Protein and AntigenicVariability among Mycoplasma hyopneumoniae Strains by SDS-PAGE and Immunoblot P.Assunc°a¬o 1 ,C.DelaFe 1 ,A.S.Ram|¨rez 1 ,O.Gonza¨lezLlamazares 2 and J.B.Poveda 1 1 Unidad de Epidemiolog|¨a y Medicina Preventiva, Facultad deVeterinaria, Universidad de Las Palmas de Gran Canaria,Trasmonta·a s/n. 35416 Arucas, Spain; 2 Departamento de I+DLaboratorios Syva, Leo¨ n, Spain Correspondence: E-mail: passuncao@becarios.ulpgc.es Assunc°a¬o, P., De la Fe, C., Ram|¨rez, A.S., Gonza¨lez Llamazares, O. and Poveda, J.B., 2005. Protein and antigenic variability among Mycoplasma hyopneumoniae strains by SDS-PAGE and immunoblot. Veter- inary Research Communications, 29(7), 563^574 ABSTRACT Porcineenzooticpneumonia(PEP),with Mycoplasmahyopneumoniae astheprimaryagent,isachronic respiratory disease that causes major economic losses to the pig industry worldwide. The aim of this work was to analyse 18 ¢eld strains of M. hyopneumoniae isolated in Gran Canaria (Spain) and the reference M. hyopneumoniae strain by SDS-PAGE and immunoblot. A monoclonal antibody (MAb) against the membrane protein p46 reacted with all the strains in this study. In contrast, a puri¢ed polyclonal antibody (PAb) against the cytoplasmic protein p36 reacted with this protein in only 10 strains. A MAb against the adhesin protein p97 stained multiple proteins of di¡erent sizes and with di¡erent intensities. Di¡erent antigenic patterns in the same M. hyopneumoniae strains were also observed after di¡erent numbers of passages in culture medium. Furthermore, variability in the staining of the 36 kDa protein was observed, depending on whether the p36 PAb or the antiserum against M. hyopneumoniae reference strain was used. It is concluded that local M. hyopneumoniae ¢eld isolates in Gran Canaria are characterized by protein diversity. Keywords: antigenic variability, immunoblot, Mycoplasma hyopneumoniae,SDS-PAGE Abbreviations: LDH, lactate dehydrogenase; MAb, monoclonal antibody; PAb, polyclonal antibody; PBS, phosphate-bu¡ered saline; PEP, porcine enzootic pneumonia; SDS-PAGE, sodium dodecyl sulphate^polyacrylamidegel electrophoresis INTRODUCTION Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia (PEP), is present in the vast majority of swine farms worldwide, causing respiratory distress resulting from pneumonia in growing pigs. It causes considerable economic losses through retarded growth, poor food conversion and increased susceptibility of pigs to infections by other organisms (Kobisch, 2000; Kobisch and Friis, 1996). It has been demonstrated that M. hyopneumoniae strains are characterized by protein, antigenic and genetic heterogeneity. Ro and Ross (1983) observed di¡erences regarding identi¢cation of M. hyopneumoniae by serological methods, since the same VeterinaryResearchCommunications,29(2005)563^574 # 2005Springer.PrintedintheNetherlands 563