Scientific Abstract

Proposal No. IBD-0262R
Principal Investigator:  Charles N. Bernstein, M.D.
Applicant Organization:  University of Manitoba (Winnipeg, Canada)
Project Title:  A population-based characterization of potential microbial etiologies of inflammatory bowel disease using geographically defined high and low rate prevalence/incidence areas in Manitoba
Period of Award:  April 1, 2009 – March 31, 2011

In 1995, we established the University of Manitoba IBD Epidemiology Database, the largest validated population-based IBD database in North America. We developed a validated, sensitive and specific administrative definition of IBD applicable to the Manitoba Health administrative health database to identify (anonymously) Manitobans with IBD. Manitoba has amongst the highest rates of IBD reported worldwide. Our aim is to analyze the microbial composition of potable water in areas of high as well as low incidence of IBD. Microorganisms present in high incidence areas but absent or less frequent in low incidence areas could be implicated as etiologic in CD or UC, whereas microbes widely present in low incidence areas but absent in higher incidence areas might be considered important in preventing the development of IBD. We are broadly pursuing an unbiased assessment of the microbial ecology of potable water in high and low incidence areas. Our study was designed to look for promising ‘leads’ rather than searching only for specific pathogens. However, momentum in IBD research increasingly suggests two candidate microbes - Mycobacterium avium subspecies paratuberculosis (MAP) and adherent/invasive E coli (AIEC).  Thus, we are now including a focus on these putative etiologic agents in this proposal. Our study program has 5 phases.

Phase 1: Defining high and low incidence areas of IBD-COMPLETED. We have mapped the city of Winnipeg (Manitoba’s main population hub) and the entire province by incidence rates for both CD and UC, identifying high and low incidence areas. The areas of high or low incidence of CD correlate highly with those of UC. We hope to harness this unique epidemiological data to pursue an etiological study of IBD, particularly the potential environmental (microbiological) component.

Phase 2: Overlapping the water distribution map of Winnipeg and other areas of the province onto the map of IBD incidence rates-COMPLETED. Working with the City of Winnipeg Waterworks Dept. we identified the supply and distribution pattern for the entire city. From a distribution map and with the cooperation of City officials we identified peripheral sample collection points across the City. Similarly, we planned water collection in 2 rural communities (1 of high and 1 of low IBD incidence).

Phase 3: Water sampling-COMPLETED, though some additional sampling might be desirable to follow up on microbiological leads uncovered, to confirm data, or to add additional sampling points in areas of interest. We collected water samples from Shoal Lake, and each water reservoir and a number of peripheral sites in the distribution system. Water (as particulates trapped on a filter) and biofilm samples (from installed pipe sections) were collected from most peripheral sites at 3 month intervals over one year. In general, fire halls or community & day care centres were used.

Phase 4: Preparing water samples for microbial analysis & cataloging of these samples-COMPLETED.Water samples, pipe sections and filters were shipped from Manitoba to Ottawa. The objective for this phase was to develop a library of cultured isolates and a clone library. Many of the identifications completed to date are for common environmental bacteria that might be expected in soil and water samples. This includes species with obvious pathogenic potential. We also undertook: DNA isolation and quantification; PCR amplification for DGGE and for cloning; cloning, plasmid isolation and plasmid insert checking; blast identifications. Microscopic examination of the sample eluates was performed for preliminary assessment of the presence eukaryotic life forms; culture for protozoa showed them at many Winnipeg sites except none were found in the area of low incidence.

Phase 5: Microbial analysis - the main thrust of this grant. We will pursue completing the complex analysis of our water samples (cultures and DNA that are stored). We will determine if novel microbes appear in Manitoba drinking water, and any phylogenetic differences between high incidence and low incidence areas. We will also determine if the AIEC already identified in human tissue from IBD cases in our previous studies, has drinking water as a potential source for Manitobans. If any samples are found to containAIEC the intent is to confirm whether the virulence factors found in the biopsy specimen isolates are also found in the water. Similarly, we will search for MAP in drinking water samples and determine if there are distribution variations of MAP in Manitoba’s drinking water. We will determine if water in Manitoba provides discrepant microbial ecologies between high & low incidence areas of IBD.