Evaluation of Avian Mycobacteriosis Diagnostic Assays: A Comparison of Culture, Acid Fast Staining, and Polymerase Chain Reaction Testing
Abstract
Mycobacteriosis is an insidious disease that causes mortality in pet birds and is of zoonotic concern. Mycobacterium avium and more recently, M. genavense have been the causative agents most commonly identified in companion avian species.5,6,8 Avian mycobacteriosis predominately affects the gastrointestinal and hepatic systems. Affected birds shed organisms in their feces, that may remain viable in the environment for months to years. This environmental contamination poses a threat to domestic animals, other birds, and human populations.7
Avian mycobacteriosis is a challenging disease to diagnose antemortem, particularly in the early stages of the infection. Antemortem clinical diagnostic procedures for screening avian patients for this disease include skin testing and hematologic, radiographic, and/or laparoscopic examinations.9 The tuberculin intradermal test has been used successfully for many years for identifying infected fowl and domestic poultry flocks, but this test has been of little use in exotic avian species.1 The disadvantages of hematologic, radiographic, and endoscopic testing are their low sensitivity, their inability to provide a definitive answer and their requirement of restraint and/or anesthesia. Reported laboratory tests for diagnosing mycobacteriosis in humans and animals include serologic tests, acid fast staining, histopathology, mycobacterial cultures, the radiometric BACTEC system, DNA probes, and nucleic acid amplification by polymerase chain reaction (PCR).2,9 The majority of these tests has been applied to birds, but their ability to detect the early stages of disease is poor. PCR has been used successfully in the identification of M. avium and M. genavense in tissues from necropsied birds;2,3 however, obtaining adequate antemortem tissue samples is more difficult. Nucleic acid amplification via PCR and its application for detecting mycobacterial DNA in fecal samples has been minimally investigated in avian species.4 Although some diagnostic methods are considered more reliable than others,2 a direct comparison of antemortem diagnostic assays has not been reported.
The purpose of this study was to compare and contrast the efficiency and reliability of three fecal diagnostic assays throughout the course of experimentally induced mycobacterial infections in Japanese quail (Coturnix coturnix japonica). The three methods utilized were acid fast staining of fecal smears, fecal mycobacterial culture, and PCR evaluation of fecal samples to detect mycobacterial DNA. These methods were chosen based on their non-invasive nature and potential for antemortem diagnosis. The clinical advantage of acid fast staining and PCR testing is that the assay results can be obtained within several days, whereas culture of the organism may take as long as 8 wk.
Fecal samples for this study were obtained from Japanese quail after IV inoculation with 3.9x107 colony forming units of M. avium. The course of the infection was followed for 12 wk. Fecal samples were collected for a 24-h period every other week for the first 6 wk post-inoculation. During weeks 7 through 12 post-inoculation, feces were collected for a 24-h period three times per week. Fecal smears were analyzed utilizing the Ziehl-Neelsen and Truant's acid fast stains. In addition, samples were analyzed via fecal culture and PCR testing.
Acknowledgments
This work was supported by a grant from the Center for Companion Animal Medicine, School of Veterinary Medicine, University of California, Davis.
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