Wisselink et al. BMC Veterinary Research (2017) 13:221 DOI 10.1186/s12917-017-1141-1 RESEARCHARTICLE Open Access Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid Henk J. Wisselink* , Jan B.W.J. Cornelissen, Fimme J. van der Wal, Engbert A. Kooi, Miriam G. Koene, Alex Bossers, Bregtje Smid, Freddy M. de Bree and Adriaan F.G. Antonis 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. Keywords: Bovine respiratory disease, PCR, Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Trueperella pyogenes * Correspondence: [email protected] Wageningen Bioveterinary Research, P.O. Box 65, 8200, AB, Lelystad, The Netherlands © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wisselink et al. BMC Veterinary Research (2017) 13:221 Page 2 of 10 Background analysis of BRD associated organisms. To offer veteri- In veal production, bovine respiratory disease (BRD) is narians a fast and complete laboratory result, a set of the most common and economically important disease three multiplex real-time PCRs was developed for de- [1–3]. BRD is a multifactorial disease, involving multiple tection of i) viral, ii) bacterial and iii) mycoplasma potentially pathogenic microorganisms that causes eco- BRD-associated pathogens in bronchoalveolar lavage nomic losses due to morbidity, mortality, medication fluid (BALF) of calves, under the name RespoCheck costs, increased time on feeding and associated labour (WBVR, Lelystad, The Netherlands). In this study the costs [4]. In the Dutch veal industry, calves at an age RespoCheck bacterial multiplex real-time PCR (abbre- between ten days and five weeks are sold by dairy viated to RespoCheck PCR) was evaluated with com- farmers to traders who transport calves to the calf col- monly used culture methods as reference. This PCR lection centres, from where they are transported to fat- has been developed for the detection of four bacterial tening farms where they are mingled with other young pathogens commonly associated with BRD [9, 13], i.e. calves, reared and fattened until slaughter at an age of Pasteurella multocida, Mannheimia haemolytica, His- 6–7 months. The young age of the calves, their inad- tophilus somni and Trueperella pyogenes.Theresults equate immunity and several stress factors including showed that the RespoCheck PCR assay is a sensitive transportation and mingling with other young calves and valuable diagnostic tool suitable for the simultan- makes that the calves are vulnerable for infections, a eous detection of four BRD associated bacterial targets scenario which is recognised as resulting in the frequent in BALF of calves. occurrence of disease problems including BRD [5]. To protect calves, at the end of the twentieth century veal Methods farmers commonly administered antibiotics prophylac- Bacterial strains and growth conditions tically immediately after arrival on the farm. Growing A total of 118 bacterial isolates, representing 46 different concerns about the increase in antibiotic resistance species, were used for evaluation of specificity of the including methicillin resistant Staphylococcus aureus RespoCheck PCR. At least five representatives of each of (MRSA) and extended-spectrum βeta-lactamase produ- the four target species, as well as bacterial strains from cing Escherichia coli in livestock resulted in a policy non-target species, consisting of isolates of phylogenetic- aimed to substantially reduce the use of antibiotics in ally closely related non-target species, and isolates of The Netherlands [6]. Part of this policy was a ban of all species from cattle with diseases other than BRD were prophylactic use of antimicrobials in the Netherlands in included. Reference strains were obtained from the 2012 (Besluit Diergeneesmiddelen, 2 November 2012; American Type Culture Collection (ATCC) and the http://wetten.overheid.nl/BWBR0032386/2015-01-01). Culture Collection of the University of Gothenburg To implement and regulate this reduction in the use of (Sweden) (CCUB) and field strains were isolated from antibiotics in the Netherlands, in recent years a collab- lungs of calves in a Dutch field study on BRD. An over- oration was set up between stakeholders in livestock view of the strains is provided in Table 1. Bacteria other production and the national government [6, 7]. This than H. somni were grown overnight at 37 °C on heart lead to registration of all antibiotics administered to infusion agar (ACU 7269C, Acumedia Manufacturers livestock at farm level [8] and to mandatory treatment Inc. Lansing, MI) supplemented with 5% defibrinated and health plans to be developed for each farm to sheep blood. H. somni was grown overnight at 37 °C enforce that antimicrobial treatment is solely based on in air with 5% CO2 on chocolate blood agar, using 7% proper diagnosis [6]. However, the availability of rapid defibrinated horse blood and Columbia blood agar and reliable diagnostic tools is limited. (CM 331, Oxoid, Badhoevedorp, The Netherlands). In BRD, several viral and bacterial pathogens may be involved [9–11]. For detection of these potential patho- Field samples gens a variety of diagnostic tests have been described, In a Dutch field study on BRD from October 2013 till including culture and molecular methods, see review by March 2014, veal calf farms (n = 10) were selected. On Fulton and Confer [12]. These methods all have their each farm, the study period started shortly after arrival benefits and limitations in terms of speed of analysis, of the calves (D0) and ended 84 days (D84) later. At the sensitivity and specificity [12]. In case of BRD, interpret- start (D0) and end (D84) of the study period and during ation of results is sometimes challenging and diagnostic outbreaks of BRD, bronchoalveolar lavage (BAL) samples laboratories usually offer diagnostic tests for a limited were taken from calves without and with clinical problems number of potential pathogens which can be involved in of BRD (as defined by severe upper respiratory tract and/ BRD and which are sometimes also time-consuming. or lower respiratory tract disease). These are drawbacks that can hamper veterinarians and BAL samples were collected as previously described farmers for sending samples to diagnostic laboratory for [14]. Between 35 and 75 mL BAL was obtained from each Wisselink et al. BMC Veterinary Research (2017) 13:221 Page 3 of 10 Table 1 List of 118 bacterial isolates and results of the RespoCheck bacterial multiplex real-time PCR Identificationa CCUG identificationb Source RespoCheck bacterial multiplex real-time PCR(Ct values) (number of isolates tested) P. multocida M. haemolytica H. somni T. pyogenes Acidovorax spp. (n =3) NAc WBVR collectiond - g --- Actinomyces spp NA WBVR collectiond -- -- Aerococcus viridans NA WBVR collectione -- -- Biberstenia trehalosi NA WBVR collectiond -- -- Brucella abortus NA WBVR collectione -- -- Comamonas kerstersii NA WBVR collectiond -- -- Corynebacterium bovis (n =2) NA WBVR collectione -- -- Corynebacterium pseudotuberculosis NA WBVR collectione -- -- Escherichia coli NA WBVR collectione -- -- Gallibacterium anatis (n = 5) NA WBVR collectiond -- -- Hafnia alvei NA WBVR collectiond -- -- Histophilus somni NA ATCC 22132f - - 15.2 - Histophilus somni (n = 4) NA WBVR collectiond - - 12.3–14.1 - Klebsiella oxytoca NA WBVR