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| Establishment and Preliminary Application of Multiplex PCR for Mycoplasma bovis, Klebsiella pneumoniae and Bovine infectious bovine rhinotracheitis virus |
| XING Xiao-Yong1, CHENG Jia-Hai1, BAO Shi-Jun1, HAO Bao-Cheng2, WEN Feng-Qin1, WU Xiao-Chun1, HU Yong-Hao1,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China |
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Abstract Bovine respiratory disease syndrome (BRDC) is a serious disease of cattle caused by a mixture or secondary infection of bacteria, viruses and environmental factors, which seriously endangers the healthy development of cattle industry. Based on the glycoprotein B (gB) gene of Infectious bovine rhinotracheitis virus (IBRV) in GenBank (GenBank No. NC0847.1), lipoprotein 48 (p48) gene of Mycoplasma bovis in GenBank (GenBank No. DQ020482.1) and Khe gene of Klebsiella pneumoniae in GenBank (GenBank No. Kx842080.1), three pairs of primers were designed to amplify the expected fragments of 727, 421 and 192 bp for Infectious bovine rhinotracheitis virus, Mycoplasma bovis and Klebsiella pneumoniae, respectively. The optimization of annealing temperature and primer concentration in PCR amplification, the specific test and clinical sample detection were carried out, and the multiplex PCR and sample evaluation were established. The results showed that the optimum annealing temperature was 52.7 ℃ in the range of 48~54 ℃, and the optimum primer concentration of IBRV, M. bovis , K. pneumoniae was 1, 1, and 15 mol/L in the range of 1~15 mol/L, and the lowest detection rate of IBRV was 103 TCID50, the lowest detectable amount of K. pneumoniae was 8.1×101 cfu and the lowest detection level of M. bovis was 6.5×101 cfu, and no cross reaction was found with Pasteurella multocida, Salmonella typhimurium, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa and Bovine diarrhea virus. The results of clinical samples were consistent with the isolation and identification of pathogens. The establishment of multiplex PCR method provides strong technical support for the diagnosis and control of bovine respiratory disease syndrome caused by these three pathogens.
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Received: 07 August 2020
Published: 01 May 2021
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Corresponding Authors:
*yhh0817@126.com
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[1] 冯娜, 高翔, 肖敏, 等. 2016. 牛源肺炎克雷伯氏菌的分离鉴定及遗传进化分析[J]. 中国兽医科学, 46(11): 1358-1364.
(Feng N, Gao X, Xiao M, et al.2016. Isolation,identification and genetic evolution analysis of Klebsiella pneumoniae strain calves[J]. Chinese Veterinary Science, 46(4): 1358-1364.)
[2] 蒙正群, 冷依伊, 任梅渗, 等. 2017. 一株牛源肺炎克雷伯氏菌的分离鉴定与耐药基因型检测[J]. 浙江农业学报, 29(4): 534-541.
(Meng Z Q, Leng Y Y, Reng M S, et al.2017. Isolation,identification and drug resistance detection of a calf Klebsiella pneumoniae strain[J]. Acta Agriculturae Zhejiangensis, 29(4): 534-541.)
[3] 秦鑫, 王亚东, 李红梅, 等. 2020. 基于mtCOI基因的三种短体线虫多重PCR检测[J]. 农业生物技术学报, 28(3): 562-570.
(Qin X, Wang Y D, Li H M, et al.2020. Multiplex-PCR detection of three pratylenchus species based on mtCOI gene[J]. Journal of Agricultural Biotechnology, 28(3): 562-570.)
[4] 辛九庆, 李媛, 郭丹, 等. 2008. 国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J]. 中国预防兽医学报, 30(09): 661-664.
(Xing J Q, Li Y, Guo D, et al.2008. First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J]. Chinese Journal of Preventive Veterinary Medicine, 30(09): 661-664.)
[5] 熊文婕, 谢芝勋, 范晴, 等. 2018. IBRV与MB二温式多重PCR检测方法的建立[J]. 中国奶牛, 12: 1-3.
( Xiong W J, Xie Z X, Fan Q, et al.2018. Study on development of two-temperature multiplex PCR for IBRV and MB[J]. China Dairy Cattle 12: 1-3.)
[6] 张颖慧, 王牧川, 张斌, 等. 2018. 检测牛传染性鼻气管炎病毒和牛支原体的双重PCR方法的建立及应用[J]. 中国兽医科学, 48(07): 824-829.
(Zhang H Y, Wang M C, Zhang B, et al.2018. Establishment and application of a dual PCR for the simultaneous detection of Infectious bovine rhinotracheitis virus and Mycoplasma bovis[J]. Chinese Veterinary Science, 48(07): 824-829.)
[7] 张玉龙, 马志宇, 崔耀成, 等. 2020. 肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立[J]. 浙江农业学报, 32(2): 210-217.
(Zhang Y L, Ma Z Y, Cui Y C, et al.2017. Isolation, identification and drug resistance detection of a calf Klebsiella pneumoniae strain[J]. Acta Agriculturae Zhejiangensis, 32(2): 210-217.)
[8] Agnon O, Thomsen J, Larsen L E, et al.2009. Respiratory disease in calves: Microbiological investigations on transtracheally aspirated bronchoalveolar fluid and acute phase protein response[J]. Veterinary Microbiology, 137(1-2): 165-171.
[9] Albayrak H, Yazici Z, Ozan E, et al.2019. Characterisation of the first bovine parainfluenza virus 3 isolate detected in cattle in Turkey[J]. Veterinary Sciences, 6(2): 56.
[10] Bell C J, Blackburn P, Elliott M, et al.2014. Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease[J]. Journal of Veterinary Diagnostic Investigation, 26(5): 631-634.
[11] Cassidy L. Klima, Rahat Zaheer, Robert E. Briggs, et al.2017. A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2 and 6[J]. Journal of Microbiological Methods, 139: 155-160.
[12] Confer A W.2009. Update on bacterial pathogenesis in BRD[J]. Animal Health Research Reviews, 10(2): 45-148.
[13] Fulton R W, Purdy C W, Confer A W, et al.2000. Bovine viral diarrhea viral infections in feeder calves with respiratory disease: Interactions with Pasteurella spp, Parainfluenza-3 virus,and Respiratory syncytial virus[J]. Canadian Journal of Veterinary Re-search, 64(3): 151-159.
[14] Fulton R W.2009. Bovine respiratory disease research (1983~2009)[J]. Animal Health Research Reviews, 10(2): 131-139.
[15] Griffin D, Chenqappa M M, Kuszak J, et al.2010. Bacterial pathogens of the bovine respiratory disease complex[J]. The Veterinary Clinics of North America. Food Animal Practice, 26(2): 381-394.
[16] Guzman E, Taylor G.2015. Immunology of Bovine respiratory syncytial virus in calves[J]. Molecular Immunology, 66(1): 48-56.
[17] Hay K E, Barnes T S, Morton J M, et al.2014. Risk factors for bovine respiratory disease in Australian feedlot cattle: Use of a causal diagram-informed approach to estimate effects of animal mixing and movements before feedlot entry[J]. Preventive Veterinary Medicine, 117(1): 160-169.
[18] Headley S A, Alfieri A F, Oliveira V H, et al.2014. Histophilus somni is a potential threat to beef cattle feedlots in Brazil[J]. Veterinary Record, 175(10): 249.
[19] Headley S A, OkanoW, Balbo L C, et al.2018. Molecular survey of infectious agents associated with bovine respiratory disease in a beef cattle feedlot in southern Brazil[J]. Journal of Veterinary Diagnostic Investigation, 30(2): 249-251.
[20] Johnson K F, Chancellor N, Burn C C, et al.2017. Prospective cohort study to assess rates of contagious disease in pre-weaned UK dairy heifers: Management practices, passive transfer of immunity and associated calf health[J]. Veterinary Record Open, 4: e000226.
[21] Kugadas A, Poindexter J, Lee M L, et al.2014. Growth of Mannheimia haemolytica: Inhibitory agents and putative mechanism of inhibition[J]. Veterinary Microbiology, 174(1-2): 155-162.
[22] Kusiluka L J, Kokotovic B, Ojeniyi B, et al.2000. Genetic variations among Mycoplasma bovis strains isolated from Danish cattle[J]. FEMS Microbiology Letters, 192(1): 113-118.
[23] Panciera R J, Confer A W.2010. Pathogenesis and pathology of bovine pneumonia[J]. Veterinary Clinics of North America: Food Animal Practice, 26(2): 191-214.
[24] Shirvani E, Lotfi M,Kamalzadeh, et al.2012. Seroepidemiological study of bovine respiratory viruses (BRSV,BoHV-1,PI-3V,BVDV,and BAV-3) in dairy cattle incentral region of Iran (Esfahan province)[J]. Tropical Animal Health and Production, 44(1): 191-195.
[25] Shanthalingam S, Goldy A, Bavananthasivam J, et al.2014. PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010[J]. Journal of Wildlife Diseases, 50(1): 1-10.
[26] Sudaryatma P E, Nakamura K, Mekata H, et al.2018. Bovine respiratory syncytial virus infection enhances Pasteurella multocida adherence on respiratory epithelial cells[J]. Veterinary Microbiology, 220: 33-38.
[27] Tegtmeier C, Angen O, Ahrens P, 2000. Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle[J]. Veterinary Microbiology, 76(4): 385-394.
[28] Timsit E, Christensen H, Bareille N, et al.2013. Transmission dynamics of Mannheimia haemolytica in newly-received beef bulls at fattening operations[J]. Veterinary Microbiology, 161(3-4): 295-304. |
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