Paranannizziopsis spp. infections in wild snakes and a qPCR assay for detection of the fungus

Abstract

The emergence of ophidiomycosis (or snake fungal disease) in snakes has prompted increased awareness of the potential effects of fungal infections on wild reptile populations. Yet, aside from Ophidiomyces ophidiicola, little is known about other mycoses affecting wild reptiles. The closely related genus Paranannizziopsis has been associated with dermatomycosis in snakes and tuataras in captive collections, and P. australasiensis was recently identified as the cause of skin infections in non-native wild panther chameleons (Furcifer pardalis) in Florida, USA. Here we describe five cases of Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and one additional case from a captive snake from Connecticut, USA. In addition to demonstrating that wild Nearctic snakes can serve as a host for these fungi, we also provide evidence that the genus Paranannizziopsis is widespread in wild snakes, with cases being identified in Louisiana (USA), Minnesota (USA), Virginia (USA), and British Columbia (Canada). Phylogenetic analyses conducted on multiple loci of the fungal strains we isolated identified P. australasiensis in Louisiana and Virginia; the remaining strains from Minnesota and British Columbia did not cluster with any of the described species of Paranannizziopsis, although the strains from British Columbia appear to represent a single lineage. Finally, we designed a pan-Paranannizziopsis real-time PCR assay targeting the internal transcribed spacer region 2. This assay successfully detected DNA of all described species of Paranannizziopsis and the two potentially novel taxa isolated in this study and did not cross-react with closely related fungi or other fungi commonly found on the skin of snakes. The assay was 100% sensitive and specific when screening clinical (skin tissue or skin swab) samples, although full determination of the assay's performance will require additional follow up due to the small number of clinical samples (n = 14 from 11 snakes) available for testing in our study. Nonetheless, the PCR assay can provide an important tool in further investigating the prevalence, distribution, and host range of Paranannizziopsis spp. and facilitate more rapid diagnosis of Paranannizziopsis spp. infections that are otherwise difficult to differentiate from other dermatomycoses.

Keywords: Chrysosporium anamorph of Nannizziopsis vriesii; Onygenales; molecular diagnostics; mycoses; reptile; wildlife disease.

FIGURE 1

Gross and histopathological images of select wild snakes with Paranannizziopsis spp. infections. (A) Ventral surface of gophersnake (Pituophis catenifer) infected with Paranannizziopsis sp. 1 (NWHC 26609-1). Arrows indicate representative gross lesions. (B) Gartersnake (Thamnophis sp.) infected with Paranannizziopsis sp. 2 (20-3833). Arrows indicate representative gross lesions. (C,D) Glossy crayfish snake (Liodytes rigida rigida) infected with P. australasiensis (NWHC 26922-5) (PAS stain; scale bar, 50 μm). (C) Areas of epithelial necrosis extend to the deep portions of the epidermis (PAS stain; scale bar, 50 μm). (D) Detail of C showing an area of necrotic epithelium containing low numbers of ∼2-μm diameter faintly PAS-positive fungal hyphae (arrows) (PAS stain; scale bar, 20 μm). (E–G) Same gophersnake as in (A) (PAS stain). (E) Within the crypt between two scales is a large area of epithelial necrosis (arrow) (scale bar, 200 μm). (F) Moderate numbers of ∼2- × 5-μm ovoid aleurioconidia (arrowhead) and ∼2-μm diameter fungal hyphae (arrows) are within the area of necrosis (scale bar, 20 μm). (G) Within a crust overlying the epithelium are many ∼2- × 5-μm ovoid aleurioconidia (arrowheads) (scale bar, 20 μm). (H) Western terrestrial gartersnake (Thamnophis elegans) infected with Paranannizziopsis sp. 2 (K22 2022-047521A). Extensive full thickness necrosis and ulceration was present along the jaws and head of this snake with the necrotic debris being widely permeated by a meshwork of silver positive fungal hyphae. An area of epithelial necrosis contains many superficial ovoid to bullet-shaped aleurioconidia (arrowhead) and deep fungal hyphae is shown (GMS stain; scale bar, 10 μm).

FIGURE 2

Best tree resulting from a maximum likelihood (ML) analysis of eight concatenated loci of Paranannizziopsis spp. strains. The consensus tree from a Bayesian analysis had an identical topology. Boldened strains are those isolated from wild snakes in this study. Two of these strains resided within the P. australasiensis clade but were not identical to existing strains belonging to that species. The remaining two strains did not cluster with described taxa and may be novel species. Support values are presented at each node (bootstrap support values from the ML analysis/posterior probabilities from the Bayesian analysis). Support values within the P. australasiensis clade are not shown due to space. Type strains are designated with “(T).” The tree is rooted with Ophidiomyces ophidiicola. Branch lengths for the root have been shortened (indicated by “/”) to make the tree easier to view.