Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid

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Abstract

Background
Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.
Results
The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.
Conclusion
It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.
Original languageEnglish
Article number221
JournalBMC Veterinary Research
Volume13
Issue number1
DOIs
Publication statusPublished - 2017

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Cattle Diseases
bovine respiratory disease
Multiplex Polymerase Chain Reaction
Bronchoalveolar Lavage Fluid
Mannheimia haemolytica
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
Arcanobacterium pyogenes
Polymerase Chain Reaction
assays
Pasteurellaceae
analytical specificity
calves
diagnostic specificity
diagnostic sensitivity
Bacterial DNA
DNA
gold
detection limit
herds

Cite this

@article{e0b0c0486d814d599ed8f98188a7309f,
title = "Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid",
abstract = "BackgroundPasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.ResultsThe analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3{\%} for M. haemolytica and 100{\%} for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.ConclusionIt was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.",
author = "H.J. Wisselink and J.B.W.J. Cornelissen and {van der Wal}, F.J. and Bart Kooi and M.G.J. Koene and A. Bossers and B. Smid and {de Bree}, F.M. and A.F.G. Antonis",
year = "2017",
doi = "10.1186/s12917-017-1141-1",
language = "English",
volume = "13",
journal = "BMC Veterinary Research",
issn = "1746-6148",
publisher = "Springer Verlag",
number = "1",

}

TY - JOUR

T1 - Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid

AU - Wisselink, H.J.

AU - Cornelissen, J.B.W.J.

AU - van der Wal, F.J.

AU - Kooi, Bart

AU - Koene, M.G.J.

AU - Bossers, A.

AU - Smid, B.

AU - de Bree, F.M.

AU - Antonis, A.F.G.

PY - 2017

Y1 - 2017

N2 - BackgroundPasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.ResultsThe analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.ConclusionIt was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

AB - BackgroundPasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.ResultsThe analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.ConclusionIt was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

U2 - 10.1186/s12917-017-1141-1

DO - 10.1186/s12917-017-1141-1

M3 - Article

VL - 13

JO - BMC Veterinary Research

JF - BMC Veterinary Research

SN - 1746-6148

IS - 1

M1 - 221

ER -