EXPERIMENTALLY INDUCED DISEASE Immunohistochemical Labelling of Cytokines in Calves Infected Experimentally with Mycoplasma bovis F. Rodr  ıguez * , P. Castro * , J. B. Poveda † , A. M. Afonso * and A. Fern andez * *Unit of Veterinary Histology and Pathology and † Unit of Epidemiology and Preventive Medicine, Institute for Animal Health, Veterinary School, Universidad de Las Palmas de Gran Canaria, Arucas, Gran Canaria, Spain Summary To gain further insight into the pathogenesis of Mycoplasma bovis-associated pneumonia, cytokine expression in different pulmonary compartments was examined. The expression of tumour necrosis factor (TNF)-a, inter- leukin (IL)-4 and interferon (IFN)-g was examined immunohistochemically in the lung of 10 calves infected experimentally with M. bovis. M. bovis antigen was located in respiratory epithelial cells and within inflamma- tory cells in the airway lumina. Immunolabelling for TNF-a, IL-4 and IFN-g was usually associated with inflammation, particularly in macrophages and lymphocytes in hyperplastic bronchus-associated lymphoid tissue (BALT), in thickened alveolar septa and in the bronchoalveolar exudate of infected animals. In M. bovis infection, macrophage and lymphocyte activation results in expression of a number of cytokines capable of inducing lung lesions and hyperplasia of the BALT. The cytokines examined likely play a role in pulmonary defence against M. bovis infection. Ó 2015 Elsevier Ltd. All rights reserved. Keywords: calf; cytokine; immunohistochemistry; Mycoplasma bovis Mycoplasma bovis is an important pathogen that causes pneumonia and arthritis in calves and young cattle and mastitis in adult cattle (Gourlay et al., 1989; Nicholas and Ayling, 2003). Lesions occurring during M. bovis pneumonia include bronchointerstitial inflammation and hyperplasia of the bronchus- associated lymphoid tissue (BALT), collectively known as ‘cuffing pneumonia’ (Radaelli et al., 2009; Hermeyer et al., 2011). A common feature of infections of the respiratory tract is persistence of the causative organisms at the mucosal surface, which may contribute to the formation of mucosal lymphoid aggregates such as seen in the lungs of animals with mycoplasma infection (Howard et al., 1987). The local release of cytokines is a key mediator of pulmonary inflammation (Kelley, 1990). Inflamma- tory cytokines activate the immune system in response to ‘danger’ and increase the efficiency of an immune response (Murtaugh and Foss, 2002). The induction of cytokines has been suggested to be a pathogenic factor during M. bovis infection (Thomas et al., 1991; Jungi et al., 1996; Vanden Bush and Rosenbusch, 2003). The aims of this study were to determine immunohistochemically the expression of cytokines in the lung of calves infected experimentally with M. bovis and to investigate the relationship between cytokine expression and the development of pulmonary lesions. Pneumonic lung tissue was obtained from ten 3- month-old Holstein calves in which M. bovis pneu- monia had been induced following the protocol described by Rodr  ıguez et al. (1996). The animals were given approximately 10 9 colony forming units/ ml of M. bovis (strain MC1750) grown in Mycoplasma broth. Calves were inoculated intratracheally with a 10 ml volume of broth and killed 14 days after inocu- lation. Lung tissue from three aged-matched calves, inoculated by the same route with 10 ml of sterile Mycoplasma broth, served as negative controls. The J. Comp. Path. 2015, Vol. -,1e5 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jcpa Correspondence to: F. Rodr  ıguez (e-mail: frodriguez@dmor.ulpgc.es). 0021-9975/$ - see front matter Ó 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcpa.2015.01.006 Please cite this article in press as: Rodr  ıguez F, et al., Immunohistochemical Labelling of Cytokines in Calves Infected Experimentally with Myco- plasma bovis, Journal of Comparative Pathology (2015), http://dx.doi.org/10.1016/j.jcpa.2015.01.006