TY - JOUR

T1 - Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

AU - Pansri, P.

AU - Katholm, J.

AU - Krogh, K. M.

AU - Aagaard, A. K.

AU - Schmidt, L. M.B.

AU - Kudirkiene, E.

AU - Larsen, L. E.

AU - Olsen, J. E.

PY - 2020

Y1 - 2020

N2 - Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93–106% and 91–104%, respectively, with assay detection limits of 10–50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135 strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71–0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

AB - Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93–106% and 91–104%, respectively, with assay detection limits of 10–50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135 strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71–0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

KW - Bovine respiratory disease

KW - Calf pneumonia

KW - Multiplex qPCR

U2 - 10.1016/j.tvjl.2020.105425

DO - 10.1016/j.tvjl.2020.105425

M3 - Journal article

C2 - 32113583

AN - SCOPUS:85078253504

VL - 256

JO - The Veterinary Journal

JF - The Veterinary Journal

SN - 1090-0233

M1 - 105425

ER -