Masters Theses

Date of Award

8-2007

Degree Type

Thesis

Degree Name

Master of Science

Major

Wildlife and Fisheries Science

Major Professor

C. A. Speer, Graham Hickling

Committee Members

Shigetoshi Eda, Kelly Robbins

Abstract

Crohn's disease is a chronic inflammatory bowel disease of unknown origin that continues to affect millions of people worldwide. Mycobacterium avium subsp. paratuberculosis (MAP) has been a suspected etiological agent of Crohn’s disease for nearly two decades. Recently, our laboratory was able to achieve a test sensitivity of 95.2% (n=21) by using a flow cytometry method (FCM) to detect anti-MAP antibodies in Johne’s diseased cattle. Here we investigate whether FCM can differentiate Crohn’s patients’ antibody titers from healthy human control antibody levels. As a second research goal, we investigated other suspected bacteria and serum-differentiating yeast using the FCM. In brief, bacteria/yeast were incubated with serum samples and washed to remove nonspecific antibodies. Antibodies bound to the surface of bacteria/yeast were then labeled with fluorescently-tagged secondary antibody and this binding evaluated by flow cytometry. Serum samples from 37 Crohn’s patients and 37 healthy human controls were tested for antibody (IgG and IgA) binding to bacteria/yeast. When targeting antibodies directed towards MAP, no significant difference was observed between the two populations (ANOVA, alpha=0.05). FCM was also unable to detect an antibody response for either group when directed against a cell-wall-deficient form of MAP, which has been reported to be near infected tissues of Crohn’s diseased patients. However, a differential antibody response to two yeasts that included S. cerevisiae and C. albicans was detected when looking for both IgG and IgA. This differentiation using yeast agrees with previous studies that have shown Crohn’s diseased subjects to have significantly higher antibody titers to this yeast than do healthy controls. This suggests that the flow cytometry method could be useful in the future for distinguishing Crohn’s subjects from healthy controls.

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