Aspergillosis in free-ranging aquatic birds.

Due to the difficulty in the access to free-ranging birds, data regarding Aspergillus infections in wild avian species is rare compared to captive wild and domestic birds.
OBJECTIVE: report three cases of Aspergillus section Fumigati causing fungal disease in free-ranging aquatic birds, with the identification of the causal agent to the species level. CASE REPORTS: The diagnosis of aspergillosis was performed by macroscopic lesions found during the necropsy and confirmed by culture. Molecular identification by partial sequencing of the calM and benA genes allowed to confirm Aspergillus fumigatus sensu stricto as the etiological agent of aspergillosis in Procellaria aequinoctialis (White-chinned petrel) (n = 1), Nannopterum brasilianus (Neotropical cormorant) (n = 1) and Chroicocephalus maculipennis (Brown-hooded gull) (n = 1).
CONCLUSION: Larger studies regarding the importance of aspergillosis in free-ranging aquatic birds are necessary, as well as it potential role in the One Heath context.

Introduction

Aspergillosis is a common fungal disease in birds, mainly due to their physiological and anatomical characteristics of the respiratory system [1]. This mycosis is widely reported in captive [[1], [2], [3]], but less frequently in free-living birds [4].

Aquatic birds are species that live in marine or freshwater environments mainly for foraging activities. Procellaria aequinoctialis (White-chinned petrel), classified as vulnerable according to Red List of the International Union for Conservation of Nature (IUCN), has a circumpolar distribution, breeds in sub-Antarctic oceanic islands, and migrates to the southern Brazilian ocean to feed, mainly during non-breeding season, in the austral winter. Chroicocephalus maculipennis (Brown-hooded gull) is classified as a “least concern” resident species according to IUCN. In Brazil, it breeds in the interior of the country and migrates to the coast in the non-breeding period. Nannopterum brasilianus (Neotropical cormorant) is, according to IUCN, a “least concern” species, non-migrant, with distribution from South America to southern United States [5,6].

Due to the difficulty in the access to free-ranging birds, data about Aspergillus infections in wild avian species is scarce. There are some reports of epizootic deaths related to feeding of contaminated food as moldy corn and rotten silage [4], beyond this, reports of aspergillosis in aquatic birds are less common than those in terrestrial birds [4].

Given that migration is a natural behavior in some species, birds could be associated with the introduction of new and foreign fungal strains in distinct regions. Since fungal reproductive structures are commonly produced in air sacs of these animals, it is possible that they can exhale conidia during breathing [1]. It is also possible that transmission could occur from decaying cadavers. Additional information about aspergillosis in free-ranging birds is an important tool for a better comprehension and monitoring of the disease in nature. Therefore, this study aimed to describe three cases of the Aspergillus infection in free-ranging aquatic birds, with the molecular identification of the causal agent to species level.

Cases

During the years 2017 and 2018, three fatal proven cases of aspergillosis in free-ranging aquatic birds from three distinct species were diagnosed in the Center of Recovery of Marine Animals (CRAM-FURG) in Rio Grande, Rio Grande do Sul, South Brazil. The three birds were severely ill, were gathered in Cassino beach (32° 9′40.71″S e 52° 5′51.71″O), Rio Grande, Rio Grande do Sul, South Brazil and died during their transportation to the Center.

Infected avian individuals were a male N. brasilianus weighing 840 g (reference weight 1200–1400g), a female P. aequinoctialis weighing 762 g (reference weight 1020–1550g), and a male C. maculipennis weighing 308 g (reference weight 290–360g). The three birds were adult and none of them showed evidence of traumatic lesions during the carcass examination.

White-yellow granulomas (ranging from 0.1cm to 0.5cm) were found in the air sacs and in the lungs of the three birds. The petrel and the cormorant showed thickening of air sacs and fungal colonies in the air sacs and in the bronchi. In addition, granulomas were also found in the kidneys of the petrel. (Fig. 1).

Fig. 1

Macroscopic lesions of aspergillosis in free-birds: A) Fungal colonies in the air sacs of Procellaria aequinoctialis; B) White-yellow granulomas in the lungs and air sacs in Chroicocephalus maculipennis; C) Fungal colonies in air sacs in Nannopterum brasilianus; D) White-yellow granulomas in kidney and peritoneum of Procellaria aequinoctialis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Tissue samples from the three birds were cultured on Sabouraud Dextrose Agar and pure blue-green fungal colonies were obtained after 48h incubation at 30 °C. Isolates were identified phenotypically (macro- and micromorphology) as Aspergillus section Fumigati. DNA extraction was performed and the identification of isolates at the species level was done by molecular sequencing of partial calM [7] and partial benA genes [8].

Sequencing of partial benA was performed by ACTGene Análises Moleculares Ltd. (Center for Biotechnology, UFRGS, Porto Alegre, RS, Brazil), and sequencing of partial calM was performed at National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal. Nucleotide sequences were aligned using the “Mega” software version 7.0 and species homology was defined by comparing the nucleotide sequences by BLAST on the website https://blast.ncbi.nlm.nih.gov/Blast.cgi. The sequencing data showed that Aspergillus fumigatus sensu stricto was the etiological agent of aspergillosis in the three birds. Sequences obtained were deposited in GenBank data base [Accession numbers (benA: MN250367, MN250368 and MN250369) and (calM: MN746387, MN746388 and MN746389)].

Discussion

It is well established that aspergillosis is a highly prevalent disease in captive birds [2,9]. However, there is no agreement in the scientific literature about the importance of this disease in free-living birds. While some authors consider aspergillosis as rare in free birds [10], others affirm that this is the most common disease among medical conditions not induced by trauma [11,12]. Our studies, focused on three different aquatic avian species found in nature (in south Brazil) and infected by Aspergillus spp., indicate the need for more investigation about aspergillosis in free-ranging birds.

Both the petrel and the cormorant were in poor physical condition at the time of the diagnosis, but we cannot assume that this condition was a predisposing factor to aspergillosis development, since it could also be a consequence of the disease progression [13]. In contrast, the gull was in good general physical condition, and no evidence of a known predisposing factor was observed, suggesting that aspergillosis could have occurred as a primary disease in this bird [12].

Some species of aquatic birds travel for long periods during the migration, even crossing oceans, and are, therefore, difficult to access [14]. The occurrence of neotropical cormorants is more common among the Patos lagoon estuary (32° 02′ 6.00″ S/52° 05′ 55.00″ W), but groups of these birds in the Cassino beach are frequently observed. It is important to highlight that the region of Patos lagoon estuary is highly populated, with animals and humans sharing the same environment. Similarly, the brown-hooded gull occurs in both environments cited previously but in the open-air garbage dump too. On the other hand, the white-chinned petrel, a migrating oceanic bird, feeds in the Brazilian waters, and reproduces in sub Antarctic islands. The occurrence of these birds in the beaches may occurs as consequence of injury, debilitation, or diseases.

Aspergillus fumigatus sensu stricto, the etiologic agent of all the aspergillosis cases from our study, remains the main causal agent of the Fumigati section involved in avian aspergillosis [2,3,9], as well as in mammals affected by the disease [1]. Besides the production of gliotoxin, an immunosuppressive mycotoxin [15], the thermotolerance of A. fumigatus sensu stricto could be a factor that promotes a better fungal growth at avian body temperatures [16].

Our data, showing the occurrence of aspergillosis in free-living birds acquired in a natural environment, stress that the dissemination of pathogens by migrating birds should also be considered [17]. With the emergent concern regarding azole resistance in Aspergillus fumigatus, the environment plays an important role in this context. Since most environmental strains can infect and cause aspergillosis in susceptible hosts, without differences in virulence attributes of clinical versus environmental isolates [18], there is a need for for thinking about the spread of potential pathogenic fungi as a global concern [19].

The human population in coastal areas is greater than inland [20], and humans share this environment with other animals, such as aquatic birds. In this context, and taking in consideration the previously mentioned concerns for Aspergillus resistance and the potential importance of dissemination of pathogens by migrating birds, free-ranging aquatic birds should be monitored for aspergillosis in order to increase the knowledge of Aspergillus epidemiology in the context of One Health, in which humans, animals and environments are understood to be all connected [19].

To our knowledge, this is the first report of aspergillosis in the bird species Procellaria aequinoctialis, Chroicocephalus maculipennis and Nannopterum brasilianus. The obtained data suggests that aspergillosis in wild birds could be more prevalent than previously thought.

Declaration of competing interest

There are none.