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 .
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 , lyons , france ) therapy was given for 7 days .
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 ) .
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 . obtaining a tracheal washing of the siberian tiger by bronchoscopy .
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 ( gen - probe 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 .
their skin test results were negative , and clinical or radiologic signs of tuberculosis were not detected .
colony morphology of the isolated mtbc strain showed dysgonic growth on lj medium and microaerophilic growth on lebek medium .
the strain was susceptible to pza ( 100 g / ml ) and thiophen-2-carboxylic acid hydrazide ( tch ; 1 g / ml ) and negative for niacin accumulation and nitrate reduction ( 911 ) . 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 ( 5,1216 ) .
susceptibility of the isolate to pza was linked with a wild - type pnca sequence .
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 3943 and 316 ( figure 2 ) , and t to g mutation at position 1,311 in the gyrb gene , characteristic of m. bovis subsp .
on the basis of these phenotypic and genetic characteristics , the strain was identified as m. bovis subsp .
spoligotype patterns of the isolate obtained from the siberian tiger : lane 1 ; spacer sequences 316 and 3943 are absent ) ; lane 2 , control strain mycobacterium tuberculosis h37rv atcc 27294 ; lane 3 , control strain m. bovis atcc 19210 ; lane 4 , m. bovis bcg atcc 27289 .
colony morphology of the isolated mtbc strain showed dysgonic growth on lj medium and microaerophilic growth on lebek medium .
the strain was susceptible to pza ( 100 g / ml ) and thiophen-2-carboxylic acid hydrazide ( tch ; 1 g / ml ) and negative for niacin accumulation and nitrate reduction ( 911 ) . 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 ( 5,1216 ) .
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 3943 and 316 ( figure 2 ) , and t to g mutation at position 1,311 in the gyrb gene , characteristic of m. bovis subsp .
on the basis of these phenotypic and genetic characteristics , the strain was identified as m. bovis subsp .
spoligotype patterns of the isolate obtained from the siberian tiger : lane 1 ; spacer sequences 316 and 3943 are absent ) ; lane 2 , control strain mycobacterium tuberculosis h37rv atcc 27294 ; lane 3 , control strain m. bovis atcc 19210 ; lane 4 , m. bovis bcg atcc 27289 .
mtbc comprises these closely related organisms : m. tuberculosis ; m. africanum ; m. bovis ; the vaccine strain , m. bovis bacillus calmette - gurin ; and three rarely seen members , m. microti , m. canettii , and the recently described seal bacillus , m. pinnipedii ( 1720 ) .
differentiation within mtbc is necessary for individual patient treatment ( i.e. , inclusion or exclusion of pza ) and for epidemiologic purposes , especially in areas of the world where tuberculosis has reached epidemic proportions or wherever the transmission of m. bovis between animals , animal products , and humans is a problem ( 10 ) .
the host range of m. bovis is wide , including many animal species and humans .
carnivores such as large felines may acquire the infection through the alimentary tract by eating infected meat ( 4 ) .
reports of tuberculosis in large captive or free - living felines are not common ( 2126 ) , however .
to our knowledge , this case is the first in which tuberculosis attributable to m. bovis subsp .
the rapid and accurate in vivo diagnosis of tuberculosis is indispensable in endangered captive animals such as the siberian tiger , not only because of the declining population of this species but also to prevent the transmission of the disease to other animals . although nasal or throat swabs are used most often , we found tracheal washing by bronchoscopy was easy to perform , rapid , and more adequate than swabs ( provided a larger sample volume from the lower airways ) for obtaining clinical specimen for mycobacterial or other microbiologic tests .
the rapid diagnosis of tuberculosis is essential for adequate antituberculosis treatment to be started as early as possible .
however , when an endangered animal is involved , early diagnosis of the disease might help control it in time to save the animal , especially with the help of a rapid in vivo diagnostic method such as tracheal washing through bronchoscopy .
tracheal washing can also be the method of choice to bacteriologically monitor the efficacy of therapy . in this case , the poor appetite and condition of the animal did not allow survival long enough for delivery of antituberculosis treatment .
the source of infection could not be conclusively identified retrospectively ; infected goat meat ( a usual diet of the animal ) is a likely possibility because the tuberculosis - related control measures are not as strict with goats as with cattle in hungary ( annual tuberculin skin testing of goats is not mandatory , for example ) ( 28 ) .
this report indicates that routine differentiation within the mtbc is indispensable for understanding the epidemiology of tuberculosis and for determining the prevalence , transmission , and clinical importance of the different members of the complex .
 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 . 
Case Report
Characterization of MTBC Isolate
Conclusions