DISSEMINATED MYCOBACTERIOSIS IN A STRANDED LOGGERHEAD SEA TURTLE (CARETTA CARETTA) Author(s): Giordano Nardini, D.M.V., Ph.D., Daniela Florio, D.M.V., Ph.D., Nicola Di Girolamo, D.M.V., Andrea Gustinelli, D.M.V., Ph.D., Francesco Quaglio, D.M.V., Ph.D., Laura Fiorentini, D.M.V., M.S., Stefania Leopardi, D.M.V., and Maria Letizia Fioravanti, D.M.V., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 45(2):357-360. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0252R1.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0252R1.1

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Journal of Zoo and Wildlife Medicine 45(2): 357–360, 2014 Copyright 2014 by American Association of Zoo Veterinarians

DISSEMINATED MYCOBACTERIOSIS IN A STRANDED LOGGERHEAD SEA TURTLE (CARETTA CARETTA) Giordano Nardini, D.M.V., Ph.D., Daniela Florio, D.M.V., Ph.D., Nicola Di Girolamo, D.M.V., Andrea Gustinelli, D.M.V., Ph.D., Francesco Quaglio, D.M.V., Ph.D., Laura Fiorentini, D.M.V., M.S., Stefania Leopardi, D.M.V., and Maria Letizia Fioravanti, D.M.V., Ph.D.

Abstract: A loggerhead sea turtle (Caretta caretta) was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, and increased respiratory effort. It died a few hours after admission, and a postmortem examination was immediately performed. Miliary yellowish nodules were evident in the liver, and a lower number in the heart, stomach, and gut wall. Hundreds of whitish nodules were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions. Histologically, all nodular lesions consisted of a small central area of necrosis with acid-fast bacilli surrounded by epithelioid cells, macrophages, and lymphocytes. Giant cells were found in the spleen and the liver. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general isolation media and selective isolation media for mycobacteria. The isolate showed a restriction pattern identical to Mycobacterium chelonae by polymerase chain reaction–restriction fragment length polymorphism. To the best of the authors’ knowledge, this is the first description of a disseminated infection caused by a potentially pathogenic mycobacteria in a stranded, freeranging loggerhead sea turtle. Veterinary staff and biologists who handle sea turtles with suspected mycobacterial disease should protect themselves appropriately. Key words: Chelonian, disseminated mycobacteriosis, dyspnea, loggerhead sea turtle, Mycobacterium chelonae.

INTRODUCTION Mycobacteriosis is the oldest known infectious disease of reptiles, with the first case of spontaneous mycobacteriosis in a snake described in 1889,11 7 yr after Robert Koch isolated Mycobacterium tuberculosis for the first time. Since then, tuberculosis has been described in a few captive sea turtles.1,3,6 The large genus Mycobacterium comprises more than 100 species.10 Besides the obligate pathogenic mycobacteria (Mycobacterium tuberculosis complex and Mycobacterium leprae), the genus includes potentially pathogenic mycobacteria (PPM) and environmental saprophytic mycobacteria.10 Although PPM are not obligate pathogens, contact with immunocompromised, chronically ill aniFrom the Fondazione Cetacea ONLUS, Viale Torino 7/ A, 47838 Riccione (RN) (Nardini); the Clinica Veterinaria Modena Sud, Piazza dei Tintori, 1, 41057 Spilamberto (MO) (Nardini, Leopardi); the Department of Veterinary Medical Sciences (DIMEVET), Alma Mater Studiorum University of Bologna, Via Tolara di Sopra, 50, 40064 Ozzano dell’Emilia (BO) (Florio, Gustinelli, Fioravanti); the Clinica per Animali Esotici, Via Sandro Giovannini 53, Roma (Di Girolamo); the Department of Comparative Biomedicine and Food Science, University of Padua, Via dell’Universita`, 16, 35020 Legnaro (PD) (Quaglio); and the Istituto Zooprofilattico Sperimentale of Lombardia and Emilia-Romagna, Via Marchini 1, 47122 Forlı` (FC) (Fiorentini), Italy. Correspondence should be directed to Dr. Girolamo ([email protected]).

mals or humans with PPM could lead to disseminated, life-treating infections.4,5,9 The purpose of the present study is to describe, to the best of the authors’ knowledge for the first time, a disseminated infection caused by a PPM in a stranded loggerhead sea turtle (Caretta caretta). A 21-kg, 54-cm (straight carapace length) loggerhead sea turtle was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, increased respiratory effort, and poor muscular strength. The turtle was brought to the Sea Turtle Hospital of Fondazione Cetacea, where it died 4 hr after admission. Postmortem examination was performed immediately after its death using standard techniques. Abundant coelomic and pericardial effusions were observed. A traumatic esophageal perforation secondary to the ingestion of a fishing hook was present. Miliary yellowish nodules were evident in the liver, with a lower number in the heart, and in mucosa and submucosa of stomach and small intestine. Hundreds of whitish nodules ranging from 1 mm to 7 cm were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions (Fig. 1). Three specimens of the anisakid nematode Sulcascaris sulcata and two specimens of the digenean Pachypsolus irroratus were found in the esophagus and in the lumen of the small intestine, respectively. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general

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Figure 1. Gross aspect of the lungs from a stranded loggerhead sea turtle (Caretta caretta) with evidence of respiratory distress. Nodular lesions replaced the majority of pulmonary parenchyma. Notice mucus accumulation. Insets—(A) Photomicrograph of three granulomas in the lungs. H&E, bar ¼ 100 lm. (B) Photomicrograph of a granuloma from the liver containing acid-fast rods. ZN stain, bar ¼ 50 lm.

and selective isolation media for mycobacteria. The bacteria isolated from the cultures were identified by phenotypic tests and Analytical Profile Index (API) Systems (API 20E, 20NE, BioMe´rieux, F-69280, Marcy l’Etoile, France) and by standard biochemical methods. Smears were obtained from the same samples and stained with Ziehl–Neelsen (ZN). Samples from major coelomic organs were collected and fixed in neutral buffered 10% formalin. Paraffin wax sections of 5 lm were stained with hematoxylin and eosin (H&E) and ZN. Histologically, all nodular lesions comprised central areas of necrosis surrounded by epithelioid macrophages and lymphocytes. Acid-fast bacilli were present within the core of these lesions and within the cytoplasm of the previously mentioned epithelioid macrophages. An outer band of fibrosis bordered the margins of the granulomas. Within the lungs, the inflammatory nodules were focal to multifocal to coalescing, and were effacing and expanding the interstitium, compressing the surrounding alveoli. Aggregates

of macrophages and bacteria were inside the alveolar lumens. Occasional giant cells were present only within the spleen and the liver (Fig. 1). The bacterial culture media tryptic soy agar plus 2% NaCl, brain–heart infusion agar plus 2% NaCl, and blood agar allowed the isolation of a strain of Aeromonas hydrophila complex from the lungs, whereas in Lo¨wenstein-Jensen and Middlebrook 7H10 medium, whitish colonies positive to ZN stain were observed after 5 days at 308C. A fragment of ;439 bp of the 65-kDa heat shock protein gene (hsp65) was amplified with the primers TB11 and TB12 and then subjected to polymerase chain reaction (PCR)–restriction fragment length polymorphism by BstEII and HaeIII enzymes (MBI Fermentas GmbH, D-68789, St. Leon-Rot, Germany).12 The isolate showed a restriction pattern identical to Mycobacterium chelonae. The free-ranging loggerhead sea turtle described in the present paper suffered a systemic infection caused by a PPM (M. chelonae). Considering both clinical (i.e., the dyspnea) and patho-

NARDINI ET AL.—MYCOBACTERIOSIS IN A LOGGERHEAD SEA TURTLE

logical findings, it appeared that the most severe lesion in this loggerhead sea turtle was the granulomatous pneumonia, although characteristic mycobacterial lesions were present in other tissues as well. Granulomatous pneumonia is a frequent pathological finding in stranded sea turtles and is typical of mycobacteriosis.2,7 In a pathological survey, Oro´s and colleagues8 found granulomatous pneumonia in 12.9% of stranded sea turtles. Interestingly, all of them were negative for mycobacteria. Aeromonas hydrophila has long been recognized as an opportunistic pathogen of reptiles. In stranded loggerhead sea turtles, A. hydrophila has been isolated from several lesions,8 including ulcerative dermatitis, granulomatous pneumonia, necrotizing splenitis, ulcerative esophagitis, and granulomatous hepatitis. Considering the opportunistic habits of A. hydrophila, its isolation in this turtle suggests a concurrent immunodeficiency. Such immunodeficiency could either be responsible for, or secondary to, the systemic mycobacteriosis. Mycobacterium chelonae, belonging to Runyon group IV of nonpigmented, rapidly growing mycobacteria, is characterized by high resistance to antibiotics10 and is an opportunistic pathogen of humans and animals.4,5 Although the first isolation of M. chelonae was made in 19032 from the lung of loggerhead sea turtles, mycobacterial infections in sea turtles have rarely been found and are mainly reported in captive individuals. Eleven cases are reported in the contemporary literature, of which nine were captive green sea turtles (Chelonia mydas),1,3,6 one was a captive loggerhead sea turtle,7 and one was a stranded Kemp’s Ridley turtle (Lepidochelys kempii) during rehabilitation.4 Of the captive green sea turtles, one was described to have mycobacteriosis in a postmortem examination,6 without details regarding pathogen identification and lesion characteristics. In another survey, two farmed green turtles out of 102 (1.9%) presented with acid-fast bacilli in the lungs, consistent with mycobacteria.3 Also in those cases, further investigations were not described. Six cases of tuberculosis caused by Mycobacterium avium were described in captive green sea turtles presenting with acid-fast positive lesions in lungs and livers.1 A captive-born hatchling loggerhead sea turtle presenting with stunted growth, emaciation, and weakness died in 1977 at the National Marine Fisheries Service in Galveston, Texas.7 On postmortem examination, the turtle presented with a granulomatous pneumonia from which Mycobacterium marinum was isolated.7 Last, a stranded Kemp’s Ridley sea

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turtle developed osteoarthritis secondary to a PPM (M. chelonae) after 4 mo in rehabilitation for epidermal lesions.4 The infection was thought to be a consequence of hematogenous spread of bacteria from epidermal lesions.4 The unique feature of this case from those described above is that it was a free-ranging individual rather than captive, suggesting that ecological aspects of PPM in aquatic vertebrates may need further investigation. Furthermore, clinicians, pathologists, and microbiologists also should consider the occurrence of PPM infections in non-captive sea turtles. The prevalence of sea turtle–associated human disease is not known.13 Potentially pathogenic mycobacteria are recognized causes of disease in debilitated or immunocompromised individuals,5 with immunocompetent individuals at risk, too.9 In a recent review,13 mycobacteria were not mentioned as zoonotic agents that may be transmitted by sea turtles. This report additionally supports the need for veterinary staff and biologists to handle sea turtles with appropriate hygienic care, especially if a systemic mycobacterial disease is suspected (e.g., evident respiratory distress, radiographic findings consistent with granulomatous pneumonia). The use of appropriate gloves, masks, eye shields, and other protective clothing should be considered.

LITERATURE CITED 1. Brock JA, Nakamura RM, Miyahara AY, Chang EM. Tuberculosis in Pacific green sea turtles, Chelonia mydas. Trans Am Fish Soc. 1976;105:564–566. 2. Friedmann F. Spontane lungentuberkulose bei schildkroten und die stellung des tuberkelbazillus im system. Z Tuberk. 1903;4:439–457. 3. Glazebrook JS, Campbell RSF. A survey of the diseases of marine turtles in northern Australia. I. Farmed turtles. Dis Aquat Org. 1990;9:83–95. 4. Greer LL, Strandberg JD, Whitaker BR. Mycobacterium chelonae osteoarthritis in a Kemp’s Ridley Sea Turtle (Lepidochelys kempii). J Wildl Dis. 2003;39: 736–741. 5. Jankovic M, Zmak L, Krajinovic V, Viskovic K, Crnek SS, Obrovac M, Haris V, Jankovic VK. A fatal Mycobacterium chelonae infection in an immunosuppressed patient with systemic lupus erythematosus and concomitant Fahr’s syndrome. J Infect Chemother. 2011;17:264–267. 6. Keymer IF. Diseases of chelonians: (2) necropsy survey of terrapins and turtles. Vet Rec. 1978;103:577– 582. 7. Leong JK, Smith DL, Revera DB, Clary JC, Lewis DH, Scott JL, DiNuzzo AR. Health care and diseases of captive-reared loggerhead and Kemp’s ridley sea

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turtles. In: Proc First Int Symp Kemp’s Ridley Sea Turtle Biol Conserv Manage. 1989;178–201. 8. Oro´s J, Torrent A, Calabuig P, De´niz S. Diseases and causes of mortality among sea turtles stranded in the Canary Islands, Spain (1998–2001). Dis Aquat Org. 2005;63:13–24. 9. Paul J, Baigrie C, Parums DV. Fatal case of disseminated infection with the turtle bacillus Mycobacterium chelonae. J Clin Pathol. 1992;45:528–530. 10. Pavlik I, Falkinham JO, Kazda J. Potentially pathogenic mycobacteria. In: Kazda J, Pavlik I, Falkinham JO, Hruska K (eds.). The ecology of mycobacteria: Impact on animal’s and human’s health. Dordrecht, The Netherlands: Springer; 2009. p. 21–80.

11. Sibley WK. Ueber Tuberculose bei Wirbelthieren. Virchows Arch. 1889;116:104–115. 12. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993;31: 175–178. 13. Warwick C, Arena PC, Steedman C. Health implications associated with exposure to farmed and wild sea turtles. J R Soc Med Short Rep. 2013;4:8. Received for publication 21 October 2013