四川省某奶牛场犊牛呼吸道疾病的细菌性病原qPCR检测

doi:10.3969/j.issn.1004-1524.2022.04.06

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (4): 706-712.DOI: 10.3969/j.issn.1004-1524.2022.04.06

四川省某奶牛场犊牛呼吸道疾病的细菌性病原qPCR检测

陈久兵¹(), 耿尚景超¹^,², 王方国¹^,², 陈娅婷¹, 周金伟³, 骆巧¹, 马莉¹, 姚学萍¹, 余树民¹, 沈留红¹, 储岳峰², 曹随忠¹^,^*()

1.四川农业大学动物医学院,四川成都 611130
2.中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃兰州730046
3.蒲江县农业农村局,四川蒲江 611630

收稿日期:2020-10-10 出版日期:2022-04-25 发布日期:2022-04-28
通讯作者: 曹随忠
作者简介:*曹随忠,E-mail: suizhongcao@sicau.edu.cn
陈久兵(1994—),男,四川广元人,硕士研究生,研究方向为动物疾病诊疗研究。E-mail: jiubingchen11@163.com
基金资助:
国家重点研发计划(2018YFD0502305);校企合作项目(002H2001)

qPCR detection of bacterial pathogens of respiratory diseases in calves of a dairy farm from Sichuan

CHEN Jiubing¹(), GENGSHANG Jingchao¹^,², WANG Fangguo¹^,², CHEN Yating¹, ZHOU Jinwei³, LUO Qiao¹, MA Li¹, YAO Xueping¹, YU Shumin¹, SHEN Liuhong¹, CHU Yuefeng², CAO Suizhong¹^,^*()

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3. Agriculture and Rural Bureau of Pujiang County, Pujiang 611630, Sichuan, China

Received:2020-10-10 Online:2022-04-25 Published:2022-04-28
Contact: CAO Suizhong

摘要/Abstract

摘要：

为了解四川省某规模化奶牛养殖场犊牛呼吸道疾病(bovine respiratory disease, BRD)的病原情况,进而制定防治措施,本研究通过TaqMan荧光定量PCR方法对2019—2020年75份有呼吸道疾病症状的犊牛鼻腔棉拭子和10份其他样品进行常见细菌性病原检测。结果显示,多杀性巴氏杆菌(Pm)、睡眠嗜血杆菌(Hs)、牛支原体(Mb)、化脓隐秘杆菌(Tp)和溶血性曼氏杆菌(Mh)的检出率分别为 57.33%(43/75)、38.67%(29/75)、13.33%(10/75)、12.00%(9/75)和8.00%(6/75)。单一感染率为36.00%(27/75),主要由Pm引发,占24.00%(18/75);混合感染率为 40.00%(30/75),尤以Hs和Pm引发的混合感染为主,占18.67%(14/75)。结果表明,5种常见BRD细菌性致病菌均有检出,且混合感染类型较多。Hs的高检出率表明其与我国BRD有一定联系,有必要在全国范围内进一步开展其流行病学调查。

关键词: 牛呼吸道疾病, TaqMan荧光定量PCR, 睡眠嗜血杆菌, 混合感染

Abstract:

In order to explore the pathogens of bovine respiratory disease(BRD) in a large-scale dairy farm in Sichuan Province, TaqMan fluorescent quantitative PCR method was used to analyze 75 nasal cotton samples from the calves with respiratory disease symptoms and 10 other samples during 2019 to 2020 for common bacterial pathogens. The results showed that the detection rates of Pasteurella multocida(Pm), Histophilus somni(Hs), Mycoplasma bovis(Mb), Trueperella pyogenes(Tp) and Mannheimia haemolytica(Mh) were 57.33%(43/75), 38.67%(29/75), 13.33%(10/75), 12.00%(9/75), 8.00%(6/75). The single infection rate was 36.00% (27/75), mainly caused by Pm, accounting for 24.00%(18/75); the mixed infection rate was 40.00%(30/75), especially the mixed infection caused by Hs and Pm, accounted for 18.67%(14/75).The results showed that all five common BRD bacterial pathogens were detected, and there were many types of mixed infections. The high detection rate of Hs indicated that it had a certain connection with BRD in China, and it was necessary to carry out further epidemiological investigations nationwide.

Key words: bovine respiratory disease, TaqMan real-time PCR, Histophilus somni, mixed infection

中图分类号:

S823

陈久兵, 耿尚景超, 王方国, 陈娅婷, 周金伟, 骆巧, 马莉, 姚学萍, 余树民, 沈留红, 储岳峰, 曹随忠. 四川省某奶牛场犊牛呼吸道疾病的细菌性病原qPCR检测[J]. 浙江农业学报, 2022, 34(4): 706-712.

CHEN Jiubing, GENGSHANG Jingchao, WANG Fangguo, CHEN Yating, ZHOU Jinwei, LUO Qiao, MA Li, YAO Xueping, YU Shumin, SHEN Liuhong, CHU Yuefeng, CAO Suizhong. qPCR detection of bacterial pathogens of respiratory diseases in calves of a dairy farm from Sichuan[J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 706-712.

图/表 4

参考文献 27

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病原 Pathogen	目标基因 Target gene	引物、探针 Primer, probe (5'-3')	片段大小 Fragment size/bp
Pm	kmt-1	F: GGGCTTGTCGGTAGTCTTT	148^[2]
		R: CGGCAAATAACAATAAGCTGAGTA P: FAM-CGGCGCAACTGATTGGACGTTATT-TAMRA
Hs	16S-rRNA	F: AAGGCCTTCGGGTTGTAAAG	93^[2]
		R: CCGGTGCTTCTTCTGTGATTAT P: FAM-CGGTGATGAGGAAGGCGATTAG-TAMRA
Mb	oppD	F: TCAAGGAACCCCACCAGAT	71^[6]
		R: AGGCAAAGTCATTTCTAGGTGCAA P: FAM-TGGCAAACTTACCTATCGGTGACCCT-TAMRA
Tp	plolysin	F: ATCAACAATCCCACGAAGAG	99^[2]
		R: TTGCAGCATGGTCAGGATAC P: FAM-TCGACGGTTGGATTCAGCGCAATA-TAMRA
Mh	sodA	F: ATTAGTGGGTTGTCCTGGTTAG	144^[2]
		R: GCGTGATTTCGGTTCAGTTG P: FAM-CTGAACCAACACGAGTAGTCGCTGC-TAMRA

病原 Pathogen	目标基因 Target gene	引物、探针 Primer, probe (5'-3')	片段大小 Fragment size/bp
Pm	kmt-1	F: GGGCTTGTCGGTAGTCTTT	148^[2]
		R: CGGCAAATAACAATAAGCTGAGTA P: FAM-CGGCGCAACTGATTGGACGTTATT-TAMRA
Hs	16S-rRNA	F: AAGGCCTTCGGGTTGTAAAG	93^[2]
		R: CCGGTGCTTCTTCTGTGATTAT P: FAM-CGGTGATGAGGAAGGCGATTAG-TAMRA
Mb	oppD	F: TCAAGGAACCCCACCAGAT	71^[6]
		R: AGGCAAAGTCATTTCTAGGTGCAA P: FAM-TGGCAAACTTACCTATCGGTGACCCT-TAMRA
Tp	plolysin	F: ATCAACAATCCCACGAAGAG	99^[2]
		R: TTGCAGCATGGTCAGGATAC P: FAM-TCGACGGTTGGATTCAGCGCAATA-TAMRA
Mh	sodA	F: ATTAGTGGGTTGTCCTGGTTAG	144^[2]
		R: GCGTGATTTCGGTTCAGTTG P: FAM-CTGAACCAACACGAGTAGTCGCTGC-TAMRA

质粒 Plasmid	序列 Sequence
Pm	CGGCAAATAACAATAAGCTGAGTAATAAATAACGTCCAATCAGTTGCGCGGTTGTCAAGGAAGCAGATTGGCTCAAAACAC- CAAACTCTGCCCAACAAAACTGTGCTTTTCTTTGCCACAAGCCAAATAAAAGACTACCGACAAGCCC
Hs	ATCAACAATCCCACGAAGAGTTCCGTGACTCAAGGACTGAACGGCCTTCTCGACGGTTGGATTCAGCGCAATAGCAAGTATCC- TGACCATGCTGCAA
Mb	TCAAGGAACCCCACCAGATATGGCAAACTTACCTATCGGTGACCCTTTTGCACCTAGAAATGACTTTGCCT
Tp	AAGGCCTTCGGGGTTGTAAAGTTCTTTCGGTCTTTCGGTGATGAGGAAGGCGATTAGTTTAAGAGATTAATTGATTGACGATAA- TCACAGAAGAAGCACCGG
Mh	GCGTGATTTCGGTTCAGTTGAAGCTTTCCAATCAGAATTTGAAAAAGCAGCGACTACTCGTGTTGGTTCAGGCTGGGCGTGGTTA- GTATTAGAAGAGGGTAAATTAGCCGTTGTTTCAACCGCTAACCAGGACAACCCACTAAT

质粒 Plasmid	序列 Sequence
Pm	CGGCAAATAACAATAAGCTGAGTAATAAATAACGTCCAATCAGTTGCGCGGTTGTCAAGGAAGCAGATTGGCTCAAAACAC- CAAACTCTGCCCAACAAAACTGTGCTTTTCTTTGCCACAAGCCAAATAAAAGACTACCGACAAGCCC
Hs	ATCAACAATCCCACGAAGAGTTCCGTGACTCAAGGACTGAACGGCCTTCTCGACGGTTGGATTCAGCGCAATAGCAAGTATCC- TGACCATGCTGCAA
Mb	TCAAGGAACCCCACCAGATATGGCAAACTTACCTATCGGTGACCCTTTTGCACCTAGAAATGACTTTGCCT
Tp	AAGGCCTTCGGGGTTGTAAAGTTCTTTCGGTCTTTCGGTGATGAGGAAGGCGATTAGTTTAAGAGATTAATTGATTGACGATAA- TCACAGAAGAAGCACCGG
Mh	GCGTGATTTCGGTTCAGTTGAAGCTTTCCAATCAGAATTTGAAAAAGCAGCGACTACTCGTGTTGGTTCAGGCTGGGCGTGGTTA- GTATTAGAAGAGGGTAAATTAGCCGTTGTTTCAACCGCTAACCAGGACAACCCACTAAT

时间 Time	数量 Quantity	病原Pathogen
时间 Time	数量 Quantity	Pm/%	Hs/%	Mb/%	Tp/%	Mh/%
2019-04	18	44.44	44.44 ab	33.33 A	16.67	22.22 a
2019-11	17	58.82	58.82 a	23.53 A	17.65	5.88 ab
2020-04	40	62.50	27.50 b	0 B	7.50	2.50 b

四川省某奶牛场犊牛呼吸道疾病的细菌性病原qPCR检测

qPCR detection of bacterial pathogens of respiratory diseases in calves of a dairy farm from Sichuan

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