Coinfection with Aspergillus fumigatus and Penicillium spp. in black-necked swan (Cygnus melancoryphus) kept under human care - case reportABSTRACT
Fungal diseases resistant to antifungal agents in animals are a growing concern, making the study of these infections crucial for early detection of antifungal resistance in both animals and humans. Aspergillosis, which affects immunocompromised humans, domestic and wild animals, birds and even in vertebrates, is one such infection. The aim of this study was to identify the causative agent of the fungal infection and assess its antifungal resistance. Using a sterile swab, a sample was collected from the lesion in the air sacs during thes post-mortem analysis of a black-necked swan (Cygnus melancoryphus). Laboratory test revealed the presence of the fungus Aspergillus fumigatus and the genus Penicillium spp. To evaluateantifungal resistance, a susceptibility test was performed using the disk diffusion method with application of four antifungals: itraconazole 10 μg , nystatin 100 IU, fluconazole 25 μg, and ketoconazole 50 μg. It was found that the fungi were resistant to the antifungals used in the study. This study is important for better therapy using antifungal drugs in patients with clinical signs or suspected fungal disease.
Keywords:
antifungal resistance; wild bird; fungus; public health
RESUMO
As doenças fúngicas resistentes aos antifúngicos em animais representam uma preocupação crescente, tornando o estudo dessas infecções de suma importância para a detecção precoce da resistência aos antifúngicos em animais e humanos. A aspergilose, que afeta humanos imunodeprimidos, animais domésticos e selvagens, aves e até invertebrados, é uma dessas infecções. O objetivo deste estudo foi identificar o agente causador da infecção fúngica e avaliar sua resistência antifúngica. Com auxílio de swab estéril, foi coletada uma amostra da lesão localizada nos sacos aéreos durante a análise post mortem de um cisne-de-pescoço-preto (Cygnus melancoryphus). Após exames laboratoriais, foi identificado o fungo da espécie Aspergillus fumigatus e do gênero Penicilium ssp. Para a avaliação da resistência antifúngica, foi realizado o teste de sensibilidade aos antifúngicos utilizando o método de difusão em disco, com a aplicação de quatro antifúngicos: itraconazol 10mcg, nistatina 100UI, fluconazol 25mcg e cetoconazol 50mcg. Identificou-se que os fungos foram resistentes aos antifúngicos utilizados na pesquisa. O presente estudo é importante para uma melhor terapeutica utilizando medicamentos antifúngicos em pacientes com sinais clínicos ou suspeita de doença fungíca.
Palavras-chave:
resistência antifúngica; ave selvagem; fungo; saúde pública
INTRODUCTION
The increasing number of resistant fungal diseases in animals has raised concerns among those involved in wildlife conservation, as these pathogens are becoming more prevalent, contributing to a rise in species extinctions and significantly contributing to the loss of biodiversity (Garvey et al., 2022). In a scenario of climate crisis and biodiversity loss, zoos emerge as important conservation centers (Spooner et al., 2023).
Aspergillosis is an opportunistic fungal disease caused by infection with Aspergillus spp., a fungus widely distributed in the environment, with the potential to affect a variety of organisms, including humans, domestic and wild animals, and even invertebrates. Clinical signs are often nonspecific such as fever, loss of appetite, reduced respiratory function, and anorexia (Xavier et al., 2011).
The manifestation of aspergillosis in environments where birds are under human care raises pertinent questions about fungal transmission and the efficacy of control measures. Although there are already studies on fungal infections in birds, the occurrence of isolated and resistant fungal coinfections remains poorly described, especially in endemic South American wild animal species, such as the black-necked swan (Cygnus melancoryphus). Moreover, data on the incidence of invasive aspergillosis in birds in Brazil remain scarce, as there are no official notifications available (Rocha, 2019).
Mycosis by Penicillium spp., or penicillosis is rare in animals. However, there are few documented studies demonstrating that these genera can cause diseases in birds. Only a few species of Penicillium are considered pathogens causing systemic penicillosis in various animal species (Caro‐Vadillo et al., 2007; Guevara-Suarez et al., 2016; Zanatta et al., 2006). Infections are usually focal and rarely become systemic (Lanteri et al., 2011; Suehara and Silva., 2023).
Given this context, this case report aims to present the post-mortem identification of a coinfection by A. fumigatus and Penicillium spp. in a specimen of black-necked swan (Cygnus melancoryphus) that was under human care in a zoo in the interior of São Paulo, and to verify antifungal resistance and address the implications of the case for the health, conservation, and management of birds under human care.
CASE DESCRIPTION
A female adult of black-necked swan (Cygnus melancoryphus), weighing 3.515kg, kept under human care at the Municipal Zoo "Quinzinho de Barros" was found in the enclosure by the keeper, already dead. In the post-mortem examination, a lesion of approximately 4cm was visualized on the external part of the cranial thoracic air sac characterized by a grayish coloration, with a whitish band around the sites of spore development, with a velvety surface. Additionally, various circular structures of approximately 0.3cm with a whitish velvety appearance were observed both in the air sacs and in the lungs. (Fig. 1).
The macroscopic appearance of circular lesions of varying sizes, with whitish edges and a greenish center (circled in the image), in the left thoracic and abdominal air sacs. Source: the authors
MATERIALS AND METHODS
A sample of the lesion was collected with the help of a sterile swab and kept in Stuart transport medium for 48 hours at a temperature of 25ºC. Subsequently, the sample was inoculated on Sabouraud Agar by streaking with a metal loop and incubated in a bacteriological incubator Q-316-14 (Quimis, Diadema, Brazil) at 35°C for 48 hours. After isolating the colonies, two distinct formations of filamentous fungi were observed, in terms of micromorphology, notably, eight colonies between 40-60 mm in diameter, flat or slightly wrinkled surface with radial grooves, not dense, velvety appearance, presenting a velvety texture and color ranging from gray-green to gray-blue (Fig. 2). Additionally, two colonies of approximately 5 mm in diameter with a slightly velvety surface, light gray in color with white edges were observed (Fig. 2). For further analysis, a microculture was performed, which consists of preparing a culture on a microscopy slide for direct observation under a microscope. A piece of Sabouraud medium approximately 20 x 20 mm was placed on the slide, the samples were streaked around the medium, covered with a cover slip, and incubated in a bacteriological incubator Q-316-14 (Quimis, Diadema, Brazil) at 35°C for 48 hours. After this step, the fungus is removed, the cover slip is positioned on top of a new slide with a drop of methylene blue dye allowing the fungal structures to be observed on the microscopy.
Macroscopic appearance of fungal colonies isolated on Sabouraud Agar medium. Light gray colonies with white edges (identified by semi-arrows), with colony color varying between greenish-gray and bluish-gray (identified by an asterisk). Source: the authors
Next, an antifungal sensitivity test was performed on Sabouraud Agar, where the fungal sensitivity discs were added according to the instructions from the manufacturer and supplier CECON (Centro de Controle e Produtos para Diagnóstico Ltda. - CECON, São Paulo, Brasil), and following the standards from Veiga et al., 2020; and Yerbanga et al., 2023. The antifungals ketoconazole (50 μg), nystatin (100 UI), fluconazole (25 μg) and itraconazole (10 μg) were chosen for the sensitivity test because they are the most commonly used in veterinary practice.
RESULTS
A preliminary microscopic analysis was performed on the two colonies. In the first analysis, a filamentous fungus of the colonies was observed. Microscopically, the presence of septate hyphae, hyaline conidia, and monophialide conidiophores resembling brushes, suggestive of Penicillium spp. (Fig. 3). Filamentous fungi were also observed with septate hyphae, presenting hyaline conidiophores with smooth walls, with pear-shaped vesicles covered by phialides and columns of spherical conidia, suggestive of Aspergillus fumigatus (Fig. 4).
Optical photomicrography of Penicillium spp. stained with lactophenol cotton blue. A, B, and C: 1000X objective. D: 40X objective. Source: the authors
Optical photomicrography of Aspergillus fumigatus stained with lactophenol cotton blue. A, B, and C: 1000X objective. D: 40X objective. Source: the authors
After incubating the inoculated plates with the discs, proceed to read the inhibition halos and compare them with those in Table 1. Regarding the results of the antifungal susceptibility test, it revealed a generalized resistance to the tested antifungals. Ketoconazole, nystatin, fluconazole, and itraconazole were ineffective, as evidenced by inhibition zones that fell below established thresholds for susceptibility, suggesting a poor therapeutic outcome (Table 2 and 3).
DISCUSSION
Regarding the exposure/nesting environment, for example, previous studies have already reported that the presence of high spore loads, a high amount of organic material, excessive shading, and poor ventilation contribute to the emergence of fungal diseases in birds kept under human care (Xavier et al., 2011).
Although Penicillium is a genus known for its antimicrobial potential, this fungus can also cause penicillosis if inhaled mainly by immunocompromised individuals. The clinical signs of aspergillosis and penicillosis generally manifest as a respiratory tract disease, which can invade the trachea, air sacs, lungs, and even result in a systemic infection, affecting even the central nervous system (Lanteri, et al., 2011; Rocha, 2019).
Climate change is not only direct threats to animal and human health but also affect one health indirectly. With the increase in the planet's temperature, for example, the natural habitats of species are threatened, but it also facilitates the natural selection of fungal specimens capable of infecting animal and human species previously not possible. Without the natural habitat, the natural fauna can approach cities and humans and possibly spread fungal diseases previouslyincapable of transmission from one species to another (Artaxo, 2020).
The fungal isolation of respiratory material has been considered of limited utility in the ante-mortem diagnosis of aspergillosis in birds. However, the results suggest that the identification and antifungal susceptibility profile may be useful for monitoring therapy in avian species and for identifying where antifungal resistance may be emerging and which conditions are associated with the event (Spanamberg et al., 2020).
Another problem is antifungal resistance, the inappropriate use of some antifungals has been contributing to the increase in the selection and incidence of resistant strains. Incorrect treatment of these infections aggravates therapeutic failures and disease remission (Fernandes and Magalhães, 2021).
The indiscriminate use of antifungals has contributed to the change in the epidemiological profile of resistant fungi. In Brazil, due to the absence of official data on notifications, there are no precise reports on the first detection of resistance to antifungals in humans. The adaptability of these fungal species is presented in an accelerated manner, due to the various methods and adaptive examples to the hostile environment, contributing to generalized antifungal resistance (Fernandes and Magalhães, 2021).
Conducting new studies on antifungal resistance will be beneficial to both veterinary and human medicine, as both can share the same yeast being a reservoir for the transmission of fungal infections resistant to commonly used drugs. Thus, it is important to highlight the importance of zoos and CETAS conducting research on fungal diversity in wild animals, since these birds can have contact with different species, possibly resistant, which in the wild would not normally have contact (Silva et al., 2023).
Our study has limitations. First, while the disk method is a cost-effective screening tool and has shown over 90% agreement with the CLSI microdilution method for azoles against Candida and Cryptococcus, it has limitations for certain species and filamentous fungi (Alastruey-Izquierdo et al., 2015; Berkow et al., 2020). Although there are studies that found a good correlation between broth microdilution and disk diffusion methods, these are small studies (Esteban et al., 2005; Gupta et al., 2021). Although broth dilution methods are recommended for accurate MIC data in managing invasive infections, this technique is cumbersome and costly, making it impractical for routine use on every patient, especially in Zoological Parks, were the time and resources are often limited (Gupta et al., 2021). Disk diffusion can serve as a quick screening method that helps identify effective treatments without inadvertently promoting antifungal resistance. Unfortunately, as this is a post-mortem case study, we could not clinically evaluate the actual effects of the fungal infections. However, this study is significant because it highlights the relevance of asymptomatic resistant fungal infections, which may pose a considerable risk to a wide range of animals under human care. Additionally, we were unable to identify the fungal species at a specific level. Nevertheless, identifying the resistant isolates is a relevant advancement, as it provides valuable information about the fungal resistance profile in clinical contexts, potentially guiding future investigations and treatment strategies. Our findings underline the need for further investigation to validate the resistance profiles of the strains tested.
CONCLUSION
The results of the antifungal susceptibility tests indicate that the microorganism subjected to analysis presents a notable resistance to the examined antifungal agents, including ketoconazole, nystatin, fluconazole, and itraconazole. This is evidenced by the minimal or absence of inhibition halo around the antifungal discs used in the tests. It was concluded that the main lesion in the bird was attributed to the presence of A. fumigatus resistant to the tested antifungals, whose confirmation was based on the combination of morphological, macroscopic, and microscopic findings characteristic of Aspergillus fumigatus. Moreover, the nodular structures were attributed to Penicillium spp., evidencing a fungal coinfection in a previously healthy and asymptomatic bird.
ACKNOWLEDGMENTS
We thank the collaborator veterinary Flora Nogueira Matos.
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Publication Dates
-
Publication in this collection
14 July 2025 -
Date of issue
Jul-Aug 2025
History
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Received
10 July 2024 -
Accepted
04 Oct 2024
