Isolation of a Mycobacterium microti-like organism from a rock hyrax (Procavia capensis) in a Canadian zoo

Abstract

A Mycobacterium tuberculosis complex organism was isolated from a zoo resident rock hyrax (Procavia capensis) imported into Canada from South Africa. The strain was identified biochemically as Mycobacterium microti. The spoligotype pattern obtained for this isolate was found to be rare. This represents the first report of isolation and spoligotyping of M. microti in North America.

A female rock hyrax (Procavia capensis) was captured in the wild and imported directly into Canada from South Africa in December 1997. The hyrax was held in quarantine for 60 d before being housed in an indoor exhibit, isolated from other zoo and indigenous wild animals. The animal (> 5 y) was euthanized following a progressive, chronic lameness that led to hind leg paralysis, approximately 30 mo after importation. Postmortem examination revealed granular pale lesions on the peritoneum, with similar nodular lesions in other organs. No lesions were found on necropsy related to the lameness, but there was some suggestion of thinning of a cranial thoracic intervertebral space on radiographs taken prior to euthanasia. The spine was not closely examined on necropsy. Large cauliflower-like pale nodules were found in the lungs. Companions were depopulated upon confirmation of the finding of tuberculosis. No similar lesions were observed on necropsy of the remaining 3 hyraxes in this colony. The exhibit in which these animals were housed was cleaned and disinfected and subsequently repopulated with birds and reptiles. There was no direct contact with other mammals, and zoo staff were monitored routinely for potential transmission. There were no reports of TB in any offspring.

Tissue from the pulmonary lesions was submitted to the Mycobacterial Diseases Centre of Expertise at the Canadian Food Inspection Agency, as a suspected tuberculosis case. Histopathological examination of the tissue revealed typical granulomatous lesions without mineralization and giant cells with large numbers of acid-fast bacilli resembling those of mycobacterial species. Cultures prepared from frozen pulmonary tissue yielded moderate growth on Lowenstein Jensen medium after 6 wk incubation, which subsequently stained acid-fast. Application of the polymerase chain reaction, using primers targeting the insertion element IS6110 (1), confirmed the identification of the colonies as Mycobacterium tuberculosis complex (MTBC) organisms. The results of biochemical testing suggested that the organism was phenotypically consistent with Mycobacterium microti.

Strain typing by the spoligotyping method (2) showed a pattern distinctly different from that of M. bovis types commonly found in Canada (Figure 1). Reactions were evident within the group of oligonucleotides 39–43. This group is unreactive for isolates of M. bovis. Conversion of the spoligotype pattern to octal code was done in accordance with the method described by the National Tuberculosis Genotyping and Surveillance Network (3). The code obtained for this isolate was 772777400001671. Examination of the data base of spoligotypes (4) indicated that this type was rare, with only 2 isolates having been reported in Great Britain. However, the data base identified these isolates as MTBC organisms and not M. microti specifically. This is the first report of M. microti isolation and identification of this spoligotype in North America.

Figure 1.

Spoligotype patterns for M. microti and recent Canadian isolates of M. bovis. Row A, rock hyrax isolate of M. microti; row B, M. bovis isolate from Ontario in 2002; rows C and D, M. bovis isolates from Manitoba in 2003. Numbers at the top indicate oligonucleotide position numbers. Note that the last 5 oligo-nucleotides do not react in rows B-D, characteristic for M. bovis (bracket).

Isolations of M. microti from mammals, such as the rock hyrax, or “dassie,” from the Cape Province of South Africa, have been documented previously in 1958 (5) and more recently in 1994 in a zoo collection in Perth, Australia (6). The Australian case and this one confirmed in Canada have some common features. In both instances, the animals originated from South Africa and were captured from the wild. The animals imported to form the colony at the Perth zoo were held in quarantine in the UK for a period of months prior to arrival in Australia, while the hyraxes intended for Canada were imported directly from South Africa. Application of spoligotyping methodology to the 2 isolates obtained from the hyraxes in Australia generated the same spoligotype profile (octal code 772777400001671) as was obtained in this case. An epidemiological link is suggested by the finding of identical spoligotypes for these isolates. Further, earlier typing had indicated that the “Australian” hyrax isolates were related to the original “dassie” isolates of the 1950s (6). These findings strongly support the hypothesis that a reservoir for this clone exists in South African rock hyraxes.

Comparison of published spoligotype patterns for M. microti isolates (4,7,8) generates 3 separate strain types: a “vole” type, displaying reactions at positions 37 and 38 with variable reaction at oligonucleotide number 4; a “llama” type, with reactions at positions 4–7, 23, 24, 26 (variable), 37, and 38 (variable); and the “dassie/rock hyrax” type shown here (Figure 1) and previously (8). However, the latter isolate, designated 68/7171, did not show hybridization signals with oligonucleotides 15 and 16 (8), while that of the present case does. Geographically, M. microti isolations have been made in The Netherlands, Belgium, the United Kingdom, and France (vole and llama types). The dassie/rock hyrax type has been isolated in South Africa and in zoo animals in the United Kingdom, Australia, and now in Canada.

Mycobacterium microti is generally thought to be a disease agent of small mammals, but infrequently isolations have been reported from other species, such as pigs, cats, llamas, dogs, and humans (7–10). Given the potential for infection of species other than the conventional host, veterinarians and laboratory diagnosticians should not rule out the possibility of M. microti infection in zoo species or their close contacts.

Importation of zoo animals, such as rock hyraxes, presents problems for tuberculosis control programs, since there are no validated tests for unusual species. The conventional tuberculin skin tests are commonly applied is such circumstances as the default option; however, these tests have not been validated in rock hyrax. Additionally, such tests may not be practical in these animals and quarantine prior to import may not provide adequate protection against importation of the disease, as evidenced by the Perth zoo experience (6). Blood tests targeting a specific gamma interferon response to tuberculin or other antigens, as has been done for badgers (11); lymphocyte stimulation tests (12); or fluorescence polarization assays (13) could be evaluated for utility in this context. The problem of finding sufficient numbers of diseased and nondiseased animals to properly validate the sensitivity and specificity of such tests remains an issue. It may be that only by the continual global accumulation of test data will diagnosticians gain confidence in the use of such tests for the diagnosis of tuberculosis in unusual species.