Helicobacter ISSN 1523-5378 172 © 2009 Blackwell Publishing Ltd, Helicobacter 14: 172–176 Blackwell Publishing Ltd Oxford, UK HEL Helicobacter 1083-4389 1523-5378 © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Helicobacter XX: xx–xx XXX Original Article Serodiagnosis of Enterohepatic Helicobacter Species Wadström et al. Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections Torkel Wadström, * Jann Hau, † Ingrid Nilsson * and Åsa Ljungh * * Department of Clinical Microbiology and Immunology, Lund University Hospital, Lund, Sweden, † Department of Experimental Medicine, University of Copenhagen and University Hospital of Copenhagen, Copenhagen, Denmark Abstract Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter-infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals. Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus, and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns. Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H. bilis, in 91% for H. hepaticus, and in 82% for H. ganmani. The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium. Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents. Keywords Serodiagnosis, immunoblot, enterohepatic Helicobacter spp, cell surface proteins, laboratory mice. Reprints request to: Torkel Wadström, Department of Clinical Microbiology and Immunology, Lund University Hospital, Sölvegatan 23, SE-223 62 Lund, Sweden. E-mail: torkel.wadstrom@med.lu.se Endemic Helicobacter infections in laboratory mice and other rodents seem to be very common [1,2]. Enterohepatic Helicobacter species (EHS) that naturally infect rodents include at least 11 formally named and some provisionally named species [3] and they are increasingly recognized as emerging infectious agents in laboratory animal facilities causing chronic gut and liver inflammation and modulating an immune-mediated intestinal inflammation [4]. There are presently no serology tests available and no optimized culture methods to study infections with these fastidious EHS in rodents and other animals. Several Helicobacter species are not possible to culture today, and culture independent immuno-based diagnostic methods should be developed to discriminate between gastrointestinal infections by diagnosing a seroconversion. The present study focused on three widely distributed Helicobacter species infecting laboratory mice; H. bilis, H. hepaticus, and H. ganmani. H. bilis was first identified in inbred mice with chronic colitis and hepatitis by Warthin–Starry silver staining of gut and liver tissues, and isolated from the liver, bile, and intestine of aged, inbred mice [5]. Infections with this spe- cies were found to be widespread among mouse colonies particularly in immuno-deficient mice [1,5]. H. bilis has also been isolated from dogs, gerbils, rats, and cats [6]. H. hepaticus was isolated from the livers of A/JCr mice with active chronic hepatitis and liver cancer [7], and is presently the most studied EHS. The genome of H. hepaticus was published 2003 [8] and this species produces a cytolethal distending toxin causing a progressive cell enlargement, cell cycle arrest, DNA mutations, and cell death [9]. H. ganmani is a urease-negative anaerobe EHS first isolated from