Detection and Differentiation of <i>Mycobacterium Avium</i> and <i>Mycobacterium Genavense</i> by Polymerase Chain Reaction and Restriction Enzyme Digestion Analysis

Abstract

Mycobacteria are acid-fast, facultative intracellular bacilli that are highly resistant to environmental conditions and can survive in soil for months.4,14 Mycobacterium avium causes a slow spreading, granulomatous infection seen predominantly in birds and in humans with AIDS.14 Once organisms are ingested, they disseminate to the intestine and internal organs, resulting in chronic weight loss, diarrhea, emaciation, and eventually death. Tubercles developing at the site of infection in the intestinal wall allow for constant shedding into the environment.4 Recently a new species of mycobacteria, Mycobacterium genavense, has been identified and shown to cause infection in birds, in immunocompromised humans, and in other animals including dogs.2,6,8,9,11 In Switzerland, an epidemiologic study undertaken between 1986 and 1995 identified M. genavense as the most common etiologic agents of mycobacteriosis in birds.7 In both birds and humans, M. avium and M. genavense result in nearly identical clinical signs following infection and have primarily been differentiated through sequencing of the 16S ribosomal RNA (rRNA) gene.2,8,10 Moreover, dual infections have been reported.10 Currently, there are many limits to the detection of M. avium and M. genavense infection. Culture may take up to 4 weeks and requires the use of special media because mycobacteria are slow growing and fastidious, making this method inefficient and expensive.4,5 Primary growth of M. genavense on conventional solid mycobacterial medium has not been demonstrated,2,8 which creates a serious problem for detection and diagnosis of M. genavense infection, particularly for veterinary diagnostic laboratories. Because of the potential biological hazards of liquid culture, standard mycobacterial protocols for most veterinary diagnostic laboratories are typically limited to solid media, such as Middlebrook or Lowenstein-Jensen. It is therefore possible that many M. genavense infections go undiagnosed in animals. Techniques that utilize the polymerase chain reaction (PCR) have recently been developed for the detection and differentiation of mycobacterial species. Several published PCR protocols rely on sequencing of amplicons from various genes, including rRNA genes.1,12,15,16 However, the need for sequencing following amplification limits the usefulness of these protocols as rapid diagnostic assays for species differ-