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Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture

Published online by Cambridge University Press:  23 February 2015

Heidi Hiitiö ,
Rauna Riva ,
Tiina Autio ,
Tarja Pohjanvirta ,
Jani Holopainen ,
Satu Pyörälä  and
Sinikka Pelkonen
Heidi Hiitiö*
Affiliation:
Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Finland
Rauna Riva
Affiliation:
Veterinary Bacteriology Research Unit, Finnish Food Safety Authority Evira, Neulaniementie 4, 70210 Kuopio, Finland
Tiina Autio
Affiliation:
Veterinary Bacteriology Research Unit, Finnish Food Safety Authority Evira, Neulaniementie 4, 70210 Kuopio, Finland
Tarja Pohjanvirta
Affiliation:
Veterinary Bacteriology Research Unit, Finnish Food Safety Authority Evira, Neulaniementie 4, 70210 Kuopio, Finland
Jani Holopainen
Affiliation:
Thermo Fisher Scientific, Ratastie 2, 01620 Vantaa, Finland
Satu Pyörälä
Affiliation:
Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Finland
Sinikka Pelkonen
Affiliation:
Veterinary Bacteriology Research Unit, Finnish Food Safety Authority Evira, Neulaniementie 4, 70210 Kuopio, Finland
*
*For correspondence; e-mail: [email protected]
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Abstract

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85·7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83·0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97·0 and 95·8% compared with BC. For Staphylococcus spp., the corresponding figures were 86·7 and 75·4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37·0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Keywords

Bovine mastitisdiagnosticsreal-time PCRbacteriological culture
Type
Research Article
Information
Journal of Dairy Research , Volume 82 , Issue 2 , May 2015 , pp. 200 - 208
Copyright
Copyright © Proprietors of Journal of Dairy Research 2015 

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References

Andersen, S, Dohoo, IR, Olde Riekerink, R & Stryhn, H 2010 Diagnosing intramammary infections: evaluating expert opinions on the definition of intramammary infection using conjoint analysis. Journal of Dairy Science 93 2966–2975Google Scholar
Bexiga, R, Koskinen, MT, Holopainen, J, Carneiro, C, Pereira, H, Ellis, KA & Vilela, CL 2011 Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing. Journal of Dairy Research 78 49–55Google Scholar
Botaro, BG, Cortinhas, CS, Março, LV, Moreno, JFG, Silva, LFP, Benites, NR & Santos, MV 2013 Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction. Journal of Dairy Science 96 6955–6964Google Scholar
Bradley, AJ, Leach, KA, Breen, JE, Green, LE & Green, MJ 2007 Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. Veterinary Record 160 253–258CrossRefGoogle ScholarPubMed
Cameron, M, McKenna, SL, MacDonald, KA, Dohoo, IR, Roy, JP & Keefe, GP 2014 Evaluation of selective dry cow treatment following on-farm culture: risk of postcalving intramammary infection and clinical mastitis in the subsequent lactation. Journal of Dairy Science 97 270–284Google Scholar
Cederlöf, SE, Toft, N, Aalbaek, B & Klaas, IC 2012 Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off. Acta veterinaria Scandinavica 54 65–71Google Scholar
Dohoo, IR, Smith, J, Andersen, S, Kelton, DF & Godden, S 2011 Diagnosing intramammary infections: evaluation of definitions based on a single milk sample. Journal of Dairy Science 94 250–261Google Scholar
Gillespie, BE & Oliver, SP 2005 Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction. Journal of Dairy Science 88 3510–3518CrossRefGoogle ScholarPubMed
Halasa, T, Huijps, K, Østerås, O & Hogeveen, H 2007 Economic effects of bovine mastitis and mastitis management: a review. Veterinary Quarterly 29 18–31CrossRefGoogle ScholarPubMed
Hein, I, Lehner, A, Rieck, P, Klein, K, Brandl, E & Wagner, M 2001 Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. Applied and Environmental Microbiology 67 3122–3126Google Scholar
Hogan, J, González, R, Harmon, R, Nickerson, S, Oliver, S, Pankey, J & Smith, K 1999 Laboratory Handbook on Bovine Mastitis. Madison, WI: National Mastitis CouncilGoogle Scholar
Honkanen-Buzalski, T & Seuna, E 1995 Isolation and identification of pathogens from milk. In Sandholm, M, Honkanen-Buzalski, T, Kaartinen, L & Pyörälä, S, (Eds.), The Bovine Udder and Mastitis. University of Helsinki, Faculty of Veterinary Medicine, pp. 121–141. Place of publication Gummerus, Jyväsklä.Google Scholar
Hovinen, MH, Simojoki, H, Pösö, R, Suolaniemi, J & Pyörälä, S 2014 N-acetyl-beta-D-glucosaminidase activity in normal bovine milk. In NMC 53rd Annual Meeting ProceedingsGoogle Scholar
Keane, OM, Budd, KE, Flynn, J & McCoy, F 2013 Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture. Veterinary Record 173 268–273Google Scholar
Koivula, M, Pitkälä, A, Pyörälä, S & Mäntysaari, E 2007 Distribution of bacteria and seasonal and regional effects in a new database for mastitis pathogens in Finland. Acta Agriculturae Scandinavica A 57 89–96Google Scholar
Koskinen, MT, Wellenberg, GJ, Sampimon, OC, Holopainen, J, Rothkamp, A, Salmikivi, L, van Haeringen, WA, Lam, TJGM & Pyörälä, S 2010 Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria. Journal of Dairy Science 93 5707–5715CrossRefGoogle ScholarPubMed
Lago, A, Godden, SM, Bey, R, Ruegg, PL & Leslie, K 2011 The selective treatment of clinical mastitis based on on-farm culture results: I. Effects on antibiotic use, milk withholding time, and short-term clinical and bacteriological outcomes. Journal of Dairy Science 94 4441–4456Google Scholar
Makovec, JA & Ruegg, PL 2003 Results of milk samples submitted for microbiological examination in Wisconsin from 1994 to 2001. Journal of Dairy Science 86 3466–3472Google Scholar
Mattila, T & Sandholm, M 1985 Antitrypsin and N-acetyl-β-d-glucosaminidase as markers of mastitis in herd of Ayrshire cows. American Journal of Veterinary Research 46 2453–2456Google Scholar
National Mastitis Council 2004 Microbiological Procedures for the Diagnosis of Bovine Udder Infection and Determination of Milk Quality, 4th edition. Madison, WI: NMCGoogle Scholar
Olde Riekerink, RGM, Barkema, HW, Kelton, DF & Scholl, DT 2008 Incidence rate of clinical mastitis on Canadian dairy farms. Journal of Dairy Science 91 1366–1377CrossRefGoogle ScholarPubMed
Oliveira, L, Hulland, C & Ruegg, PL 2013 Characterization of clinical mastitis occurring in cows on 50 large dairy herds in Wisconsin. Journal of Dairy Science 96 7538–7549Google Scholar
Paradis, M-É, Haine, D, Gillespie, B, Oliver, SP, Messier, S, Comeau, J & Scholl, DT 2012 Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens. Journal of Dairy Science 95 6436–6448Google Scholar
Persson Waller, K, Aspán, A, Nyman, A, Persson, Y & Grönlund Andersson, U 2011 CNS species and antimicrobial resistance in clinical and subclinical bovine mastitis. Veterinary Microbiology 152 112–116Google Scholar
Phuektes, P, Mansell, PD & Browning, GF 2001 Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis. Journal of Dairy Science 84 1140–1148Google Scholar
Ruegg, PL 2014 Risks, realities and responsibilities associated with mastitis treatments. In NMC Regional Meeting, Ghent, Proceedings, pp. 29–35Google Scholar
Sears, PM & McCarthy, KK 2003 Diagnosis of mastitis for therapy decisions. The Veterinary Clinics of North America, Food animal Practice 19 93–108CrossRefGoogle ScholarPubMed
Spittel, S & Hoedemaker, M 2012 Mastitis diagnosis in dairy cows using PathoProof real-time polymerase chain reaction assay in comparison with conventional bacterial culture in a Northern German field study. Berliner und Munchener tierarztliche Wochenschrift 125 494–502Google Scholar
Steeneveld, W, van Werven, T, Barkema, HW & Hogeveen, H 2014 Cow-specific treatment of clinical mastitis: an economic approach. Journal of Dairy Science 94 174–188Google Scholar
Studer, E, Schaeren, W, Naskova, J, Pfaeffli, H, Kaufmann, T, Kirchhofer, M, Steiner, A & Graber, HU 2008 A Longitudinal field study to evaluate the diagnostic properties of a quantitative real-time polymerase chain reaction–based assay to detect Staphylococcus aureus in milk. Journal of Dairy Science 91 1893–1902Google Scholar
Taponen, S, Salmikivi, L, Simojoki, H, Koskinen, MT & Pyörälä, S 2009 Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. Journal of Dairy Science 92 2610–2617Google Scholar