423.2020.06.007

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (6): 718-722.doi: 10.12140/j.issn.1000-7423.2020.06.007

深圳某引种奶牛场奶牛感染隐孢子虫的分子特征

姜岩岩¹(), 袁忠英¹, 沈玉娟¹, 徐宁¹, 张仁利², 黄达娜², 曹建平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,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
2 深圳市疾病预防控制中心,深圳 430079

收稿日期:2020-07-13 出版日期:2020-12-30 发布日期:2021-01-12
通讯作者: 曹建平
作者简介:姜岩岩（1979-）,女,博士,副研究员,从事肠道原虫检测及感染免疫研究。E-mail: jiangyy083@163.com
基金资助:
国家重点研发项目(2017YFD0501300);国家卫生行业科研专项(201502021)

Molecular characteristics of Cryptosporidium sp. isolated from imported breeding dairy cows in a dairy farm in Shenzhen, China

JIANG Yan-yan¹(), YUAN Zhong-ying¹, SHEN Yu-juan¹, XU Ning¹, ZHANG Ren-li², HUANG Da-na², CAO Jian-ping¹^,^*()

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention;Chinese Center for Tropical Diseases Research;WHO Collaborating Centre for Tropical Diseases;National Center for International Research on Tropical Diseases, Ministry of Science and Technology;Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
2 Shenzhen Center for Disease Control and Prevention, Shenzhen 430079, China

Received:2020-07-13 Online:2020-12-30 Published:2021-01-12
Contact: CAO Jian-ping
Supported by:
National Key Research and Development Program of China(2017YFD0501300);Chinese Special Program for Scientific Research of Public Health(201502021)

摘要/Abstract

摘要：

目的了解深圳市某奶牛场引进育种奶牛的隐孢子虫感染情况及隐孢子虫分离株的分子特性。方法于2018年5月收集深圳市某奶牛场奶牛新鲜粪样,粪样分装后抽提隐孢子虫基因组DNA,巢式PCR扩增隐孢子虫小亚单位核糖体核糖核酸基因（SSU rDNA）,阳性样品进行测序和BLAST比对分析,鉴定隐孢子虫基因型,并构建系统进化树。采用Graphad Prism7.0软件进行作图和卡方检验。结果共收集20头奶牛粪样,其中成年产乳牛粪样5份,断奶后犊牛粪样10份,断奶前犊牛粪样5份。检出隐孢子虫阳性8份,阳性率为40%;其中成年产乳牛隐孢子虫阳性1份（1/5）,断奶后犊牛隐孢子虫阳性5份（5/10）,断奶前犊牛隐孢子虫阳性2份（2/5）。测序分析鉴定出2个隐孢子虫基因型,芮氏隐孢子虫（n = 5）和牛隐孢子虫（n = 3）,各年龄组中均有芮氏隐孢子虫感染。经与GenBank中隐孢子虫SSU rDNA基因型比对,5个芮氏隐孢子虫分离株中4个与埃塞俄比亚牛源芮氏隐孢子虫SSU rDNA基因（GenBank登录号为KT922233）序列相似度达100%,1个与华北地区牛源芮氏隐孢子虫SSU rDNA基因（GenBank登录号为HQ179574）序列相似度达99.9%;3个牛隐孢子虫分离株与埃塞俄比亚牛源牛隐孢子虫SSU rDNA基因（GenBank登录号为KT922231）的序列相似度达100%。基于SSU rDNA基因序列构建的隐孢子虫种系发育进化树,本研究获得的芮氏隐孢子虫序列与牛隐孢子虫分别在两群分支中,与不同地区的同一隐孢子虫虫种一致。结论深圳市某奶牛场奶牛感染隐孢子虫较为普遍,以芮氏隐孢子虫为优势感染虫种。

关键词: 芮氏隐孢子虫, 牛隐孢子虫, SSU rDNA, 奶牛, 深圳市

Abstract:

Objective To investigate the status of Cryptosporidium infection in imported breeding dairy cows in a dairy farm in Shenzhen, and analyze the molecular characteristics of Cryptosporidium isolates. Methods Fresh cow fecal samples were collected from a dairy farm in Shenzhen. Genomic DNA was extracted from the samples, and the small subunit ribosomal ribonucleic acid gene (SSU rDNA) of Cryptosporidium was amplified by nested PCR. The PCR products were sequenced and blasted to determine the genotype, and construct phylogenetic tree. Data were analyzed with Chi-square test using Graphad Prism 7.0. Results A total of 20 cow fecal samples were collected, including 5 from adult cows, 10 from weaned calves and 5 from pre-weaning calves. The PCR results showed that 8 samples were positive of Cryptosporidium, with positive rate of 40%, including 1 adult cow (1/5), 5 weaned calves (5/10), and 2 pre-weaning calves (2/5). Sequencing results showed that 2 Cryptosporidium genotypes were detected, including C. ryanae (n = 5) and C. bovis (n = 3). The C. ryanae infection was found in all age groups. After blasting with the SSU rDNA gene of C. ryanae, four of the five isolates of C. ryanae were 100% similar to the SSU rDNA gene (KT922233) of bovine-borne C. ryanae from Ethiopia, and the other one was similar to that of the isolate of C. ryanae (HQ179574, 99.9%) from Northeast China. The three isolates of C. bovis were 100% similar with the SSU rDNA gene (KT922231) of bovine-borne C. bovis from Ethiopia. After constructing the phylogenetic tree of C. ryanae based on the sequence of SSU rDNA gene, the sequences of C. ryanae and C. bovis were in two branches, which were consistent with the same isolated Cryptosporidium species in different regions. Conclusion Cryptosporidium infection is common in this dairy farm in Shengzhen, with C. ryanae as a dominant species in infection.

Key words: Cryptosporidium ryanae, Cryptosporidium bovis, SSU rDNA, Dairy cattle, Shenzhen City

中图分类号:

R382.3

姜岩岩, 袁忠英, 沈玉娟, 徐宁, 张仁利, 黄达娜, 曹建平. 深圳某引种奶牛场奶牛感染隐孢子虫的分子特征[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(6): 718-722.

JIANG Yan-yan, YUAN Zhong-ying, SHEN Yu-juan, XU Ning, ZHANG Ren-li, HUANG Da-na, CAO Jian-ping. Molecular characteristics of Cryptosporidium sp. isolated from imported breeding dairy cows in a dairy farm in Shenzhen, China[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(6): 718-722.

图/表 3

参考文献 20

[1]	Gong C, Cao XF, Deng L, et al. Epidemiology of Cryptosporidium infection in cattle in China: a review[J]. Parasite, 2017,24:1. pmid: 28098070
[2]	Panciera RJ, Thomassen RW, Garner FM. Cryptosporidial infection in a calf[J]. Vet Pathol, 1971,8(5/6):479-484.
[3]	Guo BP, Lian DR. Investigation on Cryptosporidium infection in dairy cattle in Guangzhou[J]. Chin J Zoonoses, 1999,15(3):10. (in Chinese)
	( 郭步平, 连德润. 广州奶牛隐孢子虫感染调查[J]. 中国人兽共患病学报, 1999,15(3):10.)
[4]	Zhao JF, Qi M, Wang HY, et al. Species and genotype identification of Cryptosporidium in cattle in Kaifeng, China[J]. Chin J Zoonoses, 2015,31(8):733-736. (in Chinese)
	( 赵金凤, 齐萌, 王海燕, 等. 开封地区奶牛隐孢子虫种类及基因型鉴定[J]. 中国人兽共患病学报, 2015,31(8):733-736.)
[5]	Li WC, Yang HH, Kan ZZ, et al. Prevalence of Cryptosporidium spp. infections in large-scale pig farms in north of Yangtze River in Anhui Province[J]. Chin J Schisto Control, 2018,30(4):420-423, 464. (in Chinese)
	( 李文超, 杨回回, 阚珍珍, 等. 安徽省江北地区规模化猪场隐孢子虫感染现状调查[J]. 中国血吸虫病防治杂志, 2018,30(4):420-423, 464.)
[6]	Meng R, Wang X, Dong M, et al. Investigation on Cryptosporidium infection among dairy cattle in Tianjin area, China[J]. Chin J Pathogen Biol, 2007,2(3):225-227. (in Chinese)
	( 孟然, 王欣, 董明, 等. 天津地区奶牛感染隐孢子虫情况的调查[J]. 中国病原生物学杂志, 2007,2(3):225-227.)
[7]	Jiang Y, Ren J, Yuan Z, et al. Cryptosporidium andersoni as a novel predominant Cryptosporidium species in outpatients with diarrhea in Jiangsu Province, China[J]. BMC Infect Dis, 2014,14:555.
[8]	Liu H, Shen Y, Yin J, et al. Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China[J]. BMC Infect Dis, 2014,14:25. pmid: 24410985
[9]	Cai M, Guo Y, Pan B, et al. Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai, China[J]. Vet Parasitol, 2017,241:14-19.
[10]	Sulaiman IM, Lal AA, Xiao LH. Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus[J]. J Parasitol, 2002,88(2):388.
[11]	Liu AQ, Wang RJ, Li YH, et al. Prevalence and distribution of Cryptosporidium spp. in dairy cattle in Heilongjiang Province, China[J]. Parasitol Res, 2009,105(3):797-802. pmid: 19424720
[12]	Fan YY, Wang T, Koehler AV, et al. Molecular investigation of Cryptosporidium and Giardia in pre-and post-weaned calves in Hubei Province, China[J]. Parasites Vectors, 2017,10:519.
[13]	Wegayehu T, Karim R, Anberber M, et al. Prevalence and genetic characterization of Cryptosporidium species in dairy calves in central Ethiopia[J]. PLoS One, 2016,11(5):e0154647. pmid: 27135243
[14]	Liang N, Wu YY, Sun MF, et al. Molecular epidemiology of Cryptosporidium spp. in dairy cattle in Guangdong Province, South China[J]. Parasitology, 2019,146(1):28-32. pmid: 29986775
[15]	Ayinmode AB, Olakunle FB, Xiao LH. Molecular characterization of Cryptosporidium spp. in native calves in Nigeria[J]. Parasitol Res, 2010,107(4):1019-1021. doi: 10.1007/s00436-010-1972-1 pmid: 20644959
[16]	Âberg M, Emanuelson U, Troell K, et al. A single-cohort study of Cryptosporidium bovis and Cryptosporidium ryanae in dairy cattle from birth to calving[J]. Vet Parasitol Reg Stud Reports, 2020,20:100400. pmid: 32448548
[17]	Zhang XY, Jian YN, Li XP, et al. Molecular detection and prevalence of Cryptosporidium spp. infections in two types of domestic farm animals in the Qinghai-Tibetan Plateau Area (QTPA) in China[J]. Parasitol Res, 2018,117(1):233-239. doi: 10.1007/s00436-017-5697-2 pmid: 29199371
[18]	Qi M, Wang HY, Jing B, et al. Occurrence and molecular identification of Cryptosporidium spp. in dairy calves in Xinjiang, Northwestern China[J]. Vet Parasitol, 2015,212(3/4):404-407.
[19]	Feng YY, Gong XQ, Zhu KX, et al. Prevalence and genotypic identification of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves in Guangdong, China[J]. Parasites Vectors, 2019,12:41.
[20]	Zhang WZ, Wang RJ, Yang FK, et al. Distribution and genetic characterizations of Cryptosporidium spp. in pre-weaned dairy calves in northeastern China’s Heilongjiang Province[J]. PLoS One, 2013,8(1):e54857.

不同年龄 Age (months)	样品数 No. of samples	阳性数 No. of positive	基因型（数量） Species（n）
断奶前犊牛（≤ 2月） Pre-weaned calves (≤ 2 months)	5	2	芮氏隐孢子虫C. ryanae（1）,牛隐孢子虫C. bovis（1）
断奶后犊牛(> 2月) Pre-weaned calves (> 2 months)	10	5	芮氏隐孢子虫C. ryanae（3）,牛隐孢子虫C. bovis（2）
成年产乳牛 Dairy cattle	5	1	芮氏隐孢子虫C. ryanae（1）
合计Total	20	8	芮氏隐孢子虫C. ryanae（5）,牛隐孢子虫C. bovis（3）

深圳某引种奶牛场奶牛感染隐孢子虫的分子特征

Molecular characteristics of Cryptosporidium sp. isolated from imported breeding dairy cows in a dairy farm in Shenzhen, China

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