Mycobacterium liflandii Infection in European Colony of Silurana tropicalis

Mycobacterium liflandii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the first epizootic of mycobacteriosis in a European colony of clawed frogs (Silurana tropicalis), previously imported from a United States biologic supply company. Our findings suggest the emerging potential of this infection through international trade.

Mycobacterium lifl andii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the fi rst epizootic of mycobacteriosis in a European colony of clawed frogs (Silurana tropicalis), previously imported from a United States biologic supply company. Our fi ndings suggest the emerging potential of this infection through international trade.
M any species of nontuberculous mycobacteria inhabit the environment. Mycobacterium fortuitum, M. chelonae, M. marinum, and M. xenopi are some of the mycobacteria that infect amphibians, causing subcutaneous nodules, edema, and chronic wasting (1).
The aquatic, pipid frog, Silurana tropicalis, is an emerging laboratory model for genetic and embryologic/ ontogenetic research. Although smaller than the related Xenopus laevis, S. tropicalis has research advantages: diploidy and brief maturation time make this species ideal for genetic analyses over multiple generations (2).
We investigated an epizootic of M. lifl andii in a colony of African tropical clawed frogs (S. tropicalis) in a European research laboratory. With the rising popularity of this vertebrate laboratory model and the foreseen establishment of stock centers for mutant or transgenic animals, the epizootiology of this emerging disease must be defi ned so that preventive measures may be instituted.

The Study
In November 2004, we began to study an epizootic mycobacteriosis in a colony of imported captive S. tropicalis, the African tropical clawed frog. The Department of Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology of Ghent University, Belgium, had imported S. tropicalis frogs from a supplier in the United States in September 2004. Within 5 weeks, some animals became lethargic with signs similar to those described by Trott et al.: loss of diving refl ex, bloating, and ulcerative skin lesions (3). An average number of 2 deaths each week were reported in a colony of 300 specimens. Preliminary examination of 2 affected animals did not show chytridiomycosis; iridoviral infection; common bacterial infections of liver, lungs and kidneys; chlamydophila infection; or intestinal parasites.
From November 2004 through April 2005, 19 visually affected and visually unaffected specimens of S. tropicalis, 2 tadpoles, and 4 tank water samples were selected for detailed examination for mycobacteria. The frogs were euthanized and dissected, and selected organs and fl uid were removed aseptically (liver, lungs, gallbladder, gastrointestinal tract, spleen, kidneys, fat body, ovary, oviduct, tibia, and coelomic fl uid). Each of the specimens was divided into 2 equal parts, half for histopathologic analysis and half for preparation of decontaminated suspensions for culture and microscopic examination (3,(5)(6)(7). Water samples were concentrated by fi ltration as described by Iivanainen et al. (7), and suspensions were made from the complete tadpoles (6). Further analyses were performed as described for the decontaminated frog suspensions. DNA for genetic analyses was extracted from the suspensions and pure cultures as described previously (6,8). M. lifl andii was identifi ed by IS2404 nested PCR and sequence analysis of 16S rRNA gene (9,10). A combination of 4 genetic typing assays (including 3 previously investigated in M. ulcerans) was used to type M. lifl andii (3,9,11,12). A fl owchart of the performed tests is shown in Figure 1.
All visually affected specimens showed positive results for at least 1 organ and for at least 2 of the following tests: microscopy (Ziehl-Neelsen staining), in vitro cultivation (LJ medium and charcoal medium), IS2404 nested PCR, and histopathologic examination. Of note, all ovarian tissue of the 11 visually affected specimens showed positive results for at least 2 tests. Three of 8 visually unaffected specimens showed positive results for at least 1 organ (including the ovary) and for 1 test. All tadpoles and water samples showed negative results for all tests. Histopathologic evaluation showed many acid-fast bacilli (AFB) in the oviduct lumen ( Figure 2). Numerous AFB were found in the kidney tubules, on the surface epithelium, and in the lumens of the gallbladder, stomach, intestine, and oviduct. Papillary hyperplasia of the gallbladder mucosa was marked, and the lamina propria was expanded by heterophils and many AFB (Figure 2). Lung parenchyma, liver, femur, and tibia were normal and free of AFB.
We identifi ed the causative pathogen as M. lifl andii in all frogs: by growth on charcoal medium, by restriction fragment length polymorphism, or by sequence analysis. Isolate M05-0456 had a similarity value of 100% with M. lifl andii (GenBank accession no. AY845224.1). Growth on charcoal medium can be considered as an additional identifi cation criterion for M. lifl andii because growth on charcoal is better than on LJ medium (3), differentiating M. lifl andii from M. ulcerans. Clinical isolates of M. ulcerans are grown readily on LJ medium but never on charcoal medium. The antibiogram of strain M04-2878 showed resistance to isoniazid, ethambutol, rifampin, clarithromycin, and ethionamide. In each of the genotyping assays, M. lifl andii produced profi les that were distinct from those of M. ulcerans (data not shown). None of the laboratory staff who handled the anurans exhibited any signs of a mycobacterial disease.

Conclusions
The The genetic and phenotypic identifi cation of M. lifl andii as causative agent of the epizootic, the fact that cases of M. lifl andii infection have not been reported in Europe to date, the strikingly similar signs and disease progress (3), and the probability that the frogs were imported from the same supplier (3) all suggest that some members of the imported S. tropicalis colony were infected with M. lifl andii before arrival in Europe. Crowding and stress associated with captivity may have contributed to spread of infection within the colony. How and where the imported frogs became infected remains unknown (3). Additionally, during an extensive study in the Democratic Republic of Congo, Portaels (13) isolated 956 mycobacterial strains from the environment from Buruli ulcer-endemic regions. Among the unknown species, none was characterized as a M. ulcerans-like mycobacterium. To our knowledge, no M. lifl andii infection, in humans or wild anurans, has been reported from Africa. We have confi rmed that all isolates from Buruli ulcer patients and environmental samples analyzed by our laboratory were true M. ulcerans infections and not IS2404 PCR-positive M. ulcerans-like mycobacteria (unpub. data).
The apparently enzootic character of M. lifl andii infection in different S. tropicalis breeding companies in  the United States (3,4) and the exchange of transgenic or mutant S. tropicalis lines between research laboratories, may pose a serious threat for the international research community working with this emerging laboratory model. Diffi culties in detecting the pathogen in visually unaffected specimens and the high infection rate call for urgent efforts in the management of this epizootic disease. Thus far, no preventive measures or treatment for this amphibian mycobacteriosis are known (3,4,14). Resistance to antimycobacterial agents by environmental mycobacteria is not unusual and has been reported previously (15).
We propose examining the oocytes of newly imported frogs as an intervening noninvasive screening method on a regular basis, noting that all affected frogs reported in both intercontinental epizootics were females (3), oocytes from living adult S. tropicalis are easily obtained for research purposes (2), and ovarian tissue was positive for all visually affected specimens and for 1 of 3 positive visually unaffected specimens. However, further studies are needed to determine the role of oocytes in the epizootics of this emerging frog disease, especially in the evaluation of our proposed screening method. To prevent the infection of existing stocks with wild-caught frogs of unknown origin, we further recommend the importation of only certifi ed pathogen-free laboratory-bred specimens from recognized biologic suppliers. Recently, Tarigo et al. reported a frog mycobacteriosis in an adult female, albino South African clawed frog (X. laevis) in a research colony at North Carolina State University (14). The etiologic agent was identifi ed as M. marinum complex on the basis of mycobacterial culture, but genetic analyses were not performed to exclude M. lifl andii infection. To avoid further spread of this disease, every new outbreak of M. lifl andii infection in pipid frogs or other anuran species should be reported to relevant authorities and research communities. Until more is known about this epizootic and its prevention and treatment, caution must be exercised in transportation, husbandry, and human contact with these animals (zoonotic potential). We do not know at this stage whether the importation of frogs contaminated by M. lifl andii represents a danger for wild or autochthonous frogs. Further investigation is required to establish this.