# Identification of Aspergillus terreus and Aspergillus pseudonomiae as causative agents of aspergillosis in endangered Okinawa Rails

## TL;DR

This study identifies two Aspergillus species causing aspergillosis in endangered Okinawa Rails, providing insights into antifungal resistance and disease dynamics in wildlife.

## Contribution

First report of Aspergillus pseudonomiae isolated from animal hosts in Japan and genomic analysis of Aspergillus isolates in this endangered bird species.

## Key findings

- Aspergillus terreus and Aspergillus pseudonomiae were identified as causative agents of aspergillosis in Okinawa Rails.
- An A. terreus isolate showed low susceptibility to voriconazole, raising questions about intrinsic or acquired resistance.
- One A. pseudonomiae isolate lacked sclerotia and aflatoxin production, indicating possible adaptation during infection.

## Abstract

Aspergillosis is a serious infectious disease in avian species, including endangered birds. However, reports in such species remain scarce. This study focused on the Okinawa Rail (Hypotaenidia okinawae), an endangered flightless bird restricted to the northern forests of Okinawa Island, Japan.

Air sac tissues and swabs from two deceased Okinawa Rails were cultured on potato dextrose agar. Identification of isolates was confirmed by ITS and benA sequencing. Antifungal susceptibility was tested using CLSI M38 broth microdilution. Aflatoxin production of isolates was assessed by dichlorvos–ammonia method and quantified via ELISA and LC–MS. Genomic DNA from these isolates was extracted and sequenced using PacBio technology, followed by genome assembly and analysis of antifungal resistance genes and secondary metabolite clusters.

We identified and characterized Aspergillus terreus and Aspergillus pseudonomiae as causative agents of aspergillosis in two captive Okinawa Rails. In Case 1, an A. terreus isolate showed low susceptibility to voriconazole. It remains unclear whether this low susceptibility was intrinsic or acquired during antifungal treatment. Two phenotypically distinct isolates of A. pseudonomiae were obtained from the second case. One of them lacked sclerotia and aflatoxin production, suggesting a possible adaptation during infection. This is the first report of A. pseudonomiae isolation from animal hosts in Japan. Accurate identification and genomic analysis of Aspergillus isolates provide insights into antifungal resistance and the ecological dynamics of aspergillosis in Okinawa’s forest environment, with important implications for wildlife conservation.