Cross-continental emergence of Nannizziopsis barbatae disease may threaten wild Australian lizards

Abstract

Members of the genus Nannizziopsis are emerging fungal pathogens of reptiles that have been documented as the cause of fatal mycoses in a wide range of reptiles in captivity. Cases of severe, proliferative dermatitis, debility and death have been detected in multiple free-living lizard species from locations across Australia, including a substantial outbreak among Eastern water dragons (Intellagama lesueurii) in Brisbane, Queensland. We investigated this disease in a subset of severely affected lizards and identified a clinically consistent syndrome characterized by hyperkeratosis, epidermal hyperplasia, dermal inflammation, necrosis, ulceration, and emaciation. Using a novel fungal isolation method, histopathology, and molecular techniques, we identified the etiologic agent as Nannizziopsis barbatae, a species reported only once previously from captive lizards in Australia. Here we report severe dermatomycosis caused by N. barbatae in five species of Australian lizard, representing the first cases of Nannizziopsis infection among free-living reptiles, globally. Further, we evaluate key pathogen and host characteristics that indicate N. barbatae-associated dermatomycosis may pose a concerning threat to Australian lizards.

Figure 1

Gross pathology of Australian lizards with severe dermatomycosis caused by Nannizziopsis barbatae (A) Free-living Eastern water dragon (white circle indicates skin lesion; case EWD006) and (B) captive Centralian blue tongue skink (case CBT001) with proliferative skin plaques [Photo credit: Dr. S. Simpson]. (C) Free-living Eastern water dragon (case EWD005) and (D) tommy roundhead dragon (case TRH001) with ulcerative skin lesions (E) free-living Eastern water dragon (case EWD006) with digital necrosis and loss.

Figure 2

Histopathological features of cutaneous lesions from free-living Australian lizards with Nannizziopsis barbatae-associated dermatomycosis (A) severe hyperkeratosis (black double pointed arrow), papillary epidermal hyperplasia (white double pointed arrow) (case EWD009; H&E). (B) Abundant fungal hyphae and arthroconidia, morphologically consistent with Nannizziopsis spp. (Case EWD009; GMS) (C) surface tuft of arthroconidia (arrow) (case EWD009; PAS). (D) Dysplastic invaginating epithelium with intralesional fungal hyphae (arrow) (case TRH001; PAS). (E) Perivascular dermal granuloma with intralesional hyphae (arrow) in the sole animal with deep dermal mycosis (case EWD008; PAS). (F) Epidermal intracytoplasmic inclusion bodies (arrowheads) (Case EWD009; H&E).

Figure 3

Morphological characteristics of Nannizziopsis barbatae, isolated from cutaneous lesions of free-living Eastern water dragons and incubated on PDA at 30 °C. (A) Colony morphology on PDA at 21 days. (B) Scanning electron micrograph showing branched fungal hyphae bearing aleurioconidia. (C) Microscopic morphology showing pyriform to clavate, mainly sessile aleurioconidia, 2.5–10 μm in length and 1.5–3 μm in width, born along septate branching hyphae and occasionally subtended by swollen cells. (D) Fission arthroconidia, 2.8–8.3 μm in length and 1.8–3.3 μm in width. (E) Abundant undulate hyphae, characteristic of Nannizziopsis species, and intercalary chlamydospores (arrows). (F) Ascomatal initials and (G) budding cells were occasionally present. Bars 20 μm.

Figure 4

Maximum likelihood trees depicting the relationship of fungal isolates obtained in this study with other members of the Nannizziopsis genus for which similar sequences were available. Analyses were performed using (A) ITS2, (B) 28S, (C) β-tubulin, and (D) concatenated (ITS + 28S + β-tubulin) sequence alignments. Midpoint-rooted consensus trees were constructed from 750 original trees produced using PhyML, based on a generalized time reversible substitution model, bootstrapped with 1000 random re-samplings of the data. Branches are supported by consensus support threshold values > 75%. Scale bars represent the mean number of nucleotide substitutions per length.