Mycobacterium avium subspecies paratuberculosis(M. paratuberculosis) is a gram-positive, acid-fast bacillus that is the causative agent of Johne's disease, a chronic infection of ruminant animals characterized by inflammation of the digestive tract leading to nutrient malabsorption and eventually death. Dramatic changes in the intestinal environment due to clinical disease are expected to result in alterations to the normal flora, which in turn will significantly impact the health and metabolic potential of the host animal. A molecular phylogentic approach was used to identify changes in the microbiota of cows infected with M. paratuberculosis. DNA was isolated from archived fecal samples taken from cows before and after the observed onset of clinical disease, or from recent fecal samples taken as part of a dairy herd survey. The infection status of animals was determined by fecal culture and PCR. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the organisms present in the fecal DNA preparations. Clustering of the T-RFLP results indicated that regardless of the amount of fecal shedding or the source animal, infected animals grouped together and separately from uninfected animals. The T-RFLP fragment profiles were then used to identify the bacterial genera present in each fecal sample. Actinobacteria (which includes M. paratuberculosis) increased from an average bacterial population component of 6.1% in uninfected animals to 13.5% in infected animals, while Firmicutes decreased from 33.5% to 27.1%. Overall Proteobacteria levels remained relatively stable between infected and uninfected animals at 34.6% and 36.9%, respectively; however, Gammaproteobacteria comprised 12.0% of this group in uninfected animals and 17.4% in infected animals. The implications of these observed changes in the microbiota will be further studied with a sequencing-based approach to establish the functional changes associated with this population shift. Elucidating the impact of host-pathogen interactions on commensal microorganisms has the potential to enhance our understanding of the disease process as well as provide novel approaches for diagnosis and treatment.