Evaluation of clinical, histological and immunological changes and qPCR detection of Mycoplasma hyopneumoniae in tissues during the early stages of mycoplasmal pneumonia in pigs after experimental challenge with two field isolates Lauren K. Woolley a,b , Shayne Fell a , Jocelyn R. Gonsalves a , Mark J. Walker c , Steven P. Djordjevic d , Cheryl Jenkins a , Graeme J. Eamens a, * a NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Woodbridge Road Menangle, NSW 2568, Australia b School of Biological Sciences, University of Wollongong, Wollongong NSW 2522, Australia c School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia d The ithree Institute, University of Technology Sydney, NSW 2007, Australia 1. Introduction Mycoplasma hyopneumoniae is the causal agent of mycoplasmal pneumonia, a contagious pulmonary disease of swine characterised by a dry, non-productive cough, reduced growth rate and poor feed conversion efficiency Veterinary Microbiology 161 (2012) 186–195 A R T I C L E I N F O Article history: Received 21 February 2012 Received in revised form 16 July 2012 Accepted 16 July 2012 Keywords: Mycoplasma hyopneumoniae Mycoplasmal pneumonia Clinical Histopathology Cytokines Quantitative polymerase chain reaction A B S T R A C T Differences in Mycoplasma hyopneumoniae strain virulence and infection patterns will affect experimental challenge systems used to evaluate vaccine efficacy. Two strains (Hillcrest and Beaufort) were assessed by experimental pig challenge for their ability to induce clinical and pathological lesions and cytokine responses. Tracheobronchial lavage fluid (TBLF) was collected before and 17–18 days after challenge with Hillcrest (n = 8), Beaufort (n = 8) or no organisms (n = 3). Coughing was assessed twice daily, and at slaughter 21 (n = 9) or 28 (n = 10) days post-challenge, gross and histopathology of lungs were quantified and a quantitative PCR (mhp183 qPCR) was applied to detect M. hyopneumoniae DNA in tissues and TBLF. Hillcrest was clearly superior to Beaufort in its ability to induce coughing and pneumonic lesions. At 17–18 days, interleukin (IL)-1b and IL-6 concentrations in TBLF were only significantly higher (8.7 and 5.1 fold respectively) than controls (P < 0.001) in Hillcrest-challenged pigs. Lungs of all Hillcrest-challenged pigs were qPCR positive at either slaughter date, but only at day 28 in Beaufort-challenged pigs. M. hyopneumoniae DNA was highest in concentration in lungs 21 days after Hillcrest challenge, and was detected in the spleen, kidney and/or liver of Hillcrest-challenged pigs, but not in Beaufort pigs. While M. hyopneumoniae DNA concentration in TBLF was elevated following Hillcrest and Beaufort challenge, there was no significant difference in mean mycoplasmal DNA concentration detected in TBLF from pigs challenged with either isolate (P > 0.05). Thus a suitable challenge strain, coupled with lung pathology and cytokine assays, are valuable in assessing post-challenge responses. Assessment of M. hyopneu- moniae DNA in lung and abdominal tissues by mhp183 qPCR, in conjunction with histopathology, were valuable in confirming M. hyopneumoniae infection. Crown Copyright ß 2012 Published by Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +61 246406358; fax: +61246406384. E-mail address: graeme.eamens@dpi.nsw.gov.au (G.J. Eamens). Contents lists available at SciVerse ScienceDirect Veterinary Microbiology jou r nal h o mep ag e: w ww .els evier .co m/lo c ate/vetm ic 0378-1135/$ – see front matter . Crown Copyright ß 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.07.025