info@biomedres.us   +1 (502) 904-2126   One Westbrook Corporate Center, Suite 300, Westchester, IL 60154, USA   Site Map
ISSN: 2574 -1241

Impact Factor : 0.548

  Submit Manuscript

Mini ReviewOpen Access

PCR Methods for Detecting Bovine Respiratory Pathogens Volume 11 - Issue 4

Yuhui Dong, Lijia Luo and Xiangmei Zhou*

  • National Animal Transmissible Spongiform Encephalopathy Laboratory, China

Received: October 30, 2018;   Published: December 05, 2018

*Corresponding author: Xiangmei Zhou, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, Beijing, China

DOI: 10.26717/BJSTR.2018.11.002145

Abstract PDF

Also view in:

Abstract

Bovine Respiratory Disease (BRD) is an important disease in cattle production, BRD may be associated with one or more pathogens, of which Mycobacterium bovis, Mycoplasma bovis, and Klebsiella pneumoniae are three important pathogens. Fast and accurate detection methods are important for preventing and controlling BRD. This review focuses on the PCR detection methods for the above three pathogens in recent years.

Introduction

Bovine Respiratory Disease (BRD) is an important disease in cattle production, causing serious economic losses world widely [1]. The occurrence of BRD is a combination of multiple factors and may be associated with one or more pathogens [2]. Among them, Mycobacterium bovis, Mycoplasma bovis, and Klebsiella pneumoniae are three important pathogens. Mycobacterium bovis can infect many kinds of animals. Besides cattle, there are 50 kinds of vertebrates such as humans. Sick animals showed a gradual loss of body weight, anemia and cough. Cattle with active tuberculosis are the main source of infection. Their respiratory tract carrys bacteria, which are excreted from coughing and sneezing [3]. Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. It was found that 5.5% of the nasal swabs from cattle with respiratory symptoms were positive for Mycoplasma bovis [4]. Klebsiella pneumoniae, an important conditional pathogen, mainly exists in the intestine, respiratory tract and urogenital tract [5]. The incidence of respiratory and urinary tract is the highest. Aslan et al. isolated bacteria from bovine upper respiratory tract infections and found that Klebsiella pneumoniae accounted for 20% [6].

PCR Detection

Polymerase Chain Reaction

PCR technology is a molecular biotechnology in which DNA of pathogenic microorganisms is expanded to conventional detectable levels in vitro. Quan Z et al. designed a multiplex PCR with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex to differentiate Mycobacterium bovis from Mycobacterium tuberculosis and NTM species [7]. Gioia et al. developed and validated a multitarget PCR assay that can discriminate between Acholeplasma and Mycoplasma and identify Mycoplasma bovis [8]. Turton et al. identified and typed Klebsiella pneumoniae by PCR using capsular type-specific, variable number tandem repeat and virulence gene targets [9]. Fonseca et al. established a one-step multiplex PCR to identify klebsiella pneumoniae, klebsiella variicola and klebsiella quasipneumoniae in the clinical routine [10].

Quantitative Real-Time PCR

Quantitative Real-time PCR technology can achieve quantitative analysis, and it is more specific and sensitive than conventional PCR. Choi Y et al. developed a real-time PCR targeting 16S ribosomal RNA for the detection of Mycobacterium tuberculosis complex [11]. Sales et al. developed and validated two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 (RD 4) for the characterization of Mycobacterium bovis isolates. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng. The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg [12]. Cezar et al. developed a qPCR targeting the region of RD4, which showed that 0.25% milk and 2% blood samples were positive for Mycobacterium bovis [13]. Fu-Xiang et al. developed a TaqMan real-time PCR for detection of Klebsiella pneumoniae, which could be applied for early diagnosis of Klebsiella pneumoniae infection [14]. We developed a TaqMan-based multiplex real-time PCR assay primer and TaqMan probes were designed based on the specific 229 bp sequence of Mycobacterium bovis, the uvrC gene of Mycoplasma bovis and the khe gene of Klebsiella pneumoniae. The assay sensitivity was 10 copies/μL. 37 bovine nasal swabs collected from cattle were identified, of which 21.62% (8/37) was Mycoplasma bovis-positive, 18.92% (7/37) was Klebsiella pneumoniae-positive, none (0/37) was Mycobacterium bovispositive. However, Mycobacterium bovis was detected in nasal swabs of cattle with symptoms of respiratory disease.

Summary

PCR detection technology is a sensitive, specific and fast method for the detection of BRD. We believe that the establishment of a TaqMan-based multiplex real-time PCR for the simultaneous detection of Mycobacterium bovis, Mycoplasma bovis, and Klebsiella pneumoniae can contribute to the early diagnosis and control of BRD

References

  1. Tortorelli G, Carrillo GN, Mendonça Ribeiro BL, Miranda Marques L, Timenetsky J, et al. (2017) Evaluation of mollicutes microorganisms in respiratory disease of cattle and their relationship to clinical signs. J Vet Intern Med 31(4): 1215-1220.
  2. Neibergs HL, Seabury CM, Wojtowicz AJ, Wang Z, Scraggs E, et al. (2014) Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves. BMC Genomics 15(1): 1164.
  3. Chapman J (1999) Bovine tuberculosis. Vet Rec 144(21): 596.
  4. Ewelina Szacawa, Monika SC, Krzysztof Niemczuk, Katarzyna Dudek, Grzegorz Woźniakowski, et al. (2016) Prevalence of pathogens from mollicutes class in cattle affected by respiratory diseases and molecular characteristics of Mycoplasma bovis field strains. Journal of Vet Rec 60(4): 391-397.
  5. Rees CA, Nordick KV, Franchina FA, Lewis AE, Hirsch EB, et al. (2017) Volatile metabolic diversity of klebsiella pneumoniae in nutrient-replete conditions. Metabolomics 13(2): 1-11.
  6. Aslan V, Maden M, Erganis O, Birdane FM, Corlu M (2002) Clinical efficacy of florfenicol in the treatment of calf respiratory tract infections. Vet Q 24(1): 35-39.
  7. Quan, Z, Haiming T, Xiaoyao C, Weifeng Y, Hong J, et al. (2016) Development of one-tube multiplex Polymerase Chain Reaction (PCR) for detecting Mycobacterium bovis. J Vet Med Sci 78(12): 1873-1876.
  8. Gioia G, Werner B, Nydam DV, Moroni P (2016) Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among new york state dairy farms. J Dairy Sci 99(6): 4668-4677.
  9. Turton JF, Perry C, Elgohari S, Hampton CV (2010) PCR characterization and typing of klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. J Med Microbiol 59(Pt 5): 541-547.
  10. Fonseca EL, Ramos ND, Andrade BG, Morais LL, Marin MF, et al. (2017) A one-step multiplex PCR to identify klebsiella pneumoniae, klebsiella variicola and klebsiella quasipneumoniae in the clinical routine. Diagn Microbiol Infect Dis 87(4): 315-317.
  11. Choi Y, Hong SR, Jeon BY, Wang HY, Lee GS, et al. (2015) Conventional and real-time PCR targeting 16s ribosomal RNA for the detection of mycobacterium tuberculosis complex. Int J Tuberc Lung Dis 19(9), 1102-1108.
  12. Sales ML, Fonseca AA, Orzil L, Alencar AP, Hodon MA, et al. (2014) Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates. Genet Mol Res 13(2): 4607-4616.
  13. Cezar RD, Lucena-Silva N, Filho AF, Borges Jde M, de Oliveira PR, et al. (2016) Molecular detection of Mycobacterium bovis in cattle herds of the state of pernambuco, brazil. BMC Vet Res 12(1): 31.
  14. Fu-Xiang L, De-fang L, Jun Y, He-li X, Hua-chun L (2014) Development of taqman real-time PCR for detection of klebsiella pneumoniae. Chinese Vet Sci pp. 1231-1235.