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

Sara Ellinor Cederlöf³  Nils Toft²  Bent Aalbaek¹  Ilka Christine Klaas² 

[1] Department of Veterinary Disease Biology, University of Copenhagen, Ridebanevej, Frederiksberg C, Denmark

[2] Department of Large Animal Science, University of Copenhagen, Grønnegårdsvej, Frederiksberg C, Denmark

[3] Hushållningssällskapet Västernorrland, Trädgårdsgatan 7, Härnösand, Sweden

关键词: Bacteriological culture;    Mastitis;    Specificity;    Sensitivity;    Latent class analysis;    PCR;    Staphylococcus aureus;   

Others  :  789435
DOI  :  10.1186/1751-0147-54-65

 received in 2012-06-13, accepted in 2012-11-15,  发布年份 2012

【 摘 要 】

Background

Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs.

Methods

Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations.

Results

The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs.

Conclusion

Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow.

【 授权许可】

   
2012 Cederlöf et al.; licensee BioMed Central Ltd.

【 参考文献 】

• [1]Bradley AJ, Green MJ: The importance of the nonlactating period in the epidemiology of intramammary infection and strategies for prevention. Vet Clin N Am -Food Anim Pract 2004, 20(3):547-568.
• [2]Reyher KK, Dufour S, Barkema HW, Des Côteaux L, DeVries TJ, Dohoo IR, Keefe GP, Roy J, Scholl DT: The national cohort of dairy farms—a data collection platform for mastitis research in Canada. J Dairy Sci 2011, 94(3):1616-1626.
• [3]Petzer I, Lourens DC, van der Schans TJ, Watermeyer JC, van Reenen R, Rautenbach GH, Thompson P: Intramammary infection rate during the dry period in cows that received blanket dry cow therapy: efficacy of 6 different dry-cow intra-mammary antimicrobial products. J S Afr Vet Assoc -Tydskr Suid-Afr Vet Ver 2009, 80(1):23-30.
• [4]Anonymous: Announcement on the veterinary use, dispensing and prescribing of drugs for animals. 2010. [Danish (Bekendtgørelse 785 af 25/06/2010 om dyrlægers anvendelse, udlevering og ordinering af lægemidler til dyr)] [https://www.retsinformation.dk/Forms/R0710.aspx?id=132613 webcite]
• [5]National Mastitis Council: Laboratory handbook on bovine mastitis. Madison: WI: NMC; 1999.
• [6]Bradley AJ, Leach KA, Breen JE, Green LE, Green MJ: Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. Vet Rec 2007, 160(8):253-258.
• [7]Sears PM, Smith BS, English PB, Herer PS, Gonzalez RN: Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J Dairy Sci 1990, 73(10):2785-2789.
• [8]Hicks CR, Eberhart RJ, Sischo WM: Comparison of microbiologic culture, an enzyme-linked immunosorbent assay, and determination of somatic cell count for diagnosing Staphylococcus aureus mastitis in dairy cows. J Am Vet Med Assoc 1994, 204(2):255-260.
• [9]Buelow KL, Thomas CB, Goodger WJ, Nordlund KV, Collins MT: Effect of milk sample collection strategy on the sensitivity and specificity of bacteriologic culture and somatic cell count for detection of Staphylococcus aureus intramammary infection in dairy cattle. Prev Vet Med 1996, 26(1):1-8.
• [10]Dohoo IR, Smith J, Andersen S, Kelton DF, Godden S: Diagnosing intramammary infections: evaluation of definitions based on a single milk sample. J Dairy Sci 2011, 94(1):250-261.
• [11]Koskinen MT, Wellenberg GJ, Sampimon OC, Holopainen J, Rothkamp A, Salmikivi L, van Haeringen WA, Lam TJGM, Pyörälä S: Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria. J Dairy Sci 2010, 93(12):5707-5715.
• [12]Heid CA, Stevens J, Livak KJ, Williams PM: Real time quantitative PCR. Genome Res 1996, 6(10):986-994.
• [13]Caraguel CGB, Stryhn H, Gagne N, Dohoo IR, Hammell KL: Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches. J Vet Diagn Invest 2011, 23(1):2-15.
• [14]Finnzymes: Instruction Manual- PathoProofTM Mastitis PCR Assay F-870S/L, version 1.2, April 2008. Espoo, Finland, 2008.
• [15]Riffon R, Sayasith K, Khalil H, Dubreuil P, Drolet M, Lagace J: Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. J Clin Microbiol 2001, 39(7):2584-2589.
• [16]Phuektes P, Browning CF, Anderson G, Mansell PD: Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples. J Dairy Res 2003, 70(2):149-155.
• [17]Gillespie BE, Oliver SP: Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction. J Dairy Sci 2005, 88(10):3510-3518.
• [18]Taponen S, Salmikivi L, Simojoki H, Koskinen MT, Pyörälä S: Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. J Dairy Sci 2009, 92(6):2610-2617.
• [19]Friendship C, Kelton D, van de Water D, Slavic D, Koskinen MT: Field evaluation of the pathoproof mastitis PCR assay for the detection of Staphylococcus aureus infected cows using DHI samples. In NMC annual meeting proceedings. January 31-February 3 2010. Albuquerque, New Mexico: NMC; 2010:226-227.
• [20]Koskinen MT, Holopainen J, Salmikivi L: Field comparison of real-time PCR and conventional bacterial culturing in bovine mastitis testing. In Mastitis research into practice: proceedings of the 5th IDF mastitis conference, 21-24 March 2010. Edited by Hillerton JB, Eric Hillerton J. Christchurch, New Zealand: New Zealand Veterinary Association, VetLearn Staff; 2010:486-488.
• [21]Bennedsgaard TW: Personal communication. Tjele, Denmark: Aarhus University, Department of Animal Bioscience and Health, Blichers Alle' 20, 8830; 2012.
• [22]Katholm J: News for veterinarians- newsletter regarding welfare and health, no. 2. 2010. Aktuelt til dyrlæger- nyhedsbrev om velfærd og sundhed, nr. 2 2010,Videncentret for landbrug, kvæg, Danish. [https://www.landbrugsinfo.dk/Kvaeg/Sundhed-og-dyrevelfaerd/Sider/AktuelttilDyrlaeger0032010.aspx#PCRMælkeprøver webcite]
• [23]Sanford CJ, Keefe GP, Sanchez J, Dingwell RT, Barkema HW, Leslie KE, Dohoo IR: Test characteristics from latent-class models of the California Mastitis Test. Prev Vet Med 2006, 77(1–2):96-108.
• [24]Toft N, Åkerstedt J, Tharaldsen J, Hopp P: Evaluation of three serological tests for diagnosis of Maedi-Visna virus infection using latent class analysis. Vet Microbiol 2007, 120(1–2):77-86.
• [25]Hui SL, Walter SD: Estimating the error rates of diagnostic-tests. Biometrics 1980, 36(1):167-171.
• [26]Toft N, Jørgensen E, Højsgaard S: Diagnosing diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard. Prev Vet Med 2005, 68(1):19-33.
• [27]National Mastitis Council: Microbiological procedures for the diagnosis of bovine udder infections and determination of milk quality. Madison, WI: NMC; 2004.
• [28]Quinn PJ, Markey BK, Carter ME, Donnelly WJC, Leonard FC: Veterinary microbiology and microbial disease. Oxford, UK: Blackwell Science Ltd.; 2006.
• [29]Barrow GI, Feltham RKA: Cowan and Steel's manual for the identification of medical bacteria. UK: Cambridge University Press; 1993.
• [30]Koskinen MT, Holopainen J, Pyörälä S, Bredbacka P, Pitkälä A, Barkema HW, Bexiga R, Roberson J, Sølverød L, Piccinini R, Kelton D, Lehmusto H, Niskala S, Salmikivi L: Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. J Dairy Sci 2009, 92(3):952-959.
• [31]Spiegelhalter D, Thomas A, Best N, Lunn D: OpenBUGS user manual, version 3.2.1 March 2011. 2011. [http://www.openbugs.info/Manuals/Manual.html webcite]
• [32]Toft N, Innocent GT, Gettinby G, Reid SWJ: Assessing the convergence of Markov Chain Monte Carlo methods: an example from evaluation of diagnostic tests in absence of a gold standard. Prev Vet Med 2007, 79(2–4):244-256.
• [33]Wellenberg GJ, Sampimon OC, Rothkamp A, van Haeringen WA, Lam TJGM: Detection of mastitis pathogens by real-time PCR in clinical and subclinical mastitis samples. In Mastitis research into practice: proceedings of the 5th IDF mastitis conference, 21-24 March 2010. Edited by Hillerton JB, Eric Hillerton J. Christchurch, New Zealand: New Zealand Veterinary Association, VetLearn Staff; 2010:539-544.
• [34]Haveri M, Hovinen M, Roslof A, Pyorala S: Molecular types and genetic profiles of Staphylococcus aureus strains isolated from bovine intramammary infections and extramammary sites. J Clin Microbiol 2008, 46(11):3728-3735.
• [35]Lovendahl P, Bjerring MA: Detection of carryover in automated milk sampling equipment. J Dairy Sci 2006, 89(9):3645-3652.
• [36]Villanueva MR, Tyler JW, Thurmond MC: Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk. J Am Vet Med Assoc 1991, 198(8):1398-1400.
• [37]Godden SM, Jansen JT, Leslie KE, Smart NL, Kelton DF: The effect of sampling time and sample handling on the detection of Staphylococcus aureus in milk from quarters with subclinical mastitis. Can Vet J -Rev Vet Can 2002, 43(1):38-42.
• [38]Hubackova M, Rysanek D: Effects of freezing milk samples on the recovery of alimentary pathogens and indicator microorganisms. Acta Vet Brno 2007, 76(2):301-307.
• [39]Schukken YH, Smit JAH, Grommers FJ, Vandegeer D, Brand A: Effect of freezing on bacteriologic culturing of mastitis milk samples. J Dairy Sci 1989, 72(7):1900-1906.
• [40]Dinsmore RP, English PB, Gonzalez RN, Sears PM: Use of augmented cultural techniques in the diagnosis of the bacterial cause of clinical bovine mastitis. J Dairy Sci 1992, 75(10):2706-2712.
• [41]Murdough PA, Deitz KE, Pankey JW: Effects of freezing on the viability of nine pathogens from quarters with subclinical mastitis. J Dairy Sci 1996, 79(2):334-336.