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Journal of Clinical Microbiology, September 2005, p. 4640-4648, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4640-4648.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

Jacques Godfroid ,1,{dagger},{ddagger} Cathy Delcorps,2,{dagger} Leonid M. Irenge,3 Karl Walravens,1 Sylvie Marché,1 and Jean-Luc Gala2,3*

Veterinary & Agrochemical Research Center, Brussels, Belgium,1 Laboratory of Applied Molecular Technology, Université Catholique de Louvain, Brussels, Belgium,2 Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium3

Received 26 November 2004/ Returned for modification 27 May 2005/ Accepted 16 June 2005

Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.

* Corresponding author. Mailing address: Applied Molecular Technology, BP 30.46, Clos Chapelle-aux-Champs, 30, B-1200 Brussels, Belgium. Phone: 32 2 764 31 65. Fax: 32 2 764 31 66. E-mail: gala{at}lbcm.ucl.ac.be.

{dagger} These authors contributed equally to this work.

{ddagger} Present address: University of Pretoria, Faculty of Veterinary Science, Department of Veterinary Tropical Diseases, Private Bag X04, Onderstepoort 0110, South Africa.

Journal of Clinical Microbiology, September 2005, p. 4640-4648, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4640-4648.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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