Central Nervous System Mucormycosis Caused by Cunninghamella Bertholletiae in a Bottlenose Dolphin (Tursiops truncatus) Author(s): Marcos Isidoro-Ayza, Lola Pérez, F. Javier Cabañes, Gemma Castellà, Marina Andrés, Enric Vidal, and Mariano Domingo Source: Journal of Wildlife Diseases, 50(3):634-638. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/2013-10-284 URL: http://www.bioone.org/doi/full/10.7589/2013-10-284

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DOI: 10.7589/2013-10-284

Journal of Wildlife Diseases, 50(3), 2014, pp. 634–638 # Wildlife Disease Association 2014

Central Nervous System Mucormycosis Caused by Cunninghamella Bertholletiae in a Bottlenose Dolphin (Tursiops truncatus) Marcos Isidoro-Ayza,1,2 Lola Pe´rez,1,2 F. Javier Caban˜es,2,3 Gemma Castella`,2,3 Marina Andre´s,4 Enric Vidal,5 and Mariano Domingo1,2,5,6 1Servei de Diagno`stic de Patologia Veterina`ria, Veterinary School, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain; 2Departament de Sanitat i Anatomia Animals, Veterinary School, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain; 3 Veterinary Mycology Group, Veterinary School, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain; 4Centre Fundacio´n para la Conservacio´n y Recuperacio´n de Animales Marinos (CRAM), 08820 El Prat de Llobregat (Barcelona), Spain; 5Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, 08193 Bellaterra, Barcelona, Spain; 6Corresponding author (email: [email protected])

in a captive-held killer whale (Orcinus orca) (Abdo et al. 2012). However, to the best of our knowledge, it has not been described affecting the CNS of any cetacean species. A 300-cm-long, adult, male, Mediterranean bottlenose dolphin was found stranded and dead on 23 May 2012, on the Catalan Mediterranean coast (41u26937.520N, 2u14954.800E). The carcass was preserved at 4 C, and a complete necropsy was performed within 10 hr after the animal was found. At necropsy the dolphin was in a good state of preservation (2 on a scale of 1 to 5, according to Jauniaux et al. 2005). On external gross examination, the dolphin was in good body condition, with welldeveloped dorsal musculature, indicating that the animal was not affected by a chronic, debilitating disease. A hard spherical mass of approximately 18 cm in diameter was found in the caudal peduncle. The skin was unaffected at that site. On radiograph the mass was characterized by ankylosis of vertebral bodies. Brucella ceti was isolated from this lesion (data not shown). No other significant gross lesions were found. No macroscopic lesions were observed during examination of the whole fresh brain, but a slightly increased amount of cerebrospinal fluid (CSF) was noticed and collected (1 mL) from one lateral ventricle through a slit in the corpus callosum. Tissue samples, including the whole brain, were fixed in 10% buffered formalin and routinely processed for histopathology. After 24 hr of prefixation, the brain was routinely sliced to allow penetration of the fixative. Gross examination revealed two well-demarcated areas of

ABSTRACT: In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead on the Spanish Mediterranean coast. At necropsy, several areas of malacia were macroscopically observed in the periventricular parenchyma of the cerebrum. Microscopically a severe, diffuse, pyogranulomatous, and necrotizing meningoencephalomyelitis was associated with numerous intralesional highly pleomorphic fungal structures. After culture, the fungus, Cunninghamella bertholletiae, was identified by culture and PCR. To our knowledge, this is the first reported case of central nervous system mucormycosis due to Cunninghamella bertholletiae in a cetacean. Key words: Bottlenose dolphin, cetacean, Cunninghamella bertholletiae, meningoencephalitis, mycosis, mucormycosis.

Mycoses are a significant cause of stranding in bottlenose dolphins (Tursiops truncatus) and are well documented in both captive and free-ranging individuals. Common sites of infection are the skin and the respiratory system; a few reports exist of central nervous system (CNS) involvement. Several cases of necrotizing and hemorrhagic meningoencephalitis due to Aspergillus fumigatus associated with systemic infection by cetacean morbillivirus (CeMV) and one case of systemic coccidioidomycosis with CNS involvement have been described in free-ranging bottlenose dolphins (Reidarson et al. 2001). Cunninghamella bertholletiae (Order Mucorales) is a cosmopolitan, saprophytic, filamentous fungus, which has been isolated from blowhole and gastric fluid of apparently healthy free-ranging bottlenose dolphins (Morris et al. 2010). Recently, it was reported as the cause of fatal pneumonia 634

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malacia of about 1.5 cm in diameter located in the nucleus caudatus (Fig. 1a), adjoining the lateral ventricles. A third malacic lesion, 1.2 cm in diameter, was found in the nucleus accumbens of the left cerebral hemisphere. Additionally, two small 1–2 mm diameter, whitish, firm nodules were observed in the laryngeal tonsil when trimming the sample for histopathology. Routine histopathology was performed on a large set of tissues. Samples from selected organs were frozen for microbiologic and molecular studies. Histologically, the malacic lesions extended from the ventral ependymal lining of both lateral ventricles inward throughout the subjacent neuroparenchyma. They consisted of a severe inflammatory infiltration composed of macrophages and large multinucleated giant cells admixed with many neutrophils and some lymphocytes and plasma cells. In these lesions there were many extracellular, perivascular and intravascular, highly pleomorphic fungal structures (Fig. 1b). They were highlighted with Grocott’s silver staining as wide, variable in diameter (5–50 mm) hyphae (Fig. 1c). They were irregularly branched (usually at 90u angles), with poorly evident septa and thin, nonparallel walls that often appeared collapsed. When transversally sectioned, they were round to oval. In many instances, the hyphae were seen within the cytoplasm of multinucleated giant cells. Medium-sized vessels showed hyalinization of the tunica media (fibrinoid necrosis) with occasional inflammatory cells (mainly macrophages and neutrophils) (Fig. 1d), sometimes with leukocytoclastic changes (leukocytoclastic vasculitis). Thrombi obliterated the vascular lumen of some of these vessels. In the perivascular space, fibrin, interstitial edema, micro-hemorrhages, and necrotic cellular debris were consistently observed. Microscopic lesions similar to those described in nucleus caudatus and accumbens, but less intense, were also detected in medulla oblongata and the cervical portion of the spinal cord, extending

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through the ventral surface of the fourth ventricle and the adjacent neuroparenchyma. The medium-sized arteriolar network of the leptomeninges at the base of the medulla oblongata was severely affected by the inflammatory process, with multifocal fibrinoid necrosis and fungal growth in the vessel walls. The leptomeninges were severely edematous (Fig. 1e). The same pleomorphic fungal structures were also found within the crypts and the lymphoid tissue of the laryngeal tonsil, associated with focal pyogranulomatous inflammatory reaction. Fungal organisms or fungal associated lesions were not detected in any other organ investigated, either macro- or microscopically. A fresh tissue sample from basal portion of dura mater at the zone of medulla oblongata, removed later from skull, was submitted for fungal culture. The sample was inoculated on Sabouraud glucose agar (SGA) and brain heart infusion agar supplemented with chloramphenicol and incubated at 30 C. Cultures yielded rapidly growing white-grayish colonies consistent with Cunninghamella sp. The fungal isolate was cultured on SGA and potato dextrose agar (PDA) at 30, 40, and 45 C for species identification. Colonies on SGA and PDA were fast-growing, white at first, although they became tannish-gray, covering the whole plate (85 mm diameter) after 7 days of incubation at 30 and 40 C. Limited growth was observed after 7 days at 45 C (20 mm diameter). The micromorphology showed characteristic erect sporangiophores with short lateral branches in the apical region, which ended in globose to piriform vesicles, covered with globose one-spored sporangioles (Fig. 1f). The isolate was identified as C. bertholletiae on the basis of its morphologic and thermophilic characteristics (Tsui et al. 2011). Identification was confirmed by ITS-5.8S and D1/D2 26S rRNA gene sequencing. DNA was extracted and purified directly from a 7day-old culture in SGA. A search of GenBank database using BLAST revealed

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FIGURE 1. Bottlenose dolphin (Tursiops truncatus) with Cunninghamella bertholletiae infection in the central nervous section. (a) Rostral section of the cerebrum; malacia was observed in the nucleus caudatus, beneath the lateral ventricle of the left cerebral hemisphere. (b) Cerebrum (nucleus caudatus); inflammatory infiltration composed of groups of macrophages and large multinucleated giant cells containing large and pleomorphic fungal structures (arrowheads). Numerous neutrophils surround the giant cells. H&E stain. (c) Medulla oblongata (leptomeninges); highly pleomorphic, pauciseptate, thin-walled, and irregularly branching hyphae that often appear collapsed, associated with the same vessel shown in Fig. 1d. Grocott’s silver stain. (d) Medulla oblongata (leptomeninges); medium-size arteriole showing a severe hyalinization of the tunica media (fibrinoid necrosis) with inflammatory cells and leukocytoclastic changes (leukocytoclastic vasculitis). Few fungal structures similar to those shown in Fig. 1b are identified within the vascular wall (arrowheads). H&E. (e) Gross microscopic view of medulla oblongata, showing severe multifocal vasculitis and periarteriolar edema in the ventral leptomeningeal arteriolar network (arrows). More external arterioles are spared. H&E. (f) Fungal culture microphotography; Terminal portion of a sporangiophore of C. bertholletiae showing a vesicle bearing sporangioles on short stalks. Lactophenol cotton blue stain.

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that both sequences of our isolate were 99% identical to C. bertholletiae ATCC 42115. Fresh samples of lung, diaphragmatic lymph node, spleen, mediastinal lymph node, and brain were collected and submitted for routine CeMV diagnosis by RT-PCR (Soto et al. 2011) with negative results. Bengal Rose screening test for Brucella was positive in serum from blood collected by heart puncture before necropsy. Brucella ceti was isolated from a swab collected from the vertebral lesion, but not from CSF, spleen, encephalon, mediastinal lymph node, or serum (data to be described elsewhere). Only a few cases of mucormycosis have been reported in cetaceans (Wunschmann et al. 1999; Robeck et al. 2002; Abdo et al. 2012). Cases are also scarce in domestic mammals (Ginn et al. 2007). Three cases of mucormycosis causing meningitis and invasion of adjacent neuroparenchyma have been published affecting a freeranging harbor porpoise (Phocoena phocoena) (Wunschmann et al. 1999) and a bottlenose dolphin and killer whale housed in a dolphinarium (Robeck et al. 2002). Isolated agents were respectively Rhizomucor pusillus, Apophysomyces elegans, and Saksenaea vasiformis. In humans, infections caused by C. bertholletiae are uncommon but frequently fatal. They usually occur in immunocompromised patients (Gomes et al. 2011). Infection by this fungal species has been described in two domestic cats (Mayayo et al. 2011). Similar to other members of the order Mucorales, the pathogenicity of C. bertholletiae consists of its ability to invade blood vessels resulting in thrombosis and infarction. Vascular dissemination and rhino-orbito-cerebral infections are the two major routes of spreading (Gomes et al. 2011). Entry through via aerosol to the laryngeal tonsillar tissue and hematogenous dissemination to the CNS is suspected in the present case, based on the severe involvement of arterioles at the basal arteriolar network of the brain.

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In cetaceans, mycoses usually are a manifestation of underlying immune suppression (Wunschmann et al. 1999). CeMV infection is considered to cause immunosuppression in cetaceans, and cases of fungal pneumonia and encephalitis have been reported in free-ranging dolphins coinfected with this virus (Domingo et al. 1992; Kennedy 1998; Reidarson et al. 2001). In this case, no microscopic lesions compatible with CeMV infection were found in any organ, and CeMV-specific PCR from all tested samples were negative, ruling out a possible coinfection with this agent. Cunninghamella bertholletiae infection was considered to be the cause of death based on the severity of the fungus associated CNS lesions. Whether chronic stress or other underlying immune suppressive factor facilitated this CNS Cunninghamella infection is unknown. This is to our knowledge the first reported case of mucormycosis in a freeranging bottlenose dolphin and the first description of meningoencephalomyelitis caused by Cunninghamella bertholletiae in a cetacean. We thank Blanca Pe´rez and Aida Neira from the Servei de Diagno`stic de Patologia Veterina`ria, Universitat Auto`noma de Barcelona, and Carolina Go´mez from the Veterinary Mycology Group of the Universitat Auto`noma de Barcelona for valuable technical assistance. Financial support came from Direccio´ General de Medi natural i Biodiversitat, Generalitat de Catalunya, and from Servei de Diagno`stic de Patologia Veterina`ria and Servei Veterinari de Bacteriologia i Micologia (Universitat Auto`noma de Barcelona). LITERATURE CITED Abdo W, Kakizoe Y, Ryono M, Dover SR, Fukushi H, Okuda H, Kano R, Shibahara T, Okada E, Sakai H, Yanai T. 2012. Pulmonary zygomycosis with Cunninghamella bertholletiae in a killer whale (Orcinus orca). J Comp Pathol 147:94–99. Domingo M, Visa J, Pumarola M, Marco AJ, Ferrer L, Rabanal R, Kennedy S. 1992. Pathologic and immunocytochemical studies of morbillivirus

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infection in striped dolphins (Stenella coeruleoalba). Vet Pathol 29:1–10. Ginn PE, Mansell JEKL, Rakich PM. 2007. Skin and appendages. In: Jubb, Kennedy and Palmer’s pathology of domestic animals, Maxie MG, editor. Elsevier, Philadelphia, Pennsylvania, pp. 707–708. Gomes MZ, Lewis RE, Kontoyiannis DP. 2011. Mucormycosis caused by unusual mucormycetes, non-Rhizopus, -Mucor, and -Lichtheimia species. J Clin Microbiol Rev 24:411–445. Jauniaux T, Beans C, Dabin W. 2005. Stranding, necropsy and sampling: Collection data, sampling level and techniques. European Cetacean Society. http://www.europeancetaceansociety. eu/student_dls/Stranding_Necropsy_Sampling_ ECS05.pdf. Accessed March 2014. Kennedy S. 1998. Morbillivirus infections in aquatic mammals. J Comp Pathol 119:201–225. Mayayo E, Klock C, Goldani L. 2011. Thyroid involvement in disseminated zygomycosis by Cunninghamella bertholletiae: 2 cases and literature review. Int J Surg Pathol 19:75–79. Morris PJ, Johnson WR, Pisani J, Bossart GD, Adams J, Reif JS, Fair PA. 2010. Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States. J Vet Microbiol 148:440– 447.

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Submitted for publication 20 October 2013. Accepted 10 January 2014.

Central nervous system mucormycosis caused by Cunninghamella bertholletiae in a bottlenose dolphin (Tursiops truncatus).

In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead on the Spanish Mediterranean coast. At necropsy, sever...
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