Andersson, A.-M.

Aspan, A.

Gaurivaud, P.

Høgberg, Jonas

Kensø, Jane

Larsen, Helene

Lauritsen, Klara Tølbøl

Pelkonen, Sinikka

Pohjanvirta, Tarja

Ridley, Anne

Tardy, Florence

Wisselink, Henk J.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: May 03, 2019A European interlaboratory evaluation of PCR and ELISA methods for MycoplasmabovisdiagnosticsAspan, A.; Wisselink, Henk J.; Ridley, Anne; Andersson, A.-M.; Pohjanvirta, Tarja; Pelkonen, Sinikka;Lauritsen, Klara Tølbøl; Kensø, Jane; Larsen, Helene; Høgberg, Jonas; Gaurivaud, P.; Tardy, FlorencePublication date:2018Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Aspan, A., Wisselink, H. J., Ridley, A., Andersson, A-M., Pohjanvirta, T., Pelkonen, S., ... Tardy, F. (2018). AEuropean interlaboratory evaluation of PCR and ELISA methods for Mycoplasma bovisdiagnostics. Postersession presented at Medvet Pathogens 2018, Prato, Italy.www.sva.seMycoplasma bovis is known worldwide as a major bovinepathogen. Increasing prevalence has been recently reported inNorthern Europe. Control ofM. bovis infections in cattle herdsis difficult as increasing antimicrobial resistance is reported,and commercial vaccines are not available or not effective.Therefore, preventive measures such as high biosecuritystandards guided by results of a highly specific and sensitivediagnosis are essential.A consortium of six European national veterinary institutes was established to evaluate the performance of PCR and ELISA diagnostic methods currently used by these institutes. For serodiagnosis two commercial ELISA test kits were used:the Bio K302 ELISA (Bio-X Diagnostics, Rochefort, Belgium)and the ID Screen ® Mycoplasma bovis ELISA (IDvet,Grabels, France). These two tests were compared to an in-house Western blot method as to be able to use a latent classanalysis statistical approach. A sample panel (n=180) wascompiled of sera from cattle from five countries with high andlow M. bovis prevalence. Sera were distributed to the sixlaboratories and tested as recommended by the suppliers ofthe test kits. Using latent class analysis, we found that thediagnostic sensitivities of the Western blot, the ID Screen®Mycoplasma bovis and the Bio K302 ELISA were 96.6 %, 99.6% and 51.0 % respectively, and the diagnostic specificities were99.8 %, 99.0 % and 85.7 % respectively (Table 1) (Andersson etal., manuscript in preparation).For evaluation of PCR diagnostics, a sample panel was testedusing five different DNA extraction methods, seven differentreal-time and/or end-point PCR methods targeting fourdifferent genes and six different real-time PCR platforms. Onlyone commercial kit was assessed (MPBO50 kit fromThermoFisher Scientific), all other were in-house PCR tests.Three different assays were conducted to assess the specificity,sensitivity and comparability of the PCRs. The sensitivity andcomparability assays were conducted using (n=21)bronchoalveolar fluids of veal calves, artificially contaminatedor naturally infected. With a few exceptions, all methods runroutinely in the participating laboratories showed comparableperformance (Figure 1) (Wisselink et al., manuscriptsubmitted for publication).MEDVET PATHOGENS,OCTOBER 8 - 11, 2018 PRATO, ITALYA European interlaboratory evaluation of PCR and ELISA methods for Mycoplasma bovis diagnostics Table 1. Posterior median and 95% posterior credibility interval (95% PCI) of sensitivity and specificity for Western blot and the two ELISA systems obtained from latent class analysis, assuming conditional independence between tests and using informative or uniform priors.Figure 1. Comparability of results of various PCR methods in use by six laboratories (Lab 1 to Lab 6) on bronchoalveolar lavage fluid (BALF) samples (n=21) from veal calves from farms with bovine respiratory disease (BRD). BALF samples were tested twice by the PCR methods and the mean Ct values were calculated from the real-time PCRs. Viability of M. bovis in the BALF samples was verified with culture. On the basis of the culture results, the figure was split in two parts, either with BALF samples negative or positive for M. bovis by culture.A. Aspán1, H.J. Wisselink2, A. Ridley3, A-M Andersson4, T. Pohjanvirta5, S Pelkonen5, K. T. Lauritsen6, J. Kensø6, H. Larsen6, J. Høgberg6, P. Gaurivaud7, F. Tardy7.1National Veterinary Institute, Department of Microbiology, Uppsala, Sweden.2Wageningen Bioveterinary Research, Department of Infection Biology, Lelystad, The Netherlands.3Animal and Plant Health Agency, Department of Bacteriology, Weybridge, United Kingdom.4National Veterinary Institute, Department of Animal Health and Antimicrobial Strategies, Uppsala, Sweden.5Finnish Food Safety Authority Evira, Research and Laboratory Department, Kuopio, Finland.6National Veterinary Institute Technical University of Denmark, Division of Diagnostics & Scientific Advice, Lyngby, Denmark.7Anses Lyon Laboratory, JRU Ruminants Mycoplasmoses, Anses, VetAgro Sup, University of Lyon, France.This work was supported by the

A European interlaboratory evaluation of PCR and ELISA methods for Mycoplasma bovisdiagnostics

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A European interlaboratory evaluation of PCR and ELISA methods for Mycoplasma bovisdiagnostics

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