A Novel Exophiala Species Associated With Disseminated Granulomatous Inflammation in a Captive Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis)

Abstract

The genus Exophiala is composed of ubiquitous, pigmented, saprotrophic fungi and includes both terrestrial and waterborne species. Though Exophiala species are generally considered opportunistic pathogens, exophialosis can be an important cause of morbidity and mortality in aquatic and semi-aquatic species. Over a 6-year period, a captive 32-year-old male eastern hellbender (Cryptobranchus alleganiensis alleganiensis), was treated for recurring, slow growing, ventral midline cutaneous masses. Excisional biopsies were characterized histologically by granulomatous dermatitis with low numbers of intralesional, pigmented fungal conidia and hyphae. Bacterial and fungal cultures of the masses and skin were negative on two separate submissions. Polymerase chain reaction amplification of a short fragment of the fungal 28S large subunit (LSU) ribosomal RNA was positive with 100% nucleotide sequence identity to several species of Exophiala. Following recurrence after successive rounds of antifungal therapy, euthanasia was elected. At necropsy, similar dermal granulomatous inflammation and intralesional pigmented fungal elements as observed in excisional biopsies formed a thick band in the dermis and extended through the coelomic body wall. Visceral dissemination was noted in the lung and kidney. Postmortem DNA sequence analysis of a large portion of the fungal LSU as well as the internal transcribed spacer (ITS) from a portion of frozen affected dermis identified the fungus as a novel species, Exophiala sp. 1 (UTHSCSA R-5437).

Keywords: amphibian; chromoblastomycosis; chromomycosis; cryptobranchid; exophialosis; fungus; phaeohyphomycosis; salamander.

Figure 1

Granulomatous mycotic dermatitis in an eastern hellbender (Cryptobranchus alleganiensis alleganiensis). (A) The dermis is expanded by a band of nodular to diffuse granulomatous inflammation with focal epidermal attenuation and extension through underlying striated muscle fibers. Hematoxylin and eosin (HE). (B) Small, sporadic, clusters of extracellular pigmented conidia are surrounded by numerous epithelioid macrophages and multinucleated giant cells amid scattered pyknotic and karyorrhectic cellular debris (HE). (C) Fungal elements are argyrophilic; conidia are accompanied by rare hyphae with thin, non-parallel walls and sporadic septation. Gomori methenamine silver (GMS). (D) Conidia and hyphae exhibit positive cytochemical reactivity within the fungal wall, consistent with a pigmented (melanin) fungus. Fontana-Masson.

Figure 2

Multicentric granulomatous mycotic inflammation in an eastern hellbender (Cryptobranchus alleganiensis alleganiensis). (A) The ventral dermis is expanded by multinodular coalescing granulomas with ulceration of the overlying epidermis. (B) Similar nodular granulomas are present within the lungs.

Figure 3

Phylogenetic tree inferred from ITS sequences showing the relationship of Exophiala sp. UTHSCSA R-5437 to known Exophiala species. Branch lengths are proportional to phylogenetic distance. ML bootstrap support values (1000 resampling, Right) >70% and posterior probability values from Bayesian inference (Left) > 0.90 are shown above the branches. Teratosphaeria karinae CBS 128774^T was designated as outgroup. ^T, type strain; CBS, CBS-KNAW Fungal Biodiversity Center culture collection, The Netherlands; NBRC, National Biological Resource Center, Japan; NCPF, UK National Collection of Pathogenic Fungi, PHE UK National Mycology Reference Laboratory and School of Biological Sciences, University of Bristol, Bristol, United Kingdom; UTHSCSA, Fungus Testing Lab, University of Texas Health Science Center at San Antonio, Texas USA. Sequence accession numbers are indicated in front of species names.