The complete genome sequence of <em>Mycobacterium avium</em> subspecies <em>paratuberculosis</em>

Lingling Li

John Bannantine

Qing Zhang

Alongkorn Amonsin

^{Department of Microbiology,}^{Biomedical Genomics Center, and}^{Department of Medicine, University of Minnesota, St. Paul, MN 55108; and}^{National Animal Disease Center, U.S. Department of Agriculture–Agriculture Research Service, Ames, IA 50010}

^{To whom correspondence should be addressed. E-mail:}ude.nmu@rupakv.

^{L.L., J.P.B., Q.Z., A.A., S.K., and V.K. have a financial conflict of interest that results from a patent application that has been filed on some DNA sequences that are disclosed and discussed in the manuscript. The patent applications that have been filed are jointly owned by the University of Minnesota and the U.S. Department of Agriculture. As named inventors, these authors may potentially financially benefit from the commercialization of the technology. In addition, some of the technology disclosed in this paper has also been licensed to ANDX, Inc., a University of Minnesota based startup company focusing on the development of molecular diagnostic assays, in which S.K. and V.K. have financial interests and are cofounders.}

Communicated by Harley W. Moon, Iowa State University, Ames, IA, July 13, 2005

Received 2005 Mar 18

Abstract

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map; and Map-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

Keywords: genomics, Johne's disease, molecular diagnostics

Abstract

Mycobacterium avium subspecies paratuberculosis (Map) is an extremely slow-growing, acid-fast, mycobactin-dependent multispecies pathogen. Infection with this bacterium leads to a chronic granulomatous enteritis in cattle and other wild and domestic ruminants, termed Johne's disease (1). Clinical signs of Johne's disease include diarrhea, weight loss, decreased milk production, and mortality. A recent study estimated that 21% of United States dairy herds are infected, resulting in considerable economic losses to the dairy industry totaling more than $200 million per annum (2, 3). A major concern with Johne's disease is the ease with which the bacterium is spread. Subclinically or clinically infected animals shed Map in feces and milk, enabling dissemination to susceptible calves, the environment, and in retail milk (4–6). Map-containing milk may be of particular concern because the bacterium has been suggested as a possible cause of Crohn's disease in humans (7).

The detection and diagnosis of Map-infected animals poses great difficulties. Map culture can require up to 16 weeks or more, and serological tests lack sensitivity because of the seroconversion occurring relatively late during the course of the disease (8). Also, previously developed PCR-based approaches (e.g., IS900) have been shown to lack specificity (9). This result is, in part, due to the high levels of genetic similarity of Map with other mycobacteria, in particular, Mycobacterium avium (Mav) (10). Previous studies from our laboratories and elsewhere show >95% nucleotide sequence similarity between many strains of Map and Mav (11–13). Therefore, it is widely recognized that the development of rapid, sensitive, and specific assays to identify infected animals is essential to the formulation of rational strategies to control the spread of Map.

As a first step toward elucidating the molecular basis of Map's physiology and virulence, and providing a foundation for the development of the next generation of Map diagnostic tests and vaccines, we report the complete genome sequence of a common clone of Map, strain K-10.

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Acknowledgments

Funding for this project was provided, in part, by grants from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service National Research Initiative competitive grants program as well as the Agricultural Research Service (to V.K. and J.P.B.).

Acknowledgments

Notes

Author contributions: L.L., J.P.B., and V.K. designed research; L.L., J.P.B., Q.Z., A.A., B.J.M., D.A., N.B., and S.K. performed research; Q.Z. and A.A. contributed new reagents/analytic tools; L.L., J.P.B., Q.Z., A.A., B.J.M., S.K., and V.K. analyzed data; and L.L., B.J.M., S.K., and V.K. wrote the paper.

Abbreviations: Map, Mycobacterium avium subspecies paratuberculosis; Mav, Mycobacterium avium; Mtb, Mycobacterium tuberculosis.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AE016958).

Notes

Abbreviations: Map, Mycobacterium avium subspecies paratuberculosis; Mav, Mycobacterium avium; Mtb, Mycobacterium tuberculosis.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AE016958).

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