Pulmonary tuberculosis due to Mycobacterium bovis subsp. caprae in captive Siberian tiger - Dispatches

Emerging Infectious Diseases, Nov, 2003 by Akos Lantos, Stefan Niemann, Laszlo Mezosi, Endre Sos, Karoly Erdelyi, Sandor David, Linda M. Parsons, Tanja Kubica, Sabine Rusch-Gerdes, Akos Somoskovi

We report the first case of pulmonary tuberculosis caused by Mycobacterium bovis subsp. caprae in a captive Siberian tiger, an endangered feline. The pathogen was isolated from a tracheal aspirate obtained by bronchoscopy. This procedure provided a reliable in vivo diagnostic method in conjunction with conventional and molecular tests for the detection of mycobacteria.

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Mycobacterium bovis, a member of the M tuberculosis complex (MTBC), can cause tuberculosis in a wide range of domestic and wild animals and also in humans (1,2). Routine differentiation of M. bovis is based on a number of phenotypic characteristics and biochemical tests (2). M. bovis shows dysgonic growth on Lowenstein-Jensen (LJ) medium and has been described as negative for nitrate reduction and niacin accumulation (2). As a further criterion for the differentiation of M. bovis, intrinsic resistance to pyrazinamide (PZA) has been described (2). However, more recently, PZA-susceptible strains of M. bovis were found in Spain and Germany; these strains were also characterized by specific molecular techniques (3-5). As a consequence, M. bovis was split into two subspecies: M. bovis subsp. bovis, which showed resistance to PZA, and M. bovis subsp. caprae, which was sensitive to PZA (6,7). M. bovis subsp. caprae was initially isolated from sheep and goats in Spain (3,4,7); however, further studies confirm its infectivity in humans, cattle, and red deer (6,8). We report the unusual case of a M. bovis subsp. caprae infection in a captive Siberian tiger.

Case Report

An 8-year-old male Siberian tiger at the Budapest Zoological and Botanical Garden had episodes of coughing in October 2001. Because the coughing did not stop in 6 to 7 days, an expectorant (Bisolvon; Boehringer Ingelheim Vetmed Gmbh., Ingelheim am Rhein, Germany) was given for 10 days. His condition showed a temporary improvement; however, after a few weeks, the animal started coughing again, and his appetite decreased. Amoxicillin plus clavulanic acid (Amoksiklav; Lek Animal Health, Ljubljana, Slovenia) and ketoprophen (Ketofen, Merial, Lyon, France) therapy was given for 7 days. The tiger's condition did not show any notable improvement. In addition, in May 2002, the animal's respiratory rate became elevated, he became dyspneic and emaciated, and his daily activity substantially decreased. Further antibacterial treatment was administered (cefatroxil, Cefa-cure; Intervet, Boxmeer, the Netherlands) during that month without clinical effect. At that point, the animal was anesthetized, and tracheoscopy was performed with a flexible 56-cm bronchoscope (Olympus B3R; Tokyo, Japan (Figure 1). The examination found a large amount of purulent mucus in the trachea. Therefore, several tracheal washings were taken for microbiologic tests by using a commercially available tracheal suction set (Medinorm Medizintechnik GmbH, Quierschied, Germany (Figure 1). A chest radiograph showed a severe and extensive bronchointerstitial pattern with cavernous lesions in both lungs.

[FIGURE 1 OMITTED]

Nine days after the specimens were taken, cultures for mycobacteria showed growth in the broth-based MGIT 960 system (Becton-Dickinson Microbiology Systems, Sparks, MD). The acid-fast organism that was isolated was identified as MTBC by the AccuProbe TB assay (GenProbe Inc., San Diego, CA).

Since the tiger had stopped eating and his condition had dramatically deteriorated, the animal was euthanized and necropsy was performed. Hematoxylin and eosin--stained histologic sections of the lung segments showed an extensive multifocal infiltration of lymphocytes, histiocytes, and some scattered multinuclear giant cells within the framework of proliferated connective tissue and collagen fibers of the cavernous lesions. Ziehl-Neelsen staining showed an intracellular accumulation of acid-fast bacteria in several alveolar macrophages and epithelioid cells.

The keepers of the tiger also underwent pulmonary radiographs and tuberculin skin testing. Their skin test results were negative, and clinical or radiologic signs of tuberculosis were not detected.

Characterization of MTBC Isolate

Colony morphology of the isolated MTBC strain showed dysgonic growth on LJ medium and microacrophilic growth on Lebek medium. The strain was susceptible to PZA (100 [micro]g/mL) and thiophen-2-carboxylic acid hydrazide (TCH; 1 [micro]g/mL) and negative for niacin accumulation and nitrate reduction (9-11).

The genome of the isolate was analyzed for specific mutations in the pncA, oxyR, and gyrB genes by automated DNA sequencing, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) technique, and spoligotyping as described previously (5,12-16). Susceptibility of the isolate to PZA was linked with a wild-type pncA sequence. In addition, the isolate contained the M. bovis--specific G-to-A mutation at position 285 in the oxyR gene and the G-to-A mutation at position 756 in the gyrB gene. However, spoligotyping showed a pattern with the absence of spacer sequences 39-43 and 3-16 (Figure 2), and T to G mutation at position 1,311 in the gyrB gene, characteristic of M. bovis subsp. caprae, could also be detected by DNA sequencing. On the basis of these phenotypic and genetic characteristics, the strain was identified as M. bovis subsp. caprae (3,6,7,17).