Establishment and application of multiplex PCR assay for bovine pasteurellosis
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    Abstract:

    [Background] Bovine pasteurellosis, caused by serotypes A, B, and E of Pasteurella multocida (Pm), is an acute infectious disease of cattle. Polymerase chain reaction (PCR) is an effective means to diagnose and control this disease. [Objective] To establish a multiplex PCR assay for rapid detection of serotypes A, B, and E of Pm and provide technical support for the rapid and accurate clinical diagnosis of bovine pasteurellosis. [Methods] According to the conserved regions of hyaD-hyaC, bcbD, and ecbJ genes of Pm, we designed three pairs of specific primers for PCR and determined the appropriate annealing temperature (Tm) by temperature-gradient PCR assay. Chessboard test was employed to optimize the primer concentration and then a multiplex PCR assay was established. The recombinant plasmid standard and positive strain were used to determine the sensitivity (limit of detection) of the established assay. The nucleic acid samples of 8 common bovine pathogens (Mannheimia hemolytica C1655, Escherichia coli C237, Listeria monocytogenes C1597, Staphylococcus aureus C3053, Salmonella dublin C79351, Mycobacterium paratuberculosis C1625, bovine infectious rhinotracheitis virus CAV1546, and Mycoplasma bovis C65-1) were used to determine the specificity of the multiplex PCR assay. Three batches of diagnostic reagents were prepared to perform inter-batch and intra-batch tests on sensitive and specific samples to determine the repeatability of the assay. Three different models of PCR instruments were used to detect sensitive and specific samples with the established method to determine the applicability of the assay. The performance of the assay in clinical application was evaluated by detection of clinical samples and simulated infection samples. [Results] The optimal multiplex PCR assay was established under the following conditions: Tm of 55 ℃ and the three pairs of primers at the concentrations of 0.25, 0.30, and 0.20 μmol/L, respectively. The established method could simultaneously detect Pm serotypes A (821 bp), B (203 bp), and E (363 bp). The multiplex PCR assay had high sensitivity. It showed the limits of detection of 43.080, 3.710, and 4.350 copies/μL for recombinant plasmid standards pMD-A, pMD-B, and pMD-E, respectively, as well as the limit of detection of 102 CFU for the positive bacterial liquid. Moreover, the established assay had strong specificity as it only produced bands for the serotypes A, B, and E of Pm and no bands for other pathogens. The consistent results of the inter-batch and intra-batch tests indicated good repeatability of the assay. The detection results of clinical samples and simulated infection samples showed a 100% coincidence rate with the pathogen isolation and identification. [Conclusion] The multiplex PCR assay for the detection of serotypes A, B, and E of Pm was successfully established, which provided technical support for the identification and epidemiological investigation of Pm.

    Reference
    [1] 林立山, 黄秦, 才灵杰, 马家乐, 姚火春, 潘子豪. 牛源多杀性巴氏杆菌的分离与初步鉴定[J]. 微生物学报, 2018, 58(12):2240-2248. Lin LS, Huang Q, Cai LJ, Ma JL, Yao HC, Pan ZH. Isolation and diversity analysis of bovine Pasteurella multocida[J]. Acta Microbiologica Sinica, 2018, 58(12):2240-2248(in Chinese)
    [2] 《一、二、三类动物疫病病种名录》及《三类动物疫病防治规范》发布[J]. 中国水产, 2022(8):23-26. "List of Class I, Class II and Class III Animal Diseases" and "Specification for Prevention and Control of Class III Animal Diseases" were released[J]. China Fisheries, 2022(8):23-26(in Chinese)
    [3] Carter GR. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types[J]. American Journal of Veterinary Research, 1955, 16(60):481-484
    [4] Snyder E, Credille B. Mannheimia haemolytica and Pasteurella multocida in bovine respiratory disease[J]. Veterinary Clinics of North America:Food Animal Practice, 2020, 36(2):253-268
    [5] Zhang WL, Liu XD, Liu MC, Ma B, Xu L, Wang JW. Development of a multiplex PCR for simultaneous detection of Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes[J]. Acta Veterinaria Hungarica, 2017, 65(3):327-339
    [6] Cuevas I, Carbonero A, Cano D, García-Bocanegra I, Amaro MÁ, Borge C. Antimicrobial resistance of Pasteurella multocida type B isolates associated with acute septicemia in pigs and cattle in Spain[J]. BMC Veterinary Research, 2020, 16(1):222
    [7] Elsayed MSAE, Eldsouky SM, Roshdy T, Said L, Thabet N, Allam T, Mohammed ABA, Nasr GM, Basiouny MSM, Akl BA, et al. Virulence determinants and antimicrobial profiles of Pasteurella multocida isolated from cattle and humans in Egypt[J]. Antibiotics:Basel, Switzerland, 2021, 10(5):480
    [8] Cirone F, Padalino B, Tullio D, Capozza P, Lo Surdo M, Lanave G, Pratelli A. Prevalence of pathogens related to bovine respiratory disease before and after transportation in beef steers:preliminary results[J]. Animals:an Open Access Journal from MDPI, 2019, 9(12):1093
    [9] Peng Z, Wang XR, Zhou R, Chen HC, Wilson BA, Wu B. Pasteurella multocida:genotypes and genomics[J]. Microbiology and Molecular Biology Reviews, 2019, 83(4):e00014-e00019
    [10] 李霄阳, 许建, 刘文晓, 章振华, 徐福洲, 陈小玲, 李永清. 牛源多杀性巴氏杆菌主要荚膜群和脂多糖基因型两组三重PCR检测方法[J]. 中国动物检疫, 2022, 39(1):97-104
    Li XY, Xu J, Liu WX, Zhang ZH, Xu FZ, Chen XL, Li YQ. Two sets of triplicate PCR assays detecting major capsular serogroups and lipopolysaccharide genotypes of bovine Pasteurella multocida[J]. China Animal Health Inspection, 2022, 39(1):97-104(in Chinese)
    [11] Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B. Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system[J]. Journal of Clinical Microbiology, 2001, 39(3):924-929
    [12] Frucchi APS, Dall Agnol AM, Bronkhorst DE, Beuttemmuller EA, Alfieri AA, Alfieri AF. Bovine coronavirus co-infection and molecular characterization in dairy calves with or without clinical respiratory disease[J]. Frontiers in Veterinary Science, 2022, 9:895492
    [13] McFadden AMJ, Christensen H, Fairley RA, Hill FI, Gill JM, Keeling SE, Spence RP. Outbreaks of pleuritis and peritonitis in calves associated with Pasteurella multocida capsular type B strain[J]. New Zealand Veterinary Journal, 2011, 59(1):40-45
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WANG Haojie, XIN Lingxiang, REN Xiaoxia, LIU Yan, YAO Wensheng, HOU Xuexia, LI Xuni, ZHU Liangquan. Establishment and application of multiplex PCR assay for bovine pasteurellosis[J]. Microbiology China, 2022, 49(12): 5083-5091

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History
  • Received:September 10,2022
  • Revised:September 30,2022
  • Online: December 06,2022
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