RESEARCH ARTICLE Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis Victoria Naranjo 1* , Margarita Villar 2* ,M a Paz Martín-Hernando 1 , Dolors Vidal 1 , Ursula Höfle 1, 3 , Christian Gortazar 1 , Katherine M. Kocan 4 , Jesús Vázquez 2 and José de la Fuente 1, 4 1 Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain 2 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid, Spain 3 Centro de Investigación Agraria El Dehesón del Encinar, Junta de Comunidades de Castilla – La Mancha, Oropesa, Spain 4 Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and character- ize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB1) wild boars. Genes identified in MLNand tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB1 and nontuberculous (TB2) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albu- min, and apolipoprotein A1 were found at lower levels in MLN of TB1 boars. Manganese super- oxide dismutase was found up-regulated in MLN of TB1 boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB1 boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host–pathogen interactions and for diagnostics and vaccine development. Received: March 16, 2006 Revised: October 11, 2006 Accepted: October 14, 2006 Keywords: Differential expression / Mycobacterium / Tuberculosis / Wild boar 220 Proteomics 2007, 7, 220–231 1 Introduction Mycobacterium bovis (M. tuberculosis complex) causes bovine tuberculosis, a well known reemerging zoonotic disease which affects cattle worldwide [1–5]. Several wildlife species are naturally infected with M. bovis, but European wild boar (Sus scrofa) is a major reservoir in some regions of Spain [6– 11]. In this species, most of the lesions associated with tuberculosis occur in the mandibular lymph nodes (MLN), while disseminated lesions are occasionally observed in the Correspondence: José de la Fuente, Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA E-mail: jose_delafuente@yahoo.com; djose@cvm.okstate.edu Fax: 11-405-744-5275 Abbreviations: ANX, annexin; ApoA1, apolipoprotein A1; APP, acute-phase proteins; ARG1, arginase I; CK, creatine kinase; HDL, high-density lipoprotein; HSPs, heat shock proteins; IDP, NADP- dependent isocitrate dehydrogenase; LYZS, lysozyme; MHC II, MHC class II; MLN, mandibular lymph nodes; MnSOD, manga- nese superoxide dismutase; PLA2, phospholipase A2; SAA, serum amyloid A; SPP1, osteopontin; SSH, suppression-subtrac- tive hybridization; TB1, tuberculous; TB2, nontuberculous * These authors contributed equally to this work. DOI 10.1002/pmic.200600527  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com