Journal of Veterinary Diagnostic Investigation

http://vdi.sagepub.com/ Dermatophilosis in Captive Tortoises

David A. Bemis, Clark S. Patton and Edward C. Ramsay J VET Diagn Invest 1999 11: 553 DOI: 10.1177/104063879901100616 The online version of this article can be found at: http://vdi.sagepub.com/content/11/6/553

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ogenesis of canine parvovirus enteritis: sequential virus distribution and passive immunization. Vet Pathol 22:617624. 12. Olsen CW: 1993, A review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination. Vet Microbiol 36:137. 13. Takeuchi A, Binn LN, Jervis HR, Keenan KP: 1976, Electron

microscopic study of experimental enteric infection in neonatal dogs with a canine coronavirus. Lab Invest 34:539549. 14. Yasoshima A, Fujinami F, Doi K, et al.: 1983, Case report on mixed infection of canine parvovirus and canine coronavirus. Electron microscopy and recovery of canine coronavirus. Jpn J Vet Sci 45:217225.

J Vet Diagn Invest 11:553557 (1999)

Dermatophilosis in captive tortoises

David A. Bemis, Clark S. Patton, Edward C. Ramsay
Dermatophilus infections have been observed in several lizard species, a boa constrictor (Constrictor constrictor), and an American alligator (Alligator mississippiensis).1,3,5,6,10,11,13 Lesions in reptiles have been described as surface crusts comprised of necrotic cellular debris, keratin, and inammatory cells, with necrosis of the epidermis or caseous subcutaneous nodules. Dermatophilosis was diagnosed by isolation of D. congolensis in culture or identication of its unique branching, lamentous morphology in lesions. Until recently, D. congolensis was the only species assigned to the genus Dermatophilus, and phenotypic properties of the species were said to vary considerably.4 A new species designation, D. chelonae, was proposed for 3 highly divergent isolates from chelonids in Australia.10 Chelonid isolates appear to be adapted to poikilotherms, growing better at lower temperatures than D. congolensis and having low infectivity for mammals.10 There is little information available on disease caused by D. chelonae in reptiles and its recognition in the eld. The purpose of this report is to describe the isolation of D. chelonae-like bacteria from cutaneous and visceral lesions in 2 species of captive tortoises. Five bowsprit tortoises (Chersina angulata) and 3 Egyptian tortoises (Testudo kleinmanni) were housed in adjacent sections of an open tripartite wooden box. Another bowsprit tortoise was housed separately in a glass terrarium. Sphagnum moss bedding was used. Room temperature was maintained at approximately 30 C. A daily photoperiod of 12 hours was maintained with a light source.a Fecal matter was removed regularly, and the boxes were emptied and washed with detergent and half of the sphagnum moss was replaced monthly. The 5 bowsprit tortoises were wild-caught. They had been housed together since their arrival in 1991. The sixth bowsprit tortoise had been housed with the group until September 1995. The 3 Egyptian tortoises were of unknown origin. They were received from 2 separate donors in 1994 and were housed in the box from September to November 1995. SevFrom the Departments of Comparative Medicine (Bemis, Ramsay) and Pathology (Patton), College of Veterinary Medicine, University of Tennessee, Knoxville TN 37901. Received for publication August 26, 1998.

eral times during the fall and winter of 1995/1996, rain water leaked into the box. In November 1995, cutaneous lesions appeared simultaneously in 3 bowsprit tortoises, including the one in the glass terrarium. Each tortoise had multiple skin lesions (10), especially on the neck and in deep recesses around the neck and legs (Fig 1). The lesions were yellow-white, 0.21.2cm-diameter nodules covered with dry aking skin with a tract of yellowish material extending into the dermis. One tortoise was euthanized because of the extensive skin nodules and ulcerative stomatitis. Another tortoise also had bilateral septic gonitis, and a fourth bowsprit developed skin lesions and bilateral septic arthritis 5 months later. Skin and joint lesions were debrided and cleansed with dilute povidoneiodine solution. In addition, parenteral and oral antibiotics (ampicillin and amikacin or enrooxacin, and metronidazole) were administered. The skin lesions regressed in 38 months but recurred at different sites within 12 months. Two bowsprit tortoises and 1 Egyptian tortoise were found dead with no antemortem signs between January and April 1996. Tissues from these and the euthanized bowsprit were xed in 10% neutral buffered formalin, embedded in parafn, sectioned at 4 m, and stained with hematoxylin and eosin (HE), Grams, Grocotts methanamine silver (GMS), and FiteFaraco acid-fast procedures. Skin biopsies from 2 bowsprit tortoises were examined histologically. Both samples had intracorneal accumulations of heterophils with bacteria. Heterophils at the periphery had undergone coagulative necrosis with uniform eosinophilic staining of the nucleus and cytoplasm and loss of cytoplasmic granularity. The necrotic heterophils formed poorly dened layers, divided by bands of keratinocytes (Fig. 2A). The epidermis at the base of the lesion was mildly acanthotic with numerous transmigrating heterophils. The subjacent dermis contained increased broblasts, a moderate inltrate of heterophils, and a few lymphocytes (Fig. 2B). The subcutis had a mild to moderate heterophilic inltrate, perivascular lymphoid inltrates, and myxomatous metaplasia. Organisms were not visible with HE, but a Gram stain revealed gram-variable branching lamentous bacteria of varying width within the exudate (Fig. 3). The bacteria were not acid fast with Fite-Faraco but stained with GMS.

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Figure 1. Cutaneous heterophilic abscesses on the neck of a bowsprit tortoise.

The second biopsy was similar to the rst with 2 exceptions: 1) coccoid forms were also present and 2) bacteria were not demonstrable in foci of orthokeratotic and parakeratotic hyperkeratosis. Two bowsprit tortoises had granulomatous coelomitis and hepatic granulomas of unknown etiology. The Egyptian tortoise had a 2.5-cm ceolomic caseous mass adhered to the plastron; the cut surface was granular, friable, and tan with a green center. Histologically, necrotic heterophils were surrounded by a thin zone of mononuclear cells and fewer heterophils, all surrounded by a thin brous capsule. Grampositive laments and cocci (also visible with HE stains) were present at the periphery of the necrosis; divisions in 2 planes were not seen. Joint uid, dermal samples, liver, and coelomic granuloma were submitted for aerobic and anaerobic bacterial culture. Samples for aerobic culture were inoculated onto 1) brainheart infusion agarb supplemented with 1% yeast extract,b 1% heat inactivated horse serum,c and 5% debrinated sheep bloodd (EBA plate), 2) MacConkey agarb (MAC), 3) either Colistin-Nalidixic acid agarc (CNA) or phenylethyl alcohol agarb (PEA) supplemented with 5% debrinated sheep blood, and 4) thioglycolate broth b supplemented with 0.0005% hemin and 0.0001% vitamin K. EBA, PEA, and CNA plates were incubated at 35 C in a 7% CO2 atmosphere, MAC plates and thioglycolate broth were incubated at 35 C under atmospheric conditions. In addition, anaerobic cultures were inoculated onto brain-heart infusion agar supplemented with 0.5% yeast extract, 0.0005% hemin, 0.04% L-cystine, 0.001% vitamin K, and 5% debrinated sheep blood (CDC plate) and anaerobic PEA (APEA). Plates were incubated at 35 C in an atmosphere of 5% hydrogen, 5% CO2, and 90% nitrogen. Tests for catalase, urease, gelatin hydrolysis, and nitrate reduction were performed using standard laboratory procedures. Reference strains of D. congolensis (ATCC 14634) and D. chelonae (ATCC 51576) were obtained from the American Type Culture Collection.e Parafn-embedded specimens were processed for transmission electron microscopy (TEM) by removing the parafn with xylene and alcohol, rinsing in cacodylate buffer,

Figure 2. Bowsprit tortoise. A. intraepidermal heterophilic abscess with laminae due to squames and peripheral viable heterophils. HE. Bar 60 m. B. Base of intraepidermal heterophilic abscess. Dermis with heterophil and mononuclear cells on right. HE. Bar 300 m.

and postxing in osmium tetroxide. Epon-embedded sections were stained with uranyl acetate and lead citrate. For TEM of cultured organisms, a drop of medium was placed on a formvar-coated copper grid; organisms were negatively stained with potassium phosphotungstic acid and examined with a transmission electron microscope.f A D. chelonae-like bacterium was isolated as the predominant organism, often in pure culture, from 8 chelonid specimens, including 6 from cutaneous lesions in 3 bowsprit tortoises, 1 from the stie joint of a bowsprit tortoise, and 1 from the coelomic granuloma in an Egyptian tortoise. Small (1 mm) white to off-white, dry adherent colonies with prominent hemolysis appeared on blood agar after 3 days of either aerobic or anaerobic incubation at 37 C. Eight- to 21day-old cultures on blood agar were membranous and produced exfoliative-like rugose folds, which created a rose bud appearance of isolated colonies (Fig. 4). Isolates were identied as gram-positive branching laments; however, transition during growth from branching laments to cocci was often abrupt (Fig. 5). Zoospores were observed by light microscopy in aqueous suspensions of growth from blood agar plates after at least 7 days incubation at room temperature. Flagellated zoospores were observed by TEM (Fig. 6). Elongate mulberry-like clusters of cocci were abundant in thioglycolate broth cultures incubat-

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Figure 3. Branching, gram-positive lamentous bacteria within an intraepidermal heterophilic abscess in a bowsprit tortoise. Gram stain. Bar 20 m.

Figure 5. Dermatophilus chelonae-like isolate in a 7-day thioglycolate broth culture. Morphology is variable. Dark to light contrast reects positive to negative Gram staining of the organism. Bar 20 m.

ed at room temperature for at least 3 days; however, denitive laments dividing in 2 planes were rarely observed. Filaments containing longitudinal and transverse septa were occasionally found in parafn-embedded tissue from the coelomic granuloma processed for TEM (Fig. 7). The stock strain of D. congolensis consistently produced intact, easily recognizable branching laments with longitudinal and transverse septa. Biochemical and growth characteristics of the chelonid Dermatophilus isolates were similar and most closely resembled D. chelonae (Table 1). Differential characteristics included better growth at 23 C than at 35 C, nitrate reduction, and failure to produce urease or carotenoid pigment. Dermatophilosis is a widely recognized skin disease of mammals caused by the actinomycete D. congolensis. Known by such colloquial names as lumpy wool, strawberry foot rot, and rain scald, dermatophilosis affects a great variety of domestic and wild mammals and is economically important in cattle and sheep in high-rainfall areas.2,12 Mam-

malian epidermal dermatophilosis is associated with orthokeratotic and parakeratotic hyperkeratosis with layers of neutrophils and branching laments with longitudinal and transverse divisions. No organisms were visible in HE-stained sections of the epidermal lesions in the 2 bowsprit tortoises, but they were revealed with Grams and GMS stains.

Figure 4. Colony morphology of a chelonid Dermatophilus isolate after 7 days incubation on blood agar at 24 C. The conuent rst quadrant of growth is membranous, with rugose folds (arrow). After 21 days of incubation, colonies have a rose-bud appearance (inset).

Figure 6. Electron micrograph of zoospore from chelonid Dermatophilus isolate from aqueous suspension of 14-day culture. Negative stain, 0.5% potassium phosphotungstic acid, pH 7. Bar 1.0 m.

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Table 1. Comparison of chelonid Dermatophilus isolates with D. congolensis and D. chelonae.

D. chelonae (ATCC 51576) D. congolensis (ATCC 14634)

Characteristic

Chelonid isolates (n 8)

Motile (agellated zoospores produced) Branching gram-positive lament with longitudinal and transverse septa Small, dry, adherent hemolytic colonies Anaerobic and aerobic growth Catalase produced Gelatin hydrolyzed Acid-fast stain Orange colonies Urease produced Growth at 23 C faster than growth at 35 C Nitrate reduced Figure 7. Electron micrograph of chelonid Dermatophilus in ultrathin section of coelomic granuloma. Cells that have divided in longitudinal and transverse planes are seen within a fragmented bacterial lament. Parafn-embedded tissue processed for TEM. Lead citrate and uranyl acetate. Bar 1.0 m.

* This result is in disagreement with that reported for the original isolate; however, lack of urease was considered a distinctive characteristic of other chelonid isolates in the original report.

Skin lesions attributed to D. congolensis in several reptilian species differ from those presented here. Spontaneous epidermal lesions in 6 Senegal chameleons (Chamaeleo senegalensis), a collared lizard (Crotaphytus collaris), and a green iguana (Iguana iguana) consisted of hyperkeratosis, inammation, and necrosis of the underlying epidermis.6 The organisms did not stain with HE, but the Giemsa stain revealed laments with divisions in 2 planes. Nodules of epidermal hyperplasia with hyperkeratotic caps had heterophils in the underlying epidermis, and organisms were visible with HE.11 Two marble lizards (Calotes mystaceous) had nodules of hyperkeratosis without inammation; typical laments were visible with HE.1 Cutaneous dermatophilosis reported here differs in some ways from these earlier descriptions. Necrotic heterophils were present within hyperkeratosis, and numerous heterophils inltrated the underlying parakeratotic epidermis. Hyperkeratosis without heterophilic inltration was seen in one of 2 samples, but no bacteria were seen. Epidermal necrosis was not seen. Bacteria were not stained with HE; with Grams or GMS stains, divisions in 2 planes were not seen. Cocci were prominent in 1 sample. Subcutaneous caseous lesions due to D. congolensis have been reported in 3 reptilian species.5,6,13 Lesions in a bearded lizard (dragon) (Amphibolurus barbatus) consisted of either an encapsulated caseous abscess or encapsulated calcication; by inference, organisms did not stain with HE but were seen as gram-positive branching laments.13 The morphology of D. chelonae in tissue has not been described previously, and the longitudinal septum formation is difcult to observe in cultures.10 The pleomorphic microscopic morphology of Dermatophilus may be misleading in lesions and cultures from chelonids.

Dermatophilus spp. have a morphologically complex life cycle.12 In the present laments are gram variable, division in 2 planes was difcult to observe, and cocci are abundant. Under some conditions, bacteria with gram-positive cell walls may lose their ability to retain crystal violet dye in the Grams staining procedure.8 Zoospores of D. chelonae are gram negative more frequently than the lamentous forms of the organism, perhaps because the zoospores are older, less actively dividing cells. Dermatophilus chelonae-like bacteria were recovered in 6 of 7 culture attempts from cutaneous lesions. Denitive classication and more precise identication of D. chelonae-like bacteria requires further molecular characterization of these isolates. The distribution and mode of transmission of D. chelonae is not known. The relatively abrupt onset of the present Dermatophilus infections in a restricted number of chelonid species with opportunities for close contact suggested that there might have been a common source. Bowsprit and Egyptian tortoises are primarily dry habitat animals; prolonged exposure to an abnormally wet environment may have been a predisposing factor. The D. chelonae-like bacteria were not isolated from bedding material or from a sample of water that had leaked through the roof. Given the timing of infection, the Egyptian tortoises could also have been the source of infection. Dermatophilus chelonae infections most frequently involve the epidermis. Infection of a joint cavity and a coelomic mass may represent extension of infection from a body surface in a host with lowered resistance. Such a mechanism has been proposed for the occasional extraepidermal infections that occur in mammals.7,9 The effect of D. chelonae infection on overall reptile health deserves further study. Acknowledgements. We thank Mary Jean Bryant, Mary

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Lambert, Jason Scott, Dee Stevenson, Dr. Jim Dugan, Dr. Jonathan Sleeman, Melanie Gregory, Bern Tryon, and Kreis Weigel for technical assistance in the preparation of this manuscript.

Sources and manufacturers

a. b. c. d. e. f. VitaLite, Duro-Test Corp., New Bergen, NJ. Becton Dickinson Microbiology Systems, Cockeysville, MD. GIBCO/BRL, Grand Island, NY. Hemostat Laboratories, Dixon, CA. American Type Culture Collection, Rockville, MD. Phillips 300, Phillips Electronic Instruments, Mahwah, NJ.

References
1. Anver MR, Park JS, Rush HG: 1976, Dermatophilus in the marble lizard (Calotes mystaceus). Lab Anim Sci 26:817823. 2. Biberstein EL: 1990, The skin as a microbial habitat: bacterial skin infections. In: Review of veterinary microbiology, ed. Biberstein EL, Zee YC, pp. 268270. Blackwell, Cambridge, MA. 3. Chineme CN, Addo PB: 1980, Pathologic changes in lizards (Agama agama) experimentally infected with Dermatophilus congolensis. J Wild Dis 16:407412. 4. Gordon MA: 1964, The genus Dermatophilus. J Bacteriol 88: 509522.

5. Jacobson ER: 1989, Dermatophilosis in reptiles. Int Colloq Pathol Reptiles Amphib 3:47. [Abstr.] 6. Jacobson ER: 1991, Diseases of the integumentary system of reptiles. In: Dermatology for the small animal practitioner, exotics, feline, canine, ed. Nesbitt GH, Ackerman LJ, pp. 225 239. Veterinary Learning Systems, Trenton, NJ. 7. Jones RT: 1976, Subcutaneous infection with Dermatophilus congolensis in a cat. J Comp Pathol 86:415421. 8. LaScola B, Raoult D: 1998. Molecular identication of Gemella species from three patients with endocarditis. J Clin Microbiol 36:866871. 9. Lloyd DH: 1984, Immunology of Dermatophilus: recent developments and prospects for control. Prev Vet Med 2:93102. 10. Masters AM, Ellis TM, Carson JM, et al.: 1995, Dermatophilus chelonae sp. nov., isolated from chelonids in Australia. Int J Syst Bacteriol 45:5056. 11. Montali RJ, Smith EE, Davenport M, et al.: 1975, Dermatophilosis in Australian bearded lizards. J Am Vet Med Assoc 167: 553555. 12. Roberts DS: 1961, The life cycle of Dermatophilus dermatonomus, the causal agent of ovine mycotic dermatitis. Aust J Exp Biol Med Sci 39:463476. 13. Simmons GC, Sullivan ND, Green PE: 1972, Dermatophilus in a lizard (Amphibolurus barbatus). Aust Vet J 48:465466.

J Vet Diagn Invest 11:557560 (1999)

Prevalences of some virulence genes among Escherichia coli isolates from swine presented to a diagnostic laboratory in Iowa
Harley W. Moon, Lorraine J. Hoffman, Nancy A. Cornick, Sheridan L. Booher, Brad T. Bosworth
Escherichia coli strains that carry genes encoding for specic virulence attributes cause diarrhea and edema disease in swine.1,2,8 Enterotoxigenic E. coli (ETEC) have genes for enterotoxins that stimulate secretion of electrolytes and water by the small intestine.6 To colonize the small intestine and cause diarrhea, ETEC must also produce mbriae (pili).16 Escherechia coli strains that cause edema disease produce E. coli Shiga toxin (Verotoxin) and are designated as STEC.7 Shiga toxin is absorbed from the intestine into blood and causes systemic vascular damage resulting in edema disease. STEC must also produce mbriae to colonize the small intestine and cause disease.2 Some E. coli strains are designated as attaching/effacing E. coli (AEEC) because of their ability to attach intimately to the surface of intestinal epithelial cells and efface microvilli.10 The attaching/effacing
From the Departments of Veterinary Pathology (Moon) and Veterinary Diagnostic and Production Animal Medicine (Hoffman) and the Veterinary Medical Research Institute (Moon, Cornick, Booker), College of Veterinary Medicine, Iowa State University, Ames, IA 50011, and the National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50011 (Bosworth). Current address (Bosworth): Pig Improvement Company, Franklin, KY 42134. Received for publication September 4, 1998.

attribute is encoded by a series of chromosomal genes located in a pathogenicity island called the locus of enterocyte effacement. ETEC, STEC, and AEEC are considered to be different pathotypes of E. coli. However, some of the virulence genes that characterize them can be located on mobile genetic elements (plasmids, transposons, bacteriophages), and combinations of pathotypes occur. For example, some AEEC such as the human pathogen E. coli O157:H7 also have genes for Shiga toxin production,11,14 and some strains associated with edema disease of swine have genes for both Shiga toxin and enterotoxin production.2 The objectives of the work reported here were to determine 1) the prevalences of ETEC, STEC, and AEEC among swine E. coli isolates obtained at the Iowa State University Veterinary Diagnostic Laboratory, 2) the comparative prevalences of genes for different enterotoxin and pilus types among such isolates, and 3) whether there are differences in the prevalences of toxin and mbrial gene types isolated from pigs in different age groups. Escherichia coli isolates recovered from 539 swine fecal or tissue samples submitted to the Iowa State University Veterinary Diagnostic Laboratory from August 1996 through December 1997 were analyzed. More than 95% of the specimens were obtained from swine herds in the midwestern

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