Abstract
Mycobacteriosis is a bacterial disease that affects many species of birds. This disease predominately affects the gastrointestinal and hepatic systems, although respiratory and skeletal involvement may occur.12 Mycobacterium avium, M. tuberculosis, and more recently M. genavense are the species most commonly isolated from birds.8,9 Ante-mortem clinical diagnostic procedures used for screening avian patients include hematologic, biochemical, and serologic testing.13 In addition, radiographic studies, endoscopic examinations and acid-fast staining of feces and tissue biopsies may be utilized. The disadvantages of these tests are their low specificity, sensitivity, and general inability to provide a definitive etiologic diagnosis. The tuberculin intradermal skin test has been used successfully for identifying infected fowl and domestic poultry flocks, however, this test has been of little use for exotic avian species.1 Mycobacterial cultures and the BACTEC AFB system (Becton Dickinson Diagnostic Instruments, Sparks, MD, USA) are more definitive ways of diagnosing mycobacteriosis.2 The disadvantages to these techniques are that a limited number of laboratories have the capabilities for culturing these organisms, it takes an extended period of time to get a positive diagnosis, and there is a potential for false negatives (the organism may fail to grow).
The newest development in diagnosing mycobacterial infections involves nucleic acid amplification and identification via polymerase chain reaction (PCR). Preliminary work (six cases) has shown PCR to be successful in the identification of M. avium and M. genavense in tissues from necropsied birds.2,3 Mycobacterial deoxyribonucleic acid (DNA) amplification and PCR identification have been described for direct detection in human clinical specimens (sputum and feces),4,5,10,11 however, ante-mortem molecular diagnostic testing for birds has been limited.7
In order to develop a sensitive PCR assay for identifying mycobacterial organisms in tissue or fecal samples from birds, it is imperative that an optimal DNA extraction technique be identified. The challenge with mycobacteria is the high concentration of lipid within their cell wall, therefore, traditional DNA extraction procedures do not work or are less than optimal. Reported methods for extracting mycobacterial DNA include boiling,10 sonication,6 heat shock,14 and intensive enzymatic lysis6,10. To date, only one study has addressed differences between extraction techniques utilizing M. lepare, M. lepraemurium, and M. bovis.14 No work has been done to determine which DNA extraction technique is optimal for Mycobacterium avium or M. genavense.
The goals of this study were to compare and contrast four different DNA extraction methods (mechanical disruption, boiling, heat shock, enzymatic lysis) utilizing M. avium and M. genavense derived from synthetic media. M. fortuitum (a fast-growing ubiquitous mycobacterial species) was also utilized for initial development of the extraction techniques.
Acknowledgments
This work was supported by a grant from the Center for Food Animal Health, School of Veterinary Medicine, University of California, Davis.
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