Comparative Medicine Vol 54, No 3 Copyright 2004 June 2004 by the American Association for Laboratory Science Pages 309-317

Characterization of a Mycobacterium ulcerans-Like Infection in a Colony of African Tropical Clawed ( tropicalis)

Kristin A. Trott,1 Brian A. Stacy, DVM,2 Barry D. Lifland,3 Helen E. Diggs, DVM,4 Richard M. Harland, PhD,1 Mustafa K. Khokha, MD,1 Timothy C. Grammer, PhD,1 and John M. Parker, DVM4,*

A nontuberculous Mycobacterium ulcerans-like organism was identified as the causative agent of an epizootic of mycobacteriosis in a colony of African tropical clawed frogs, Xenopus (Silurana) tropicalis, at the University of Cali- fornia, Berkeley. Diverse clinical signs of disease were observed, including lethargy, excess buoyancy, coelomic effu- sion, cutaneous ulcers, and granulomas. Visceral granulomas, ulcerative and granulomatous dermatitis, coelomitis, and septicemia were common findings at necropsy. Identification of M. ulcerans-like organisms was based on molecu- lar and phenotypical characteristics. The findings of this investigation indicate that this M. ulcerans-like organism is a primary cause of morbidity and mortality in aquatic anurans and should be considered in the differential diagnosis of coelomic effusion in . Furthermore, if this Mycobacterium species ultimately is identified as M. ulcerans, X. tropicalis should be considered a potential source of this important public health pathogen.

The African tropical clawed , Xenopus (Silurana) tropicalis, characterized by the formation of cutaneous and visceral granulo- is indigenous to tropical regions of western Africa, including mas and/or cutaneous ulcers. To date and to the authors’ knowl- Senegal, Nigeria, Cameroon, the Democratic Republic of Congo, edge, treatment of frog mycobacteriosis has not been standardized. and Côte d’Ivoire, and is an emerging vertebrate model system Multiple species of nontuberculous mycobacteria, including M. for biological studies (1, 12, 15). Xenopus tropicalis is similar to chelonae (11, 20), M. xenopi (27), and M. marinum (6, 7, 19, 23, its larger relative, X. laevis, but has a diploid chromosome num- 28), cause disease in amphibians. Most of these cases occur in ber and shorter generation time, allowing design of comparative captive species of anurans, including species commonly used in genetic studies. research, such as X. laevis (6, 11, 27), Rana pipiens (23), and Bufo Mycobacterium ulcerans, an atypical representative of the ge- spp. (7, 20, 28). Although M. ulcerans was reported to grow in X. nus, is a water-borne pathogen endemic to tropical regions of Af- laevis stem cells at low temperatures (8), to our knowledge, there rica, Australia, Central America, and eastern Asia. In otherwise are no reported cases of M. ulcerans infections in amphibians. healthy humans, the most common manifestation of this disease The objective of the study reported here was to determine the is a necrotizing skin lesion known as Buruli or Bairnsdale ulcers clinical and pathologic findings, course of disease, and manage- (14, 17, 35). In Africa, Buruli ulcer disease is the third most com- ment of an epizootic of frog mycobacteriosis in a colony of captive mon mycobacterial ailment, the others being tuberculosis and X. tropicalis. In addition, the etiologic agent, an M. ulcerans-like leprosy. To the authors’ knowledge, a definitive reservoir for this mycobacteria, was differentiated from M. marinum by use of mo- organism has not been determined, but epidemics are closely as- lecular and phenotypical analysis. Differentiating between these sociated with tropical wetlands. Marsollier and co-workers (18) species may have important public health implications and may recently reported isolating M. ulcerans from aquatic insects in augment future studies aimed at the eradication of this pathogen Africa; this is one of a few reports of actual isolation of viable M. from colonies. ulcerans in the wild. Mycobacterium ulcerans is phylogenetically related to the ubiquitous waterborne M. marinum, and can be dif- Materials and Methods ferentiated on the basis of phenotypical characteristics, slight . All frogs were obtained from a laboratory breeding nucleotide variations in targeted genes, and the presence of two colony of X. tropicalis that is maintained in the ectotherm facility insertion sequences, IS2404 and IS2606 (14, 30, 31, 34). at the University of California, Berkeley (UCB). The colony in- Mycobacterial pathogens are commonly implicated as the cludes more than a thousand animals, propagated from a com- cause of anuran diseases. Amphibian mycobacteriosis is often bined stock of animals purchased from several domestic vendors. The UCB program is fully approved by the Association for As- Received: 7/30/03. Revision requested: 10/09/03. Accepted: 1/26/04. sessment and Accreditation of Laboratory Animal Care (AAALAC), 1Department of Molecular and Cell Biology, University of California, Berkeley, International. All frogs are covered under an approved UCB Insti- California 94720; 2Anatomic Pathology Service, Veterinary Medical Teaching Hos- pital, University of California, Davis, California 95616; 3Department of Compara- tutional Animal Care and Use Committee protocol. tive Medicine, Stanford University, Stanford, California 94305; and 4Office of Labo- Housing and care. The X. tropicalis colony was distributed ratory Animal Care, University of California, 203 Northwest Animal Facility, Ber- among four rooms housing only this species. All affected frogs keley, California 94720. *Corresponding author. were limited to two of the rooms, and were housed in 190-L fiber- 309 Vol 54, No 3 Comparative Medicine June 2004 glass tanks at a stocking density of 1.25 to 1.5 frogs/L. All aquaria Pathology Service at the University of California, Davis, Veteri- were maintained on a flow-through water system at a rate of 12 nary Medical Teaching Hospital. Paraffin-embedded tissues were water changes every 24 h. All tanks were drained, cleaned, and processed in routine manner and were stained with hematoxylin refilled weekly. Frogs were not removed from the tanks during and eosin (H&E) to provide 5-µm-thick sections for histologic ex- this cleaning process. The supply water entered at 24°C and amination. For each case, selected sections were stained with equilibrated between 21.5 and 24°C. Ambient room temperature Fite’s acid-fast and Brown and Brenn (B&B [Gram]) stains. was maintained at 28°C. Chlorine and chloramine were removed Microbiological culture. Over a period of three months, from the supply water by use of carbon filtration. Frogs were fed specimens (n = 12) from the same subset were submitted for mi- postmetamorphic frog brittle (NASCO, Fort Atkinson, Wis.) daily. crobiological examination. These generally consisted of coelomic Case history. During the summer of 2001, an increase in mor- fluid, liver and/or spleen, and heart blood from each individual bidity and mortality was detected in a single aquarium housing animal. Specimens were inoculated on blood agar plates with 5% adult female X. tropicalis. The affected animals had developed sheep blood (BAP), chocolate agar, MacConkey agar, Rose agar generalized cutaneous ulcers. Within a month, lesions appeared (Rose agar consists of half Columbia agar base with colistin and in adult female X. tropicalis in three other tanks in the room. At naldixic acid and half phenylethyl alcohol agar base [PEA] with that time, three affected animals were submitted to a commercial 5% sheep blood), Middlebrook 7H11 agar with OADC- (oleic acid, laboratory (IDEXX Veterinary Services, Sacramento, Calif.) for albumin, dextrose and catalase) enriched non-selective and selec- histopathologic examination. Mycobacteriosis was diagnosed on tive biplates (Hardy Diagnostics, Santa Maria, Calif.), and Bordet the basis of observation of large numbers of acid-fast bacilli Gengou (BG) agar (Remel, Lenexa, Kans.). The inoculated me- within cutaneous and visceral granulomas, but culture was not dium plates were individually bagged in sterile twist-tie bags performed. Subsequently, all animals involved were depopulated (Nasco West, Modesto, Calif.), alternately with and without CO2 (a combined total of 500 frogs) and the tanks were cleaned with generators (BBL, Sparks, Md.). Initial setups were completed in diluted sodium hypochlorite (0.3%) and allowed to air dry for a triplicate and were incubated at room temperature (22 to 25°C), month. The colony remained disease free for a year. 28 to 30°C, and 35°C. Cultures were examined grossly and micro- In the fall of 2002, several animals from one tank in a second scopically, using a dissecting microscope, every day for six weeks. room developed cutaneous lesions identical to the lesions ob- All specimens were examined by use of wet preparation, phase-con- served during the summer of 2001; again, the diagnosis was myco- trast light microscopy, Gram staining, Kinyoun-Gabet cold acid- bacteriosis. In an attempt to control transmission, the aquarium fast staining, and ARF staining at 40× (high dry) and 100× (oil was quarantined, and all affected animals were culled. Subse- immersion) magnifications. All staining methods previously de- quently, animals from other tanks in the same room developed scribed were performed according to manufacturers’ instructions. similar lesions. As more frogs were examined, it became apparent Egg-based Lowenstein Jensen (LJ) medium (Hardy Diagnostics) that clinical signs and lesions of the disease varied. Affected was used for standardized growth rate and photochromogenicity frogs, however, consistently developed coelomic distention, ulcer- testing (24). Photochromogenicity testing was performed by sub- ative skin lesions, or a combination of the two. Initially, coelomic jecting 30-day growth on 7H11 plates and LJ slants to an incan- effusion was attributed to gram-negative septicemia on the basis descent 60-W light source at 12-in. distance for 1 h and 6 h, of growth of gram-negative bacteria from the coelomic aspirate of respectively, and re-incubating for a duration of up to 20 addi- one moribund frog. tional days. Cytologic examination of coelomic aspirates, however, revealed Genetic examination. Affected tissue (liver and spleen), co- large numbers of free and intrahistiocytic acid-fast bacilli in the elomic exudate, and cultures were selected for DNA extraction coelomic exudate, but no histologic evidence of secondary or con- (n = 10). Samples were boiled at 100°C for 30 min in 500 µl of dis- founding bacterial infection by non-mycobacterial species. Within tilled water or placed in 500 µl of sterile Elvis Buffer (2) contain- seven days of the onset of clinical signs of disease, mortality of ing 10 µg of proteinase K/ml (Roche, Basel, Switzerland) and were infected frogs reached approximately one death per thousand incubated for 24 h at 55°C. Samples were stored at –20°C. frogs per day. Genotyping was performed by use of polymerase chain reaction Pathologic examination. Selected affected frogs were (PCR) amplification of highly conserved regions from multiple euthanized by immersion in 1.0% benzocaine (Sigma Chemical genes commonly used for mycobacterial speciation. Specifically, Co., St. Louis, Mo.) for 10 min, and the bodies were submitted for we amplified the 65-kDa heat shock protein (hsp65), three highly diagnostic testing. The ventrum was cleansed with 70% ethanol. conserved regions of the 16s rRNA gene (16sA, 16sB, and 16sC), Coelomic exudate was aspirated percutaneously for cytologic ex- the internal spacer between the 16s and 23s rRNA coding regions amination (n = 25), and the coelomic cavity was incised for inspec- (ITS), RNA polymerase B subunit (rpoB), and two insertion se- tion of visceral organs (n = 40). Selected tissues (liver and coelomic quences specific to M. ulcerans, IS2404 and IS2606 (16, 30, 33, fluid) were removed aseptically for molecular analysis (n = 5). Im- 36). Oligonucleotide information is given in Table 1. Amplification pression smears of skin, liver, spleen, and kidney were collected of hsp65 was achieved according to the method of Telenti and co- (n = 4), and the remaining tissues were fixed in buffered 10% for- workers, and direct sequencing was then performed rather than malin. All impression smears were treated with auromine- the less-specific restriction fragment digestion described by those rhodamine fluorescent (ARF; Becton, Dickinson and Company, researchers (33). Three overlapping regions of 16s rRNA were Franklin Lakes, N.J.) stain according to the manufacturer’s instruc- amplified to obtain a full-length, high-quality sequence (36). The tions. Coelomic exudate was treated with either ARF (n = 20) or ITS gene was amplified because of reported high interspecific se- Ziehl-Neelson (ZN) (n = 5) stains, according to manufacturer’s in- quence variations (36). The rpoB gene was sequenced to compare structions or established protocols, respectively. the isolate with the three-base pair difference known to exist be- Histologic examination (n = 17) was performed at the Anatomic tween M. marinum and M. ulcerans (16). Finally, amplification of 310 Mycobacterium ulcerans-like infection in African tropical clawed frogs

Table 1. Specific primer pair names, sequences, and numerical positions in gene for each gene amplified and reference for each primer for Mycobacterium sp. Gene Oligonucleotide Reference Sequence (5’ → 3’) Position in gene hsp65 Tb11 33 ACCAACGATGGTGTGTCCAT 398-417 Tb12 33 CTTGTCGAACCGCATACCCT 817-836

16sA T39 36 CGAACGGGTGAGTAACACG 94-112 Roc1r 36 CCACCTACGAGCTCTTTACG 539-558 16sB Roc1f 36 CGTTGTCCGGAATTACTG 519-536 T13 36 TGCACACAGGCCACAAGGGA 997-1016

16sC 16s5’ CGATGCAACGCGAAGAACCTT 908-928 16s3’ CGATCCCACCTTCGACAGCT 1391-1410

ITS ITSF-16S 36 GACGAAGTCGTAACAAGG 1461-1478 ITSR2 36 ATGCTCGCAACCACTATCCA 1710-1729 rpoB RPO5’ 16 TCAAGGAGAAGCGCTACGA 902-920 RPO3’ 16 GGATGTTGATCAGGGTCTGC 1242-1261

IS2404 MU5 30 AGCGACCCCAGTGGATTGGT 383-401 MU6 30 CGGTGATCAAGCGTTCACGA 852-871

IS2606 MU7 30 GGCCTGGCGGATTGCTCAAGG 213-233 MU8 30 CGTAGATGTGGGCGAAATGG 542-523 the M. ulcerans-associated insertion sequences, IS2404 and tinct cutaneous granulomas and coelomic effusion. Additionally, IS2606, was performed to determine the relatedness of the iso- in some cases of each form, there was granuloma formation in the lates to M. ulcerans (30). liver, spleen, and kidneys (Fig. 1C). The presentation of cutaneous The PCR amplifications for all primer sets were performed in and visceral granulomas was typical of previously reported cases 50-µl volumes containing 0.15 µl of Amplitaq gold DNA poly- of mycobacteriosis in anurans (10, 28). merase (Applied Biosystems, Foster City, Calif.), 5.0 µl of 10× A spectrum of lesions was observed in the integument and un- Amplitaq PCR buffer, 1 µl of 100 µM dNTPs, 0.5 µl of each primer derlying tissues, and ranged from raised plaques to cutaneous (100 µM), and 1 µl of DNA sample. The PCR reaction cycle pa- ulcers measuring 1-4 mm in diameter, with a focal to generalized rameters were modified slightly from those of Whipps and co- distribution and prominent crateriform appearance (Fig. 1A and workers (36), Telenti and co-workers (33), Lee and co-workers 1C). Some frogs had larger, regionally extensive, ulcerated areas, (16), and Stinear and co-workers (30). Reactions for all primer and the apex of the rostrum was a site of predilection, often re- sets were preceded by a 15-min denaturation at 95°C, consisting sulting in exposure of the underlying maxillary bone. In addition, of 40 cycles of 1-min denaturation at 94°C, 1 min of annealing, many frogs had focal non-ulcerated swellings of the distal portion and a 1-min extension at 72°C, then a final 10-min extension at of the extremities. 72°C. Annealing temperatures varied by primer set, and were: Visceral lesions consisted of granulomas that principally in- hsp65 at 60°C; 16sA, 16sB, and ITS at 56°C; 16sC and rpoB at volved the liver and spleen. Hepatic granulomas were well-de- 58°C; and IS2404 and IS2606 at 55°C. marcated, 0.1- to 4.0-mm-diameter, white, raised nodules. In Samples were visualized on a 1.6% agarose gel, and products severe cases, granulomas often extended into the adjacent me- were excised and purified using the QIAquick gel extraction kit sentery and pancreas. Splenomegaly was another common find- (Qiagen Inc., Valencia, Calif.) or were purified directly from PCR ing; however, distinct splenic granulomas were less common than products using the QIAquick PCR purification kit (Qiagen Inc.). hepatic granulomas. Mural granulomas in the alimentary tract Sequences were aligned using MacVector (Accelrys, San Diego, were observed in a small number of cases. In addition, large Calif.), and were compared using the BLAST algorithm (National granulomas of the kidney (mesonephros) were present in frogs Center for Biotechnology Information) to identify the percentage with granulomas in the pelvic limbs and those with disseminated of homology to known mycobacterial species. infections. Nucleotide sequence accession numbers. The nucleotide Coelocentesis performed on frogs with coelomic effusion sequences for 16s rRNA, hsp65, ITS, and rpoB have been as- yielded up to 3 ml of non-viscous fluid, which ranged in appear- signed the GenBank accession No. AY500838, AY500839, ance from clear and straw-colored to turbid and serosanguineous. AY500840, and AY500841, respectively. The exudate contained fibrin that was adhered to serosal sur- faces in some animals. Results Acid-fast bacteria were detected in impression smears from in- Gross pathologic findings. Affected frogs were in fair to fected frogs with granulomas in the liver, spleen, kidney, and der- good nutritional condition, on the basis of the state of adipose mis, in decreasing order of frequency. Cytologic examination of stores and skeletal muscle, and exhibited two manifestations of coelomic exudate revealed free and intrahistiocytic rod-shaped, disease in roughly equal proportions. The first form consisted of non-filamentous, acid-fast bacteria (Fig. 1D). cutaneous ulcers and granulomas within the integument and/or Histopathologic findings. Histologic examination was per- deep skeletal muscle and fascia (Fig. 1A). The second form was formed on 16 frogs with representative systemic lesions, from characterized by septicemia, massive coelomic effusion, and some of which selected organs were omitted, as they were re- coelomitis (Fig. 1B). Many frogs with coelomic effusion also had quired for ancillary tests. Moribund frogs with fulminant disease generalized edema and lymph sac effusion. These manifestations commonly had severe multisystemic, granulomatous inflamma- of disease were not mutually exclusive, as some animals had dis- tion with large numbers of intralesional, 2- to 7-µm–long, slightly 311 Vol 54, No 3 Comparative Medicine June 2004

Figure 1. Adult female Xenopus tropicalis, one with severe cutaneous mycobacteriosis (A), and the other with fulminant mycobacteriosis (B). (A) Notice multifocal cutaneous ulcers distributed throughout the integument. (B) Notice marked coelomic distension and generalized edema. (C) Adult X. tropicalis with liver exteriorized; notice generalized hepatic granulomas of left liver lobe and focal cutaneous ulceration. (D) Cytologic examina- tion findings in coelomic fluid from an X. tropicalis with severe coelomitis and massive effusion. Numerous paired and individual mycobacteria are packed within macrophages and free within the effusion. Ziehl-Neelson acid-fast stain; bar = 14 µm. curved, beaded, acid-fast bacilli. In most cases, mycobacteria Hypertrophy and hyperplasia of the melanomacrophage cen- stained faintly on H&E- and gram positive on B&B-stained sec- ters was present in some cases, but was not a consistent finding tions. On histologic examination, lesions were present in all ani- (6/16) (Fig. 2B). In the spleen, there was reactive lymphoid prolif- mals, many of which had hepatic granulomas composed of eration and histiocytosis (13/13), in addition to distinct granulo- histiocytes and epithelioid macrophages (11/16) (Fig. 2A). The mas (6/13). In severe cases, parenchymal organs often contained larger granulomas were surrounded and infiltrated by thin bands a less organized, more diffuse histiocytic infiltrate, as well as dis- of fibrous connective tissue and often had extensive central necro- tinct granulomas. Splenic and renal granulomas (8/14) were simi- sis; in one case, a granuloma contained multinucleated giant cells. lar to those described in the liver. In most cases (14/16), the hepatic hematopoietic tissue and paren- Frogs with coelomic effusion often had severe coelomitis (11/13). chyma surrounding the granulomas were expanded by variable Many of these animals had extensive hepatic granulomas, with numbers of immature mononuclear leukocytes, histiocytes, and involvement of the adjacent hepatic limb of the pancreas. In these fewer acidophilic granulocytes and lymphocytes (Fig. 2B); The cases, the pancreas and surrounding mesentery were extensively former cell type often had a blastic appearance and frequent mi- infiltrated and covered by a thick layer of viable and degenerate totic figures. This finding was interpreted as reactive myeloid histiocytes and necrotic debris (Fig. 2C). Such lesions included hyperplasia, which has been observed in X. laevis with mycobac- large numbers of free and phagocytized mycobacteria (Fig. 2D). teriosis (3) and has been misinterpreted as lymphoid neoplasia. Serosal infiltrates also covered other visceral organs. 312 Mycobacterium ulcerans-like infection in African tropical clawed frogs

Figure 2. (A) Photomicrograph of a section of liver from a X. tropicalis with mycobacteriosis. The hepatic parenchyma is expanded by a small granuloma (asterisk). There is proliferation of myeloid cells (myeloid hyperplasia) of the surrounding hematopoietic tissue (white arrows), which separates the hepatic cords (black arrows). H&E stain; bar = 80 µm. (B) High-power photomicrograph of a section of liver from an X. tropicalis with myeloid hyperplasia. Notice the frequent mitotic figures (white arrows). H&E stain; bar = 45 µm. (C). Photomicrograph of a section of pancreas from an X. tropicalis with severe coelomitis. A thick layer of histiocytes (black arrows), fibrin, and necrotic cellular debris covers the coelomic surface. Exocrine pancreatic acini are present at the bottom of the section. H&E stain; bar = 30 µm. (D). Same section as that in (C) stained with Fite’s acid- fast stain. Numerous mycobacteria are packed within histiocytes and scattered within the surrounding debris. Bar = 30 µm.

Seven of the frogs also had pneumonia that ranged from mod- visceral granulomas, with the exception of one frog that had a erate to severe. In these cases, the inflammatory infiltrate princi- large, locally extensive ulcer with necrosis of the underlying skel- pally emanated from the pulmonary serosal surfaces and etal muscle and fascia that contained numerous organisms. One vasculature, and the pulmonary interstitium was markedly thick of the four animals had concurrent coelomic effusion, whereas with large numbers of histiocytes containing mycobacteria. His- visceral lesions in the others were limited to hepatosplenic my- tiocytes also were present in the airways, and in one case, formed eloid proliferation without granuloma formation. multinucleated giant cells. To identify other bacterial species, sections from three frogs Histologic examination was performed on four frogs with rep- with severe coelomitis were stained with B&B and examined side resentative cutaneous gross lesions. Three frogs had multifocal or by side with those stained with Fite’s acid-fast. In all frogs, myco- focally extensive cutaneous ulcers, and one frog had focal, non- bacteria were numerous within the lesions and exhibited the ulcerated, deep, granulomatous myositis, fasciitis, and deep der- characteristic beaded morphology (Fig. 2D). Other bacteria were matitis of one hind foot. A thick layer of histiocytes interspersed not detected on B&B-stained sections. with fibroblasts and congested vessels lined the ulcerated areas. Microbiological culture. All specimens had mycobacteria as All of the lesions were relatively paucimicrobial, compared with 2+ to 4+ beaded bacilli by use of phase-contrast, wet-prep micros- 313 Vol 54, No 3 Comparative Medicine June 2004

Figure 3. (A) Gram staining of coelomic fluid from an affected frog demonstrating few gram-negative rods. (B) Phase-contrast illumination of the same field demonstrating numerous fat shadow organism spaces that are actually acid-fast organisms. copy; 2+ to 4+ acid-fast bacilli with beading and banding, 2+ to 4+ Table 2. Sequencing results for each gene amplified, compared with known fluorescent beaded rods by use of ARF staining, and only a few Genbank sequences of M. ulcerans and M. marinum gram-negative bacilli or rare gram-positive bacilli (non-mycobac- Gene M. ulcerans M. marinum teria) by use of Gram staining. These rare gram-stained organ- hsp65 99.25% 100% isms typically coincided with specimens that had positive growth (398/401)* (401/401) of the gram-negative organisms. Cultures grown from blood- 16sA 99.54% 99.77% tinged coelomic exudate more often were overgrown with gram- (436/438) (437/438) negative bacteria than were those cultured from straw-colored 16sB 100% 100% exudate. When phase-contrast techniques were applied to the (460/460) (460/460) + + Gram-stained smears, however, 2 to 4 relief stained large, fat, 16sC 99.79% 100% shadow organism spaces were observed, consistent with the rela- (477/478) (478/478) tive numbers observed for that particular specimen’s wet prep ITS 98.94% 98.94% phase-contrast microscopic results. These most likely were the (186/188) (186/188) mycolic acid-based coats of the organisms, which are impervious rpoB 5’† 100% 98.41% to Gram staining by use of standard techniques (Fig. 3). (189/189) (186/189) + + All specimens yielded 2 to 4 confluent growth of acid-fast or- rpoB 3’† 99.08% 100% ganisms. Optimal temperature and conditions were 28°C plus (108/109) (109/109)

CO2 to yield the fastest growth and the largest colonies. Growth IS2404 + – was not observed at 35°C on any medium, with or without CO , 2 IS2606 + – and incubation for 45 days. Optimal media and incubation for colony growth were as follows: Bordet Gengou at seven to 14 *Percentage of conservation (identical nucleotides/total nucleotides) for each gene and species. days; BAP at 11 days; 7H11 nonselective developed a light film of †No complete complementary rpoB 360 base pair sequences in Genbank, so 5'- growth in the initial inoculation area at 11 days, but did not grow and 3'-end sequences were compared. to individual colonies until 25 to 30 days; and 7H11 selective took 35 days until individual colonies were macroscopically observ- of colony-forming units, relative to the greater numbers of the able. Acid-fast colonies were non-pigmented and were character- slower growing mycobacteria recovered. ized as slightly buff-colored, with rough morphology on the 7H11 Genotyping results. Bands of appropriate lengths across all medium. All acid-fast colonies were non-photochromogenic and primer sets were obtained repeatedly from abdominal viscera, had no difference in pigment production compared with non– coelomic exudate, and pure cell culture isolates. Negative-control light-exposed duplicate cultures. The growth rate on LJ medium lanes (water) had no evidence of amplification. Specific homology was 30 to 35 days for macroscopic colony formation. data are provided in Table 2. Sequences from hsp65 and 16s Most samples yielded pure colonies of Mycobacterium sp. Occa- rRNA had closest homology to M. marinum, but both sequences sionally, however, specimens also had positive growth at 18 h for were > 99% homologous to both M. marinum and M. ulcerans. typical frog pathogens, including Chryseobacterium (Flavobacte- The rpoB sequence was 100% homologous to M. ulcerans (al- rium) meningosepticum, Aeromonas hydrophilia, Flavobacterium though the entire region sequenced for M. ulcerans or M. spp., Aeromonas spp., Klebsiella sp., Citrobacter sp., and other marinum was not available in Genbank). The ITS sequence had gram-negative rods. These bacteria were never more than occa- three nucleotide substitutions from either M. marinum or M. sional to 1+ colonies and were discounted as the etiologic agent in ulcerans. The M. ulcerans-associated insertion sequences, IS2404 these events due to their rapid growth rate and smaller numbers and IS2606, were consistently present in the isolates and se-

314 Mycobacterium ulcerans-like infection in African tropical clawed frogs

quenced out accordingly. Specificity for the isolates in question bacterial isolates are probed for IS2404 and IS2606 in laboratories was supported by simultaneous sequencing of M. marinum cul- worldwide, those insertion sequences may be identified in other tures and of M. chelonae from a cane toad, B. marinus. mycobacterial species as described by Chemlal and co-workers (5). As this manuscript went to press, personal communication of re- Discussion cent molecular work conducted at the University of Tennessee fur- Mycobacterium ulcerans is recognized by the World Health Or- ther supported this putative agent as an evolutionary intermediate ganization as an important public heath problem, in part because between M. marinum and M. ulcerans, and a provisional name has of the horrific lesions produced and the high prevelance of disease been proposed by Mve-Obiang and co-workers (21). in endemic areas (4). In human medicine, differentiating between As exemplified in this case, the sequence homology between the disease caused by M. ulcerans and the closely related M. 16s rRNA and heat shock proteins of closely related mycobacteria marinum has marked prognostic consequences. Likewise in vet- warrants caution in using limited genomic studies alone for identi- erinary medicine, determining the species of mycobacterial fication of atypical species. Such studies should be interpreted in pathogen responsible for disease affects the clinical management the context of phenotypical features of the organism, clinical and and can have appreciable public health implications as well. My- pathologic presentations, and more specific target genes. cobacterium ulcerans has been detected by use of PCR analysis in Although Mycobacterium spp. have been isolated from wild water samples from sites of human epidemics, as well as in fish amphibians, clinical disease is principally associated with captiv- and aquatic insects, including a recently reported isolation of M. ity (3, 6, 7, 10, 11, 13, 19, 20, 23, 27, 28). One novel clinical sign of ulcerans from African aquatic insects (18). The organism has disease commonly observed during this outbreak was marked co- rarely been cultured from the environment, and to our knowl- elomic distension due to an effusive syndrome, with or without edge, a natural reservoir has not been definitively identified (22, cutaneous ulcers and granuloma formation. This presentation 25). In general, human cases of M. ulcerans in western Africa are had gross similarities to cases of Chryseobacterium spp. septice- geographically restricted to tropical regions and are associated mia in X. laevis described in literature (9) and in the recent clini- with wetlands and slow-moving water (14, 25, 26, 35). The over- cal experience of these authors. Thus, moribund frogs with lap between the geographic locations of human cases and the coelomic effusion were erroneously presumed to have gram-nega- natural range of X. tropicalis is provocative and warrants further tive septicemia. Collectively, the pathologic and microbiological investigations. Furthermore, to our knowledge, there are no pub- results from this epizootic indicate that X. tropicalis infected with lished reports of M. ulcerans or a M. ulcerans-like organism from this M. ulcerans-like can present as a primary effusive syndrome animals in the United States. It is suspected that the imported, and/or cutaneous ulcerative disease, with or without visceral wild-caught founding members of the X. tropicalis colony at UCB granulomas. In some cases, frogs developed focal swellings in the carried the M. ulcerans-like organism. appendages, raised plaques, or large, ulcerated, necrotic lesions During the recent decade, genotypic analysis has greatly facili- similar to those described in Buruli ulcer disease in humans (14, tated phylogenetic studies of mycobacteria. In particular, inter- 35). Experimentally induced infections to fulfill Koch’s postulate specific comparisons of the hsp65, 16s rRNA, ITS, and rpoB are needed to further investigate the course of M. ulcerans-like sequences have helped determine relationships among species. infections in X. tropicalis. Caution must be taken, however, in the identification of mycobacte- Presumably, this M. ulcerans-like infection is endemic in the rial species by use of molecular methods (16, 29, 30, 34, 35). In the UCB frog colony; however, only a subset of frogs has developed initial investigation conducted at UCB, the isolates were classified clinical signs of mycobacteriosis. It has been documented that in- as M. marinum on the basis of the acid-fast reaction, anuran isola- fection with M. marinum causes chronic subclinical granulomas tion source, and 16s rRNA and hsp65 sequencing results. Classic in R. pipiens, but becomes lethal when the animals are immuno- phenotypic features, however, including slow growth rate classifica- suppressed (23). Stress may indeed have been a factor in this epi- tion and non-photochromogenic characteristics were not consistent zootic. Common stressors in captive aquatic animals, such as with most M. marinum strains. The isolate was sent to a nationally overcrowding, poor water quality, artificial lighting, and poor nu- recognized mycobacterial laboratory for a second opinion. There, trition, may result in immunosuppression (32). In the epizootic the organism was identified as M. marinum on the basis of near- reported here, clinical mycobacteriosis only developed in mature complete 16s rRNA and hsp65 sequencing; both genes were slightly frogs that were at least four months old. The colony at UCB in- more homologous to those of M. marinum. After correspondence cludes animals in all stages of development, ranging from early relating the growth rate and non-photochromogenic characteristics, tadpoles to mature frogs. All are maintained under identical hus- the identification was amended to M. marinum subspecies. bandry conditions. The absence of clinical mycobacteriosis during The inconsistencies between the phenotypic characteristics, tadpole and juvenile stages may suggest an extended incubation genomic studies, and unusual pathologic features prompted addi- period as previously reported for other mycobacterial diseases of tional genetic studies to be performed at UCB. Those studies tar- amphibians (32). Multiple factors, however, are likely involved. geted the rpoB, IS2404, and IS2606 genes, all of which were Eradication of the M. ulcerans-like organism from the UCB X. found to be identical to those of M. ulcerans. The authors, how- tropicalis colony has not been attempted. On the basis of results ever, decided not to report the organism in this epizootic as M. of clinical trials done on other vertebrate groups, chemotherapeu- ulcerans, because studies supporting the specificity of IS2404 and tic treatment of mycobacteriosis in frogs is unlikely to be effective IS2606 are based on analysis of limited laboratory control strains once overt disease has manifested. Thus, preventive measures and species on hand, with fewer than 60 isolates tested (30). As aimed to avoid introduction and spread of this disease are sug- additional mycobacterial isolates are probed for IS2404 and gested (14, 35). All X. tropicalis at UCB are colony reared, share IS2606 in laboratories worldwide, these insertion sequences may the same water supply, and are raised under identical conditions; be identified in other mycobacterial species. As additional myco- however, clinical mycobacteriosis has been confined to a limited 315 Vol 54, No 3 Comparative Medicine June 2004 number of tanks. The uninfected members of the colony were pro- References duced from oocytes manually collected from females (from tanks 1. Amaya, E., M. F. Offield, and R. M. Grainger. 1998. Frog genet- with infections) and reared in separate enclosures. Raising em- ics: Xenopus tropicalis jumps into the future. Trends Genet. 14:253- bryos in an environment free of the M. ulcerans-like organism 255. and culling infected and exposed animals may be a means of con- 2. Amsterdam, A., S. Burgess, G. Golling, W. Chen, Z. Sun, K. trolling or eradicating the M. ulcerans-like organism from a Townsend, S. Farrington, M. Haldi, and N. Hopkins. 1999. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. closed colony. Work is underway at UCB to establish a specific- 13:2713-2724. pathogen-free colony of X. tropicalis and develop a sentinel sys- 3. Asfari, M. 1983. Mycobacterium-induced infectious granuloma in tem to screen the colony for such pathogens. Xenopus: histopathology and transmissibility. Cancer Res. 48:958- Because of the public health importance of M. ulcerans, specific 963. 4. Asiedu, K., R. Scherpbier, and M. Raviglione. 2000. The care must be taken when working with X. tropicalis. The suscep- Yamoussoukro declaration on Buruli ulcer. Buruli ulcer: Mycobac- tibility of humans to the isolate described in this report has not terium ulcerans infection. WHO/GBUI, Geneva. been ascertained, and there have been no cases of cutaneous ul- 5. Chemlal, K., G. Huys, F. Laval, V. Vincent, C. Savage, C. cers among those in contact with X. tropicalis at the UCB facility. Gutierrez, M. A. Laneelle, J. Swings, W. M. Meyers, M. Daffe, Until a specific-pathogen-free line of frogs is established, it and F. Portaels. 2002. Characterization of an unusual Mycobac- terium: a possible missing link between Mycobacterium marinum should be assumed that X. tropicalis are potential carriers of a M. and Mycobacterium ulcerans. J. Clin. Microbiol. 40:2370-2380. ulcerans-like organism. General precautions, such as protective 6. Clothier, R. H. and M. Balls. 1973. Mycobacteria and laboratory clothing and proper hygiene, should be practiced when lymphoreticular tumours in Xenopus laevis, the South African handling and raising X. tropicalis and handling contaminated clawed toad. I. Isolation, characterization and pathogenicity for Xenopus of M. marinum isolated from lymphoreticular tumour cells. water. It should be noted that all frog species have zoonotic poten- Oncology 28:445-457. tial to be carriers of opportunistic pathogens for humans. In addi- 7. Done, L. B., C. L. Willard-Mack, G. Ruble, and M. Cranfield. tion, there is a substantial threat of this organism spreading to 1993. Diagnostic exercise: ulcerative dermatitis and cellulitis in captive X. laevis colonies. At UCB, three X. laevis with marked American toads. Lab. Anim. Sci. 43:619-621. coelomic effusion had histologic lesions consistent with M. 8. Drancourt, M., V. Jarlier, and D. Raoult. 2002. The environ- mental pathogen Mycobacterium ulcerans grows in amphibian cells ulcerans-like infection. Two of the three frogs had positive results at low temperatures. Appl. Environ. Microbiol. 68:6403-6404. of PCR analysis for IS2404 and IS2606. This finding warrants 9. Green, B. T. and P. E. Nolan. 2001. Cellulitis and bacteraemia concern for facilities housing the two Xenopus species and em- due to Chryseobacterium indologenes. J. Infect. 42:219-220. phasizes the need for strict adherence to traffic patterns and pro- 10. Green, D. E. 2001. Pathology of amphibia, p. 427-439. In K. M. Wright, and B. R. Whitaker (ed.), Amphibian medicine and captive cedures within the animal facility. husbandry, original edition. Krieger Publishing Co., Malabar, Fla. In conclusion, we have reported the findings of an epizootic of 11. Green, S. L., B. D. Lifland, D. M. Bouley, B. A. Brown, R. J. mycobacteriosis in X. tropicalis. Specifically, we have character- Wallace, Jr., and J. E. Ferrell, Jr. 2000. Disease attributed to ized the clinical signs of disease associated with mycobacteriosis Mycobacterium chelonae in South African clawed frogs (Xenopus and identified the causative agent of the epizootic, M. ulcerans- laevis). Comp. Med. 50:675-679. 12. Hirsch, N., L. B. Zimmerman, and R. M. Grainger. 2002. Xeno- like organism. The organism was identified as M. ulcerans-like pus, the next generation: X. tropicalis genetics and genomics. Dev. on the basis of growth characteristics, the consensus of current Dyn. 225:422-433. reports by other investigators pertaining to the presence of 13. Inoue, S. and M. Singer. 1970. Experiments on a spontaneously IS2404 and IS2606, and close sequence homology in hsp65, 16s originated visceral tumor in the newt, Triturus pyrrhogaster. Ann. N.Y. Acad. Sci. 174:729-764. rRNA, ITS, and rpoB. However, this identification is subject to 14. Johnson, P. D., T. P. Stinear, and J. A. Hayman. 1999. Mycobac- change as more molecular targets are identified and additional my- terium ulcerans—a mini-review. J. Med. Microbiol. 48:511-513. cobacterial isolates are probed for various known targets, such as 15. Khokha, M. K., C. Chung, E. L. Bustamante, L. W. Gaw, K. A. IS2404 and IS2606. Of interest is the geographic overlap between Trott, J. Yeh, N. Lim, J. C. Lin, N. Taverner, E. Amaya, N. the region of western Africa where X. tropicalis are native and the Papalopulu, J. C. Smith, A. M. Zorn, R. M. Harland, and T. C. Grammer. 2002. Techniques and probes for the study of Xenopus region where cases of human infection have developed. Individuals tropicalis development. Dev. Dyn. 225:499-510. responsible for the care of X. tropicalis should be alert to the poten- 16. Lee, H., H. J. Park, S. N. Cho, G. H. Bai, and S. J. Kim. 2000. tial presence of this Mycobacterium species, its zoonotic potential, Species identification of mycobacteria by PCR-restriction fragment and protocols needed to prevent the spread of disease. length polymorphism of the rpoB gene. J. Clin. Microbiol. 38:2966- 2971. 17. Maccallum, P., J. C. Tolhurst, G. Buckle, and H. A. Sissons. 1948. A new mycobacterial infection in man. J. Pathol. Bacteriol. Acknowledgments 60:92-122. We thank Maura Lane, Stephen Friet, Sam Cunningham, Loren 18. Marsollier, L., R. Robert, J. Aubry, J. P. Saint Andre, H. Johnson, Jeanine Hamel, and Magdalena Mleczko for their contribu- Kouakou, P. Legras, A. L. Manceau, C. Mahaza, and B. tions. Also, we thank Pam Small, Nina Hahn, Greg Timmel, Linda Carbonnelle. 2002. Aquatic insects as a vector for Mycobacterium Lowenstine and Tanja Zabka for their much-welcomed commentary ulcerans. Appl. Environ. Microbiol. 68:4623-4628. and support. We are especially grateful to Sherril Green and Stanford 19. Maslow, J. N., R. Wallace, M. Michaels, H. Foskett, E. A. University, without whose help this paper would not exist. Author KAT Maslow, and J. A. Kiehlbauch. 2002. Outbreak of Mycobacte- was supported by the Undergraduate Research Apprenticeship Pro- rium marinum infection among captive snakes and bullfrogs. Zoo gram and Biology Fellows Program. Author MKK was supported by Biol. 21:233-241. the Pediatric Scientist Development Program of the NICHD (K12- 20. Mok, W. Y. and C. M. Carvalho. 1984. Occurrence and experimen- HD00850) and a K08-HD42550 award from the NICHD/NIH. Author tal infection of toads (Bufo marinus and B. granulosus) with Myco- RMH is supported by NIH grant (GM66684-01). bacterium chelonei subsp. abscessus. J. Med. Microbiol. 18:327-333.

316 Mycobacterium ulcerans-like infection in African tropical clawed frogs

21. Mve-Obiang, A., R. E. Lee, K. A. Trott, T. C. Grammer, J. M. 29. Stinear, T., J. K. Davies, G. A. Jenkin, F. Portaels, B. C. Ross, Parker, B. Ranger, R. M. Grainger, B. D. Lifland, and P. L. C. F. Oppedisano, M. Purcell, J. A. Hayman, and P. D. Johnson. Small. In preparation. A new mycobacteria species, M. liflandii, 2000. A simple PCR method for rapid genotype analysis of Myco- isolated from diseased Xenopus tropicalis and X. laevis frog colo- bacterium ulcerans. J. Clin. Microbiol. 38:1482-1487. nies in U.S. research laboratories produces a potent macrolide toxin 30. Stinear, T., B. C. Ross, J. K. Davies, L. Marino, R. M. Robins- similar to plasmid-encoded mycolactone A/B produced by M. Browne, F. Oppedisano, A. Sievers, and P. D. Johnson. 1999. ulcerans, the causative agent of Buruli ulcer: implications for hu- Identification and characterization of IS2404 and IS2606: two dis- man disease and plasmid-mediated transfer of virulence determi- tinct repeated sequences for detection of Mycobacterium ulcerans nants. by PCR. J. Clin. Microbiol. 37:1018-1023. 22. Portaels, F., K. Chemlal, P. Elsen, P. D. Johnson, J. A. Hayman, 31. Stinear, T. P., G. A. Jenkin, P. D. Johnson, and J. K. Davies. J. Hibble, R. Kirkwood, and W. M. Meyers. 2001. Mycobacte- 2000. Comparative genetic analysis of Mycobacterium ulcerans and rium ulcerans in wild animals. Rev. Sci. Technol. 20:252-264. Mycobacterium marinum reveals evidence of recent divergence. J. 23. Ramakrishnan, L., R. H. Valdivia, J. H. McKerrow, and S. Bacteriol. 182:6322-6330. Falkow. 1997. Mycobacterium marinum causes both long-term 32. Taylor, S. K., D. E. Green, K. M. Wright, and B. R. Whitaker. subclinical infection and acute disease in the leopard frog (Rana 2001. Bacterial diseases, p. 159-179. In K. M. Wright, and B. R. pipiens). Infect. Immun. 65:767-773. Whitaker (ed.), Amphibian medicine and captive husbandry, origi- 24. Roberts, G. D., E. W. Koneman, and Y. K. Kim. 1991. Mycobac- nal edition. Krieger Publishing Co., Malabar, Fla. terium. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. 33. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bottger, and T. Isenberg and H. J. Shadomy (ed.), Manual of clinical microbiology, Bodmer. 1993. Rapid identification of mycobacteria to the species 5th ed. American Society for Microbiology, Washington, D.C. level by polymerase chain reaction and restriction enzyme analy- 25. Ross, B. C., P. D. Johnson, F. Oppedisano, L. Marino, A. sis. J. Clin. Microbiol. 31:175-178. Sievers, T. Stinear, J. A. Hayman, M. G. Veitch, and R. M. 34. Tortoli, E. 2003. Impact of genotypic studies on mycobacterial Robins-Browne. 1997. Detection of Mycobacterium ulcerans in taxonomy: the new mycobacteria of the 1990s. Clin. Microbiol. Rev. environmental samples during an outbreak of ulcerative disease. 16:319-354. Appl. Environ. Microbiol. 63:4135-4138. 35. van der Werf, T. S., W. T. van der Graaf, J. W. Tappero, and K. 26. Schulze-Robbecke, R., B. Janning, and R. Fischeder. 1992. Asiedu. 1999. Mycobacterium ulcerans infection. Lancet 354:1013- Occurrence of mycobacteria in biofilm samples. Tuberc. Lung Dis. 1018. 73:141-144. 36. Whipps, C. M., V. G. Watral, and M. L. Kent. 2003. Character- 27. Schwabacher, H. 1957. A strain of Mycobacterium isolated from ization of a Mycobacterium sp. in rockfish, Sebastes alutus (Gil- skin lesions of a cold-blooded animal, Xenopus laevis, and its rela- bert) and Sebastes reedi (Westrheim & Tsuyuki), using rDNA se- tion to atypical acid-fast bacilli occurring in man. J. Hyg. 57:57-67. quences. J. Fish Dis. 26:241-245. 28. Shively, J. N., J. G. Songer, S. Prchal, M. S. Keasey III, and C. O. Thoen. 1981. Mycobacterium marinum infection in Bufonidae. J. Wildl. Dis. 17:3-8.

317