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

doi:10.3969/j.issn.1004-1524.2022.04.06

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (4): 706-712.DOI: 10.3969/j.issn.1004-1524.2022.04.06

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S823

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.

Figures/Tables 4

References 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

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

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