Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/31/2003
Publication Date: 5/1/2003
Citation: Motiwala, A.S., Strother, M., Amonsin, A., Byrum, B., Naser, S., Stabel, J.R., Shulaw, W., Bannantine, J.P., Kapur, V., Sreevatsan, S. 2003. Molecular epidemiology of mycobacterium avium subspecies paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis. Journal of Clinical Microbiology. Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in ruminants characterized by diarrhea, reduced feed intake, weight loss, and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd and to other susceptible populations of animals. Little is known about the transmission of this disease between different species of animals. This study was designed to examine the genetic diversity of Mycobacterium paratuberculosis between different species of ruminants and nonruminants (cattle, sheep, goats, deer, mouse, human) and then evaluate regional effects on the diversity within and between animal species. The data presented here show a high degree of similarity within bovine isolates but genetic differences between human and sheep isolates and the bovine isolates. This study indicates that genetic diversity of this bacterium between host species is probable.
Technical Abstract: The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis (MAP) isolated in the United States and identify MAP-specific diagnostic molecular markers to aid in disease detection, prevention and control. A multiplex PCR of 28 IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analysis were used to fingerprint 203 animal and 7 human MAP isolates from diverse geographic localities. A polymorphism in IS1311 was used to differentiate bovine and ovine strains. Six-hundred bacterial cultures including MAP (n = 303), non-MAP mycobacteria (n = 169), and other non-mycobacterial species (n = 128) were analyzed to evaluate MAP-specificity of two IS900 integration loci as diagnostic targets, in a duplex PCR-analyzed. Specificity of the two integration loci was confirmed by analyzing a subset of the isolates for a nucleotide substitution in hsp65 gene and for presence of a MAP-specific sequence (locus 251). Fingerprinting by MPIL clustered 81% of bovine isolates together into a major node, while the human and ovine isolates showed a larger degree of diversity. MPIL analysis also identified that the ovine strains were more closely associated with the bovine subtypes from the same state. AFLP-derived fingerprints showed similar pattern with most bovine isolates clustering into two major nodes while the ovine and human strains clustered on distinct nodes, respectively. Overall, the study identified a high degree of genetic similarity within bovine strains. Evidence for strain sharing among isolates from a variety of hosts and geographic localities was also identified. Additionally, a relatively higher degree of diversity among human and ovine strains indicates that they may have emerged before the bovine specialists.