Estimation of the sensitivity of four sampling methods for Mycoplasma hyopneumoniae detection in live pigs using a Bayesian approach.

Four sampling techniques for Mycoplasma hyopneumoniae detection, namely nasal swabbing, oral-pharyngeal brushing, tracheo-bronchial swabbing and tracheo-bronchial washing, were compared in naturally infected live pigs. In addition, a quantitative real-time PCR assay for M. hyopneumoniae quantification was validated with the same samples. 60 finishing pigs were randomly selected from a batch of contemporary pigs on a farm chronically affected by respiratory disorders. Each pig was submitted to nasal swabbing, oral-pharyngeal brushing, tracheo-bronchial swabbing and tracheo-bronchial washing. Nested-PCR and real-time PCR assays were performed on all samples. A Bayesian approach was used to analyze the nested-PCR results of the four sampling methods (i.e. positive or negative) to estimate the sensitivity and specificity of each method. M. hyopneumoniae was detected by nested-PCR in at least one sample from 70% of the pigs. The most sensitive sampling methods for detecting M. hyopneumoniae in live naturally infected pigs were tracheo-bronchial swabbing and tracheo-bronchial washing, as compared to oral-pharyngeal brushing and nasal swabbing. Swabbing the nasal cavities appeared to be the least sensitive method. Significantly higher amounts of M. hyopneumoniae DNA were found at the sites of tracheo-bronchial sampling than in the nasal cavities or at the oral-pharyngeal site (p<0.001). There was no difference between the tracheo-bronchial washing and the tracheo-bronchial swabbing results (p>0.05). Our study indicated that tracheo-bronchial swabbing associated with real-time PCR could be an accurate diagnostic tool for assessing infection dynamics in pig herds.

[1]  D. Berkvens,et al.  A Bayesian evaluation of three diagnostic assays for the detection of Giardia duodenalis in symptomatic and asymptomatic dogs. , 2008, Veterinary parasitology.

[2]  P. Wallgren,et al.  Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene , 1995, Journal of clinical microbiology.

[3]  P A Lachenbruch,et al.  Comparisons of two‐part models with competitors , 2001, Statistics in medicine.

[4]  M. Calsamiglia,et al.  Profiling Mycoplasma hyopneumoniae in farms using serology and a nested PCR technique , 1999 .

[5]  S. Kwok,et al.  40 – DETECTION OF HUMAN IMMUNODEFICIENCY VIRUS , 1990 .

[6]  David B. Dunson,et al.  Bayesian Data Analysis , 2010 .

[7]  D B Dunson,et al.  Commentary: practical advantages of Bayesian analysis of epidemiologic data. , 2001, American journal of epidemiology.

[8]  M. Calsamiglia,et al.  Application of a Nested Polymerase Chain Reaction Assay to Detect Mycoplasma Hyopneumoniae from Nasal Swabs , 1999, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[9]  Emmanuel Lesaffre,et al.  Estimating Disease Prevalence in a Bayesian Framework Using Probabilistic Constraints , 2006, Epidemiology.

[10]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[11]  H. Nathues,et al.  Detection of respiratory pathogens in porcine lung tissue and lavage fluid. , 2008, Veterinary journal.

[12]  L. Joseph,et al.  Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard. , 1995, American journal of epidemiology.

[13]  D. Berkvens,et al.  A Bayesian approach for the evaluation of six diagnostic assays and the estimation of Cryptosporidium prevalence in dairy calves. , 2006, Veterinary research.

[14]  E. Verdin,et al.  A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs. , 2000, Veterinary microbiology.

[15]  R. Goodwin.  Isolation of Mycoplasma suipneumoniae from the nasal cavities and lungs of pigs affected with enzootic pneumonia or exposed to this infecion. , 1972, Research in veterinary science.

[16]  T. Asai,et al.  Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods. , 2005, The Journal of veterinary medical science.

[17]  G. Kennedy,et al.  Duration of Mycoplasma hyopneumoniae infection in gnotobiotic pigs. , 1980, The Canadian veterinary journal = La revue veterinaire canadienne.

[18]  C. Marois,et al.  Development of a quantitative Real‐Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs , 2010, Journal of applied microbiology.

[19]  S. Dee,et al.  An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs. , 2009, Veterinary microbiology.

[20]  E. Thacker,et al.  Use of a Mycoplasma Hyopneumoniae Nested Polymerase Chain Reaction Test to Determine the Optimal Sampling Sites in Swine , 2002, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[21]  M. Ganter,et al.  Detection of Mycoplasma hyopneumoniae in Bronchoalveolar Lavage Fluids of Pigs by PCR , 1998, Journal of Clinical Microbiology.

[22]  J. Segalés,et al.  Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection , 2008, The Veterinary Journal.

[23]  M. Hewicker-Trautwein,et al.  Occurrence of Mycoplasma hyopneumoniae in coughing piglets (3-6 weeks of age) from 50 herds with a history of endemic respiratory disease. , 2009, Transboundary and emerging diseases.

[24]  C. Marois,et al.  Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets. , 2007, Veterinary microbiology.

[25]  Xiao-Hua Zhou,et al.  Statistical Methods in Diagnostic Medicine , 2002 .

[26]  A. Cavalier,et al.  Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. , 1992, Veterinary microbiology.

[27]  E. Verdin,et al.  Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs. , 2000, Molecular and cellular probes.