Scientific Abstract

Proposal No.  IBD-0095
Principal Investigator:  Laura W. Lamps, M.D.
Applicant Organization:  University of Arkansas for Medical Sciences (Little Rock, U.S.A.)
Project Title:  Pathogenic Yersinia DNA detection in Crohn’s disease
Period of Award:  January 1, 2004 – March 31, 2006

Background. The pathogenesis of Crohn’s disease (CD) remains enigmatic. Although numerous microorganisms have been implicated in the pathogenesis of Crohn’s disease (CD), no direct links between specific infectious agents and CD have been established. Yersinia enterocolitica (YE) and Y. pseudotuberculosis (YP) are food and water-borne bacteria that may cause appendicitis, enterocolitis, and mesenteric lymphadenitis. They are one of the most common causes of bacterial enteritis in Western and Northern Europe, and geographic areas that have a high incidence of CD have some of the highest rates of Yersinia infection. Studying Yersinia is challenging, as it is difficult to culture and serologic studies show cross-reactivity with other organisms. We developed the first molecular assay designed explicitly to detect pathogenic Yersinia DNA and perform serotype analysis in archival pathology materials. Our goals are to: 1) confirm and extend preliminary data by analyzing additional archival CD cases from multiple geographical areas; 2) subject Yersinia-positive cases to molecular serotyping to evaluate possible geographic and food-related associations; and 3) develop a complementary in situ hybridization assay in order to visualize bacteria in CD tissue.

Materials and Methods. Archival CD specimens will be retrieved from multiple institutions. Following DNA extraction, amplification will be performed by PCR, and primer pairs have been selected to detect single-copy virulence gene fragments present in pathogenic strains of Yersinia. As in our pilot studies, normal bowel, acute appendicitis, and active colitides of other causes will serve as negative controls. Following initial identification of Yersinia DNA, positive cases will be subjected to serogroup analysis by PCR and to in situ hybridization. When available, a limited medical record review will be conducted, including patient age, gender, clinical presentation, and disease course.

Anticipated Results. In our preliminary studies, 31% of 54 CD cases tested were positive for pathogenic Yersinia DNA by PCR analysis as compared to none of 120 tested control cases. We anticipate that we will continue to detect Yersinia DNA in a significant subset of CD patients and that we will be able to correlate positive cases with specific Yersinia serotypes. We also anticipate development of an in situ hybridization assay that will allow us to visualize the bacteria within CD tissue samples.

Conclusions. Our preliminary data raise the possibility that Yersinia plays a role in the pathogenesis of CD. Genetically susceptible individuals may be vulnerable to bacterial "triggers" in their environment, which initiate an abnormal immune response. It is also conceivable that this bacterial trigger varies with population, geographic location, and food sources. In this study we hope to further assess the prevalence of Yersinia DNA in CD, correlate CD with specific serotypes of Yersinia and to visualize the organism within CD tissues. It is hoped that our results will aid in the development of larger epidemiological studies with possible therapeutic implications, including prospective studies combining molecular analysis of tissues with stool culture and serologic assays in newly diagnosed CD patients.