Mycobacterium lifl andii Infection in European Colony of Silurana tropicalis
Patrick Suykerbuyk,* Kris Vleminckx,†‡
Frank Pasmans,‡ Pieter Stragier,* Anthony Ablordey,*§ Hong Thi Tran,†‡ Katleen Hermans,‡ Michelle Fleetwood,¶ Wayne M. Meyers,¶
and Françoi Portaels*
Mycobacterium lifl andii caus a fatal frog dia 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 fi ndings sug-gest the emerging potential of this infection through interna-tional trade.
支付的英文
M any species of nontuberculous mycobacteria inhabit the environment. Mycobacterium fortuitum, M. che-lonae, M. marinum, and M. xenopi are some of the my-cobacteria 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 rearch. Although smaller than the related Xenopus laevis, S. tropicalis has
rearch advantages: dip-loidy and brief maturation time make this species ideal for genetic analys over multiple generations (2).
In 2004, Trott et al. characterized a new mycobacterial pathogen in pipid frog colonies (3), now named M. lifl andii (4). On Middlebrook 7H11 agar supplemented with oleic acid, albumin, dextro, and catala, M. lifl andii form rough, nonpigmented, slightly buff-colored colonies. Vis-ible colonies develop after 30 to 35 days at 28°C on Löw-enstein-Jenn (LJ) medium (3). This M. ulcerans–like mycobacterium produces a plasmid-encoded toxin, myco-lactone E, which is less cytopathogenic than mycolactone A/B, produced by African M. ulcerans (4). M. lifl andii in-fection in frogs manifests as cutaneous lesions, coelomitis, and bloating, with a high death rate (3).
科研兴校We investigated an epizootic of M. lifl andii in a colony of African tropical clawed frogs (S. tropicalis) in a Euro-pean rearch laboratory. With the rising popularity of this vertebrate laboratory model and the foreen establishment of stock centers for mutant or transgenic animals, the epi-zootiology of this emerging dia 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. tropi-calis, the African tropical clawed frog. The Department of Molecular Biomedical Rearch, Flanders Interuniversity Institute for Biotechnology of Ghent University, Belgium, had imported S. tropicalis frogs from a supplier in the Unit-ed States in September 2004. Within 5 weeks, some ani-mals became lethargic with signs similar to tho 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 chytridio-mycosis; iridoviral infection; common bacterial infections of liver, lungs and kidneys; chlamydophila infection; or in-testinal parasites.
From November 2004 through April 2005, 19 visually affected and visually unaffected specimens of S. tropica-lis, 2 tadpoles, and 4 tank water samples were lected for detailed examination for mycobacteria. The frogs were eu-thanized and discted, and lected organs and fl uid were removed aptically (liver, lungs, gallbladder, gastrointes-tinal tract, spleen, kidneys, fat body, ovary, oviduct, tibia, and coelomic fluid). 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 (3,5–7). Water samples were concentrated by fi ltration as described by Iivanainen et al.
汤圆馅的做法大全(7), and suspensions were made from the complete tadpoles (6). Further analys were performed as described for the decontaminated frog suspensions. DNA for genetic analy-s was extracted from the suspensions and pure cultures as described previously (6,8). M. lifl andii was identifi ed by IS2404 nested PCR and quence analysis of 16S rRNA gene (9,10). A combination of 4 genetic typing assays (including 3 previously investigated in M. ulcerans) was ud to type M. lifl andii (3,9,11,12). A fl owchart of the per-formed tests is shown in Figure 1.
All visually affected specimens showed positive re-sults for at least 1 organ and for at least 2 of the following tests: microscopy (Ziehl-Neeln staining), in vitro culti-vation (LJ medium and charcoal medium), IS2404 nested PCR, and histopathologic examination. Of note, all ovarian tissue of the 11 visually affected specimens showed posi-tive results for at least 2 tests. Three of 8 visually unaffect-ed 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.
*Institute of Tropical Medicine, Antwerp, Belgium; †Flanders In-teruniversity Institute for Biotechnology of Ghent University, Zwij-naarde, Belgium; ‡Ghent University, Merelbeke, Belgium; §Nogu-chi Memorial Institute for Medical Rearch, Legon, Ghana; and ¶Armed Forces Institute of Pathology, Washington, DC, USA
DISPATCHES
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 epi-thelium, and in the lumens of the gallbladder, stomach, intestine, and oviduct. Papillary hyperplasia of the gall-bladder mucosa was marked, and the lamina propria was expanded by heterophils and many AFB (Figure 2). Lung p
arenchyma, liver, femur, and tibia were normal and free of AFB.
We identi fi ed the causative pathogen as M. li fl andii in all frogs: by growth on charcoal medium, by restriction fragment length polymorphism, or by quence analysis. Isolate M05–0456 had a similarity value of 100% with M. li fl andii (GenBank accession no. AY845224.1). Growth on charcoal medium can be considered as an additional identi fi cation criterion for M. li fl andii becau growth on charcoal is better than on LJ medium (3), differentiating M. li fl andii from M. ulcerans . Clinical isolates of M. ulcer-ans are grown readily on LJ medium but never on charcoal
medium. The antibiogram of strain M04–2878 showed re-sistance to isoniazid, ethambutol, rifampin, clarithromycin, and ethionamide. In each of the genotyping assays, M. li-fl andii produced pro fi les that were distinct from tho of M. ulcerans (data not shown). None of the laboratory staff who handled the anurans exhibited any signs of a mycobac-terial dia.
Conclusions
The fi rst epizootic of M. li fl andii infection was reported by Trott et al. in 2004 in pipid frog colonies in the United States (3). To our knowledge, our report is the fi rst account of M. li fl andii dia in a colony of captive S. tropicalis frogs in Europe. We do not know the prevalence of M. li-fl andii infecti
on in the colony, but we believe it was very high becau 3 of 8 clinically healthy frogs were positive for M. li fl andii by at least 1 test.
The genetic and phenotypic identi fi cation of M. li fl an-dii as causative agent of the epizootic, the fact that cas of M. li fl andii infection have not been reported in Europe to date, the strikingly similar signs and dia progress (3), and the probability that the frogs were imported from the same supplier (3) all suggest that some members of the im-ported S. tropicalis colony were infected with M. li fl 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 be-came 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. ulcer-ans –like mycobacterium. To our knowledge, no M. li fl an-dii infection, in humans or wild anurans, has been reported from Africa. We have con fi rmed that all isolates from Bu-ruli ulcer patients and environmental samples analyzed by our laboratory were true M. ulcerans infections and not IS 2404 PCR-positive M. ulcerans –like mycobacteria (un-pub. data).
The apparently enzootic character of M. li fl andii in-fection in different S. tropicalis
breeding companies in
Figure 1. Flowchart of performed tests. ZN, Ziehl-Neeln staining;
LJ, Löwenstein-Jenn medium; Middlebrook 7H11 acidi fi ed with phosphoric acid, supplemented with sheep blood and charcoal; RFLP , restriction fragment length polymorphism; VNTR, variable number of tandem repeats; MIRU, mycobacterial intersperd repetitive unit.
Figure 2. A) Oviduct, focally expanded by collections of macrophages and yolk (Y) material. Note acid-fast bacilli (AFB) throughout specimens, but concentrated at the periphery (Ziehl-Neeln [ZN] stain ×25). B) High-power magni fi cation of periphery of oviduct containing macrophages and Y material; AFB are concentrated at the periphery (ZN stain ×300). C) Gallbladder with papillary hyperplasia of the mucosa. Note mass of AFB in the lamina propria (LP) of the mucosa (ZN stain
×50). D) High-power magni fi cation of the LP of the gallbladder mucosa showing large numbers of AFB and heterophils (H) (ZN stain ×
300).
Mycobacterium lifl andii Infection in Silurana tropicalis
the United States (3,4) and the exchange of transgenic or mutant S. tropicalis lines between rearch laboratories, may po a rious threat for the international rearch 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 dia. Thus far, no preventive measures or treatment for this amphibian myco-bacteriosis are known (3,4,14). Resistance to antimycobac-terial agents by environmental mycobacteria is not unusual and has been reported previously (15).
We propo 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 re-arch purpos (2), and ovarian tissue was positive for all visually affected specimens and for 1 of 3 positive visu-ally
unaffected specimens. However, further studies are needed to determine the role of oocytes in the epizootics of this emerging frog dia, especially in the evaluation of our propod screening method. To prevent the infection of existing stocks with wild-caught frogs of unknown ori-gin, 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 rearch colony at North Caro-lina State University (14). The etiologic agent was identi-fi ed as M. marinum complex on the basis of mycobacterial culture, but genetic analys were not performed to exclude M. lifl andii infection. To avoid further spread of this dis-ea, every new outbreak of M. lifl andii infection in pipid frogs or other anuran species should be reported to relevant authorities and rearch communities. Until more is known about this epizootic and its prevention and treatment, cau-tion must be exercid in transportation, husbandry, and human contact with the animals (zoonotic potential). We do not know at this stage whether the importation of frogs contaminated by M. lifl andii reprents a danger for wild or autochthonous frogs. Further investigation is required to establish this.
Acknowledgments
We thank K. Fistte, C. Uwizeye, P. De Rijk, and M. Claes for their excellent technical assistance.
This work was supported by the Damien Foundation (Brus-ls, Belgium) and by a grant from the Flemish Interuniversity Council (VLIR) (grant no. NDOC2005UA0006).
P.S. is currently supported by a PhD grant (no. NDOC 2005UA0006) of the Flemish Interuniversity Council (VLIR).
Mr Suykerbuyk is a doctoral student at the Mycobacteriol-ogy Unit, Institute of Tropical Medicine in Antwerp, Belgium. His rearch interests include microbiology and ecology of M. ul-cerans, herpetology, and geographic information systems. References
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DISPATCHES
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Nationalestraat155,2000Antwerp,Belgium;email:***************西地碘
