Final Progress Report

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

Summary of Project Aims, Accomplishments Towards Meeting Those Aims, and Significant Results

The overall hypothesis driving this project is that microorganisms could act as  "triggers" in Crohn’s disease (CD), causing an abnormal immune response in susceptible individuals. It is possible that more than one organism could serve as such an environmental trigger, depending on geographic location and food/water sources. The specific hypotheses to be tested by this project are that Yersinia will be demonstrated in lesional CD tissue, both by PCR assay and in situ hybridization, and that we will be able to link Yersinia-positive CD patients to specific Yersinia biotypes that might be related to geographic areas and/or food sources. We requested funding from the BMRP for the three related but not interdependent specific aims listed below. Major achievements/significant results are discussed below under the specific aim with which they correlate.

Specific aim #1: To confirm and extend preliminary data by collecting and analyzing additional archival CD cases (target case accrual of 100-150 cases) from multiple geographical areas and various age groups.

We met our target goal for case accrual, as we were able to analyze 142 cases from four institutions in three states (Arkansas, Michigan, and Ohio), spanning the years 1986-2004.  We analyzed three genes (ail, Yop, and YadA) present only in pathogenic Yersinia strains, and found that a total of 52% of the cases we analyzed were positive for Yersinia DNA.  This confirmed and extended the data we obtained in our original pilot project, prior to the submission of this grant proposal [American Journal of Surgical Pathology 27: 2003], in which we detected pathogenic Yersinia DNA in 31% of the CD bowel resections that we analyzed.

Specific aim #2: To subject Yersinia-positive cases to molecular serotyping in order to evaluate possible geographic and food-related associations.

We developed and completed initial testing on a molecular biogrouping assay that, using PCR technology, can differentiate between Group 1B Yersinia enterocolitica (the high virulence serotypes) and Group 2-5 Yersinia enterocolitica (the low virulence serotypes. Evaluation of the Yersinia enterocolitica-positive CD cases using this novel assay indicated that all but one case contained a low virulence strain of Yersinia, which are believed to have a possible intracellular persistence advantage in tissue, as opposed to the high virulence strains. We were also able to test cases of other granulomatous gastrointestinal disease, such as granulomatous appendicitis and malakoplakia, which were also positive for the low virulence strains, indicating that low virulence Yersinia are associated with a granulomatous pattern of inflammation in several diseases. A control small group of cases with fulminant Yersinia infection tested positive for high virulence serotypes. This suggests a correlation between low virulence YE serotypes and chronic granulomatous inflammatory diseases. A similar test for Yersinia pseudotuberculosis is under development.

Several documented epidemic Yersinia infections have been linked to particular biogroups (each of which is composed of several serotypes) and specific geographic regions. For this reason, development of a molecular assay that effectively biogroups Yersinia (including Yersinia-positive CD cases) could yield important information regarding the epidemiology of Yersinia and CD as well as the correlation between biogroups and geographic areas.

Specific aim #3: To develop a complementary in situ hybridization assay as a confirmatory method for our PCR studies, and to visualize the bacteria within CD specimens.

This assay is still under development.

i.   If there have been no major achievements, explain why.

Not applicable-see above

ii.   Describe any negative results or failed experiments during the period.

Although we did not have any negative results or failed experiments, the in situ hybridization assay has proven to be extremely technically challenging to develop in the two year time period of our grant. However, we will continue to work on it.

Lay Summary

Crohn’s disease (CD) is a chronic inflammatory disease of the intestines for which there is no known cause. Many different bacteria have been investigated as causes, but no direct links between specific bacteria and CD have been proven.  Yersinia enterocolitica (YE) and Y. pseudotuberculosis (YP) are bacteria that are commonly found in water, milk, and meat. They are one of the most common causes of gastroenteritis in Western and Northern Europe, and countries with many cases of CD have some of the highest rates of Yersinia infection. Studying Yersinia is challenging, as there are not many ways to reliably detect it. Our group developed a novel test (called a PCR assay) for detection of Yersinia, which can be used on tissues received in a pathology laboratory. Our goals for this grant were 1) to confirm our preliminary findings by testing additional CD cases from multiple areas of the United States; 2) to find out if certain strains (called biogroups) of Yersinia can be linked to specific areas of the country or food sources; and 3) to develop an additional test called in situ hybridization that would allow us to actually see the bacteria in CD tissue.

We collected 142 CD cases from four hospitals in three states, and spanning an almost 20 year period. We found that 52% of the cases that we tested contained Yersinia genes. We also found that only certain strains (biogroups) of Yersinia are present in Crohn’s disease, whereas other strains are not. We are still developing the special in situ hybridization test in an attempt to see the bacteria within the CD tissue.

These data support our original hypothesis that Yersinia, particularly certain strains of Yersinia, play a role in the causation of CD, at least in some patients. Some individuals may be vulnerable to bacterial “triggers” in their environment, which initiate an abnormal immune response, leading to CD.