Aspán, Anna

Lauritsen, Klara Tølbøl

Pelkonen, Sinikka

Ridley, Anne

Tardy, Florence

Wisselink, Henk J.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Apr 23, 2019CoVetLab: working together to strengthen European collaboration on Mycoplasmabovis and compare available diagnostic toolsRidley, Anne; Tardy, Florence; Wisselink, Henk J.; Pelkonen, Sinikka; Lauritsen, Klara Tølbøl; Aspán,AnnaPublication date:2019Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Ridley, A., Tardy, F., Wisselink, H. J., Pelkonen, S., Lauritsen, K. T., & Aspán, A. (2019). CoVetLab: workingtogether to strengthen European collaboration on Mycoplasma bovis and compare available diagnostic tools.Poster session presented at European Mycoplasma Confernce 2019, Colindale, London, United Kingdom.BACKGROUNDDifferent clinical presentations of disease caused by Mycoplasmabovis predominate in European countries with significant economicand welfare impacts. M. bovis disease control relies on goodhusbandry and an early and reliable diagnosis. However, a lack ofstandardisation of approaches and diagnostic methods applied makescomparison of disease prevalence between countries difficult.AIMS• With assistance from CoVetLab.org a consortium of six Europeannational veterinary institutes was established to develop a networkof scientists and share tools and expertise on Mycoplasma bovis.• Objectives included hosting workshops and developing ring trials,including collating panels of DNA and serum samples, to evaluateavailable serological and PCR-based diagnostic tests.CoVetLab: working together to strengthen European collaborationon Mycoplasma bovis and compare available diagnostic toolsCONCLUSIONS • This CoVetLab project has enabled scientists from veterinary institutes in Europe undertaking M. bovis diagnostics to collaborate on mutually agreed priorities. • A joint CoVetLab -Nordic Workshop extended opportunities to widen our network of scientists and present preliminary data.• The comparison of PCR tests has provided reassurance regarding the quality of diagnosis, despite the different target genes and assays used in our laboratories.• Although only commercial ELISA kits were included, differences in the sensitivity and specificity were obtained. • Highlights the importance of inter-laboratory studies to assess performance of current and newly available tests.• References: Wisselink et al. "A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis" accepted BMC Veterinary Research.Andersson et al. (in preparation). • Analytical specificity, sensitivity and comparability of seven different PCR methods used to detect M. bovis were assessed. • All methods were in use by at least one of the participants.• Five different DNA extraction methods, seven PCRs targeting four different genes and six different real-time PCR platforms. • One commercial kit, all other PCR assays were in-house tests. M. bovis ELISA RING TRIAL• Two commercial ELISA systems (ID screen ELISA (Idvet, Grabels, France) and BIO K302 ELISA (Bio-X Diagnostics, Rochefort, Belgium)) were assessed by inter-laboratory comparison. • The sample panel (n=180) comprised sera from cattle from five countries with high and low M. bovis prevalence. • Standardised set of samples designed to reflect the range of samples encountered in all partner countries• Sera were distributed to the six laboratories and tested as recommended by the suppliers of the test kits.• Inter-laboratory variation associated with transferability of in-house assays precludes meaningful comparisons and so were not included in the analyses.• Immunoblot enabled statistical evaluation by latent class analysis. Anne Ridley1, Florence Tardy2, Henk Wisselink3, Sinikka Pelkonen4 , Klara Tølbøl Lauritsen5, Anna Aspan6 (joint authors)1Animal and Plant Health Agency, Department of Bacteriology, Weybridge, United Kingdom.2Anses Lyon Laboratory, JRU Ruminants Mycoplasmoses, Anses, VetAgro Sup, University of Lyon, France.3Wageningen Bioveterinary Research, Department of Infection Biology, Lelystad, The Netherlands. 4Finnish Food Safety Authority, Ruokavirasto, Research and Laboratory Department, Kuopio, Finland.5National Veterinary Institute Technical University of Denmark, Division of Diagnostics & Scientific Advice, Lyngby, Denmark.6National Veterinary Institute, Department of Microbiology, Uppsala, Sweden.Anna-Maria Andersson6 is acknowledged for her epidemiological analysis of the data• The ID Screen ELISA showed highest agreement with Western blot analysis and performed with higher precision and accuracy than the Bio K302 ELISA (Fig 2). • The diagnostic sensitivities of the ID Screen® Mycoplasma bovisand the Bio K302 ELISA were 95.6 % and 48.8 % respectively, with specificities of 99.3 % and 88.3 %, respectively.M. bovis PCR RING TRIALWORKSHOPSA BA. At Ruokavirasto in Kuopio to develop PCR and ELISA ring trials.B. Joint CoVetLab - Nordic Workshop on M. bovis in March 2018 at DTU, Lyngby was attended by 45 participants from the veterinary and scientific community from 10 countries.• Analytical specificity of the PCR methods was comparable, although only PCR-DGGE identified other bovine mycoplasmas.• Limits of detection varied from 10 to 103 CFU/ml to 103 and 106CFU/ml for real-time and end-point assays, respectively. • Ct values varied with naturally infected samples, both between laboratories and tests, without affecting interpretation (Fig 1).  Fig 2. Assessing sensitivity and specificity of the ELISA and immunoblot testsFig 1. 

CoVetLab: working together to strengthen European collaboration on Mycoplasma bovis and compare available diagnostic tools

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CoVetLab: working together to strengthen European collaboration on Mycoplasma bovis and compare available diagnostic tools

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