Aspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018-20: all isolates were counted and characterized by sequencing. When identified as A. fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for MICs determination. In the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains.

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Aspergillosis is a fungal infection caused mainly by Aspergillus fumigatus that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental A. fumigatus infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (Spheniscus humboldti). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Escherichia coli Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti-A. fumigatus Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in E. coli Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti-E. coli IgY and anti-α-Gal IgY in the E. coli Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that E. coli Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Aspergillosis is an opportunistic fungal infection due to Aspergillus spp., and primarily A. fumigatus. The disease is pervasive in avian populations, especially those under managed care. The incidence and the management of avian aspergillosis can be critically impacted by exposure to high levels of spores via environmental contamination and by strains resistant to azole drugs. The present study aimed at assessing the impact of environmental contamination on the clinical incidence of aspergillosis in penguins managed under human care. ZooParc de Beauval was chosen for the investigations, because this zoological facility hosts a large colony of Humboldt penguin (Spheniscus humboldti) and it is geographically located in the rural countryside of the Loir-et-Cher district, France, surrounded by numerous large crop fields Material and methods: The fungal contamination in the environment was assessed through three serial sessions of surface sampling in bird nests: all isolates were counted, identified by DNA sequencing, and then systematically screened for resistance mutations and MICs (minimal inhibitory concentrations) elevation for the A. fumigatus strains. In addition, the clinical incidence of aspergillosis was evaluated in the penguin population over a 3-year period. A microsatellite-based analysis tracked A. fumigatus isolates circulation. Results Environmental investigations highlighted a substantial increase in the fungal load during the Autumn season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati (ranging from 22.7 to 94.6%) (Fig. 1). Only one cryptic species (A. nishimurae) and one isolate exhibiting resistance mutation (G138S in the cyp51A gene; MIC itraconazole >4 μg/ml) were detected. The overall incidence of aspergillosis was measured at ∼3.4% case-years and was observed mostly in juvenile penguins (Fig. 1). The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus isolates, except for one strain that was largely over-represented during the Autumn sampling session. A limited number of isolate genotypes was collected from clinical cases and from the environment. Conclusions: Fungal environmental contamination and incidence of aspergillosis in penguins appeared variable depending on seasons, but the rural location of the penguin habitat did not seem to influence the emergence of resistant strains.