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.
This work was supported by a grant from the Center for Food Animal Health, School of Veterinary Medicine, University of California, Davis.
1. Angus RD. Tuberculins for use in animals. In: Mycobacterial Infections of Zoo Animals. Washington, DC: Smithsonian Institution Press; 1978:109–114.
2. Aranaz A, Liebana E, MateosA, Dominguez L. Laboratory diagnosis of avian mycobacteriosis. In: Seminars in Avian and Exotic Pet Medicine. Philadelphia, PA: WB Saunders Co.; 1997:9–17.
3. Boian M, Avaniss-Aghajani E, Walker R, Aronson T, Tran T, Glover N, et al. Identification of Mycobacterium genavense in intestinal tissue from a parakeet using two polymerase chain reaction methods: Are pets a reservoir of infection in AIDS patients? Aids. 1997;11:255–256.
4. Devallois A, Picardeau M, Goh KS. Comparative evaluation of PCR and commercial DNA probes for detection and identification to species level of Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol. 1996;34:2756–2759.
5. Eriks IS, Munch KT, Besser TE, Cantor GH, Kapur V. Rapid differentiation of Mycobacterium avium and M. paratuberculosis by PCR and restriction enzyme analysis. J Clin Microbiol. 1996;34:734–737.
6. Folgueira L, Delgado R, Palenque E, Noriega AR. Detection of Mycobacterium tuberculosis DNA in clinical samples by using simple lysis method and polymerase chain reaction. J Clin Microbiol. 1993;31:1019–1021.
7. Ford S, Eriks I. Improved detection method for Mycobacterium avium infection in birds. In: Proceedings of the Association of Avian Veterinarians Annual Conference. 1997:383–385.
8. Hoop RK, Botgger EC, Ossent P, Salfinger M. Mycobacteriosis due to Mycobacterium genavense in six pet birds. J Clin Microbiol. 1993;31:990–993.
9. Hoop RK, Bottger EC, Pfyffer GE. Etiological agents of mycobacteriosis in pet birds between 1986 and 1995. J Clin Microbiol. 1996;334:991–992.
10. Kocagoz T, Yilmaz E, Ozkara S, Kocagoz S, Hayran M, Sachedeva M, Chambers HF. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using simplified procedure. J Clin Microbiol. 1993;31:1435–1438.
11. Li Z, Bai GH, Fordman von Reyn C, Marino P, Brennan MJ, Gine N, Morris SL. Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR. J Clin Microbiol. 1996;34:1903–1907.
12. Montali RJ, Bush M, Thoen C, Smith E. Tuberculosis in captive exotic birds. J Am Vet Med Assoc. 1976;169:920–927.
13. Van DerHeyden N. Clinical manifestations of mycobacteriosis in pet birds. In: Seminars in Avian and Exotic Pet Medicine. Philadelphia, PA: WB Saunders Co.; 1997:18–24.
14. Zhang ZQ, Ishaque M. Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria. Int J Lepr Other Mycobact Dis. 1997;65:469–476.