Case report of respiratory aspergillosis and candidiasis in wild Magellanic penguins (Spheniscus magellanicus), Brazil

Abstract

Magellanic penguins (Spheniscus magellanicus) migrate to the continental shelf of southern-southeastern Brazil during austral winter. Stranded penguins are directed to rehabilitation centers, where they occasionally develop fungal diseases. Aspergillosis, a mycosis caused by Aspergillus spp., is one of the most important diseases of captive penguins, while Candida sp. has been detected in penguins undergoing rehabilitation. Nevertheless, their occurrence in the wild is poorly understood. This study surveyed the occurrence of mycoses in free-ranging Magellanic penguins wintering in southeastern Brazil. These penguins were either found dead or stranded alive and died during transport to a rehabilitation center. Overall, 61 fresh to moderate autolyzed carcasses were necropsied. Upon necropsy, three juvenile males (4.9%) presented mycotic-consistent gross lesions. Histopathology and panfungal PCRs confirmed the mycoses. Major microscopic findings were marked chronic necrotizing multifocal to coalescent pneumonia, airsacculitis, and esophageal/gastric serositis with two types of intralesional fungal structures: (a) septated acute-angled branching hyphae (n = 2) and (b) yeast structures (n = 1), both PAS- and Grocott-positive. Sequences identical to Aspergillus sp. were retrieved in two cases, while the third had sequences identical to Candida palmioleophila. This study describes two cases of aspergillosis and one of candidiasis in free-ranging Magellanic penguins, confirming the species’ susceptibility in the wild. These mycoses could be associated with the animals’ poor body condition, and/or impaired immunity, and natural and anthropogenic challenges related to migration. To the authors’ knowledge, this is the first report of aspergillosis in free-ranging Magellanic penguins in the Atlantic Ocean and of candidiasis in penguins worldwide.

Introduction

Seabirds are top predators, considered sentinels of the marine environmental health [1]. This group, especially Procellariiformes (albatrosses and petrels) and Sphenisciformes (penguins), is the most threatened among seabirds [2]. According to the International Union for Conservation of Nature Red List, ten of the 18 species of penguins are listed as vulnerable or endangered and three as near threatened [3]. Magellanic penguin (Spheniscus magellanicus) is currently classified as near threatened [3]. This species inhabits the southern coast of South America, breeding in colonies located on the coasts of Chile, Argentina, and the Falkland (Malvinas) Islands [4]. During austral autumn and winter (non-breeding period), Magellanic penguins from the Atlantic/Argentinean colonies migrate along the continental shelf off the coast of northern Argentina, Uruguay, and southern Brazil, following their main food item in wintering grounds—the Argentine anchovy (Engraulis anchoita) [5, 6].

The study of diseases affecting penguins is relevant in the elaboration of adequate conservation management [7]. Among these diseases, aspergillosis is of major concern, leading to high morbidity and mortality—especially in penguins in zoos, aquariums, and rehabilitation centers [8–11]. This airborne mycosis is caused by saprophytic ubiquitous mold species of the genus Aspergillus (class Eurotiomycetes, order Ascomycetes) [12]. Among the 339 Aspergillus species described to date, those belonging to section Fumigati, especially Aspergillus fumigatus stricto sensu, are the main cause of aspergillosis in birds [13, 14]. Nevertheless, other Aspergillus species have been described in birds: A. flavus, A. niger, A. glaucus, A. nidulans, and A. restrictus [13, 15]. The lesions are usually observed in the respiratory system (lung and air sacs), although other tissues can also be affected [16]. Aspergillosis has been reported in a variety of penguin species, including emperor penguin (Aptenodytes forsteri), king penguin (Aptenodytes patagonicus) [17], Gentoo penguin (Pygoscelis papua) [18], ringed penguin (Pygoscelis antarcticus) [19], yellow-eyed penguin (Megadyptes antipodes) [20], little penguin (Eudyptula minor) [15, 17], African penguin (Spheniscus demersus) [8], Humboldt penguin (Spheniscus humboldti) [21], and Magellanic penguin [8]. Aside from Aspergillus spp., reports of fungal mycoses in penguins are scarce, limited to osteolytic mucormycosis by Mucor sp. (class Zygomycetes, order Mucorales) in a captive Humboldt penguin [22], and mucormycosis by Rhizomucor pusillus in the air sacs, lung, and intracelomic nodules of a captive Magellanic penguin [23]. Furthermore, the yeast Candida sp. (class Saccharomycetes, order Saccharomycetales) was morphologically described in cultures from tracheal swabs of Magellanic penguins undergoing rehabilitation [24]. Candida spp. are opportunistic yeast pathogens responsible for infection and disease in birds [12]. Finally, Paracoccidioides brasiliensis (class Eurotiomycetes, order Onygenales) was detected in the feces of an Adélie penguin (Pygoscelis adeliae) from the King George Island, Antarctica [25].

Based on the limited information about mycoses in free-ranging penguins, our goals were to (1) survey the presence of respiratory mycoses in Magellanic penguins that either were found dead or stranded alive and died during transport from the beach to a rehabilitation center in the southern São Paulo state coast (southeastern Brazil), between July 15 and August 15, 2018, (2) employ histopathology to characterize the lesions associated with those fungi, and (3) use molecular diagnostics (PCR) to identify the mycotic agents.

Materials and methods

In the austral winter of 2018, between July 15 and August 15, 325 Magellanic penguins were found dead in the Cardoso, Comprida, and Iguape islands or died during transport to the rehabilitation center, in Cananéia county, southern São Paulo state, Brazil. Rescue and postmortem examinations were performed by the Instituto de Pesquisas Cananéia (IPeC) and Coastal Monitoring Program – Santos Basin (Projeto de Monitoramento de Praias da Bacia de Santos - PMP-BS). All penguins with low to moderate autolysis were necropsied following standard procedures (19%; 61/325), while carcasses considered in advanced autolysis were not included in this study. Out of 61 individuals, three presented gross postmortem lesions compatible with mycotic disease (4.9%; 3/61) and were further selected for this study. Necropsies followed standard procedures [26]. The body condition was determined based on a four-category classification (good, moderate, poor, or cachectic), according with pectoral muscle development and the presence of internal and subcutaneous fat deposits [27]. Age class was established based on plumage pattern [28]. Sex was determined upon visualization of the gonads. Representative tissue samples of the main organs and tissues were collected and fixed in 10% formalin, or frozen at −20/−80 °C.

Gross and microscopic study

Histopathological evaluation by light microscopy was performed in selected tissues (heart, aorta, trachea, lungs, brain, thyroid, parathyroid, tongue, esophagus, proventriculus, intestines, liver, spleen, gall bladder, pancreas, kidneys, skeletal muscle, bursa of Fabricius, Harderian gland, uropygial gland, and supraorbital gland), solely on penguins with gross findings suggestive of mycotic disease. Formalin-fixed paraffin-embedded tissues were sectioned at 5 μm and stained with hematoxylin and eosin (HE). Additionally, when required, periodic acid-Schiff (PAS) and Grocott’s methenamine silver (GMS) were used to characterize fungal structures.

Molecular study

Following histopathologic examination, total DNA from manually homogenized samples of frozen lung and air sacs of confirmed mycosis cases was extracted using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo, Irvine, CA, USA), according to the manufacturer’s protocol. The internal transcriber spacers 1 and 2, and 5.8S rRNA gene (ITS 5.8S rRNA) were amplified by panfungal PCR using the primers ITS1 (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [29], while D1 and D2 variable regions of 26S rRNA gene were amplified by PCR using primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′), as previously described [30, 31].

Both end-point PCR reactions were as follows: a final volume of 25 μl contained 2.5 μl of 10X buffer, 2.5 mM of MgCl₂, 0.2 mM of dNTPs, 0.2 μM of each primer, 1.25 U of Platinum Taq DNA polymerase (Invitrogen, Life Technologies, Brazil), 15.25 μl of ultrapure diethyl pyrocarbonate (DPEC) sterilized water, and 4 μl of DNA template. The thermocycler program for ITS 5.8S rRNA amplification was set at 95 °C for 5 min, followed by 35 amplification cycles of 95 °C 30 s, 55 °C 30 s, and 72 °C 1 min. The final extension step was performed at 72 °C 7 min. The PCR conditions for 26S rRNA gene PCR were 95 °C for 5 min, followed by 35 amplification cycles of 95 °C 30 s, 51 °C 1 min, and 72 °C 1 min. The final extension step was performed at 72 °C 7 min. Both reactions were conducted in a Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad Laboratories, CA, USA). PCR products were read on a 1.5% agarose gel electrophoresis. The expected size of the amplicons was of approximately 600 bp and 563–642 bp for ITS 5.8S rRNA and 26S rRNA PCR protocols, respectively. Amplicons with the expected size were purified with ExoSap-IT (USB Corporation, OH, USA). When multiple bands were present, those of the expected size were excised and purified using GFX gel extraction Kit (GE Healthcare, IL, USA). Positive purified samples were directly sequenced in both directions on a ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Sequence reads were assembled using ClustalW alignment in Mega7.0 [32]. After excluding primers, consensus sequences were compared with those from GenBank/EMBL/DDBJ databases using online BLASTn search (http://www.ncbi.nlm.nih.gov/blast). The genetic distance to the closest sequences was calculated based on p-distance ((1 - p-distance) × 100).

Results

Three juvenile males, one that stranded alive and died during transport (case 1) and two that were found dead (cases 2 and 3), presented gross lesions consistent with mycotic disease (e.g., air sac thickening and plaques, pulmonary granulomas). Muscle wasting and absence of subcutaneous fat indicated overall poor body condition, ranging from thin (case 2) to cachectic (cases 1 and 3, Fig. 1). Cases number 1 and 3 were classified as fresh carcasses, and case 2 was in a moderate stage of autolysis. These three Magellanic penguins stranded in Ilha Comprida, southeastern Brazil (Table 1). All three carcasses showed anatomopathologic findings suggestive of fisheries interaction (abrasion, erythema, and bruising of the medial wing surface (cases 1, 2, and 3) and marked pulmonary congestion (cases 1 and 2), Fig. 1). The main anatomopathologic findings are described below.

Fig. 1.

Pathological findings of three Magellanic penguins (Spheniscus magellanicus) with deep mycoses. a Cachectic penguin with abrasion, erythema and bruising (yellow arrow) of the medial wing surface (case 1). b.1 Lungs with multifocal pale yellow caseous masses (case 1) affecting serosa surface and parenchyma. b.2 Incised right lung (case 1) with a multifocal to coalescent pale yellow caseous mass affecting most of the parenchyma. c Anterior thoracic, posterior thoracic, and abdominal air sacs of case 2 were thickened, with marked multifocal to coalescent, yellowish to greenish, raised, granulomatous nodules. d Multifocal yellowish raised nodules up to 0.3 cm in diameter on pleura of case 3

Table 1.

Magellanic penguin (Spheniscus magellanicus) identification (ID), age class (J, juvenile; A, adult), sex (M, male; F, female), nutritional condition (NC), status (stranded alive and died during transport, found dead), stranding location, and date

ID	Sex	Age class	NC	Status	Stranding location	Date
Case 1 098273	M	J	Cachectic	Stranded alive and died during transport	Ilha Comprida (SP) 24.95° S, 47.81° W	27-Jul-18
Case 2 099821	M	J	Poor	Found dead	Ilha Comprida (SP) 24.80° S, 47.62° W	08-Aug-18
Case 3 094181	M	J	Cachectic	Found dead	Ilha Comprida (SP) 24.94° S, 47.80° W	31-Jul-18

Case 1

The main gross findings were bilateral mild abrasion, erythema, and bruising of medial wing surfaces, generalized vascular congestion, mild to moderate central nervous system congestion, capsular thickening, and focal edema of the right scapulohumeral joint with presence of a moderate volume of friable yellowish fluid. Multifocal to coalescent whitish yellow to light-tanned, elevated friable viscous irregular masses up to 2 cm in the visceral serosal surfaces of the coelomic cavity and external surface of the air sacs were observed. Chronic, disseminated, raised, light-tanned, firm, nodular caseous masses up to 0.5 cm in diameter were observed in the pulmonary tissue, affecting the serosal surface and parenchyma (Fig. 1b.1). Once incised, such lesions revealed coalescent, light-tanned, firm caseous masses filling the alveoli and bronchi, affecting the majority of the parenchymal area (Fig. 1b.2). Moderate to marked generalized pulmonary congestion and edema were also observed. Thickening of the cervicocephalic air sac, presenting multifocal to coalescent whitish firm granulomatous nodules of approximately 0.1 cm, was reported. Presence of light-tanned granulomatous nodules of 0.25 cm in diameter adjacent to the syrinx and cranial to the heart, adhered to the esophageal serosa, was also found. Additionally, 0.1 cm in diameter pearl-like firm masses were observed in the serosa of the kidneys, syrinx, and esophagus. Mild left ventricle myocardial hypertrophy and presence of focal firm light pink nodules of approximately 1.3 cm infiltrated in the epicardial tissue, adjacent to the left ventricle. Presence of ulcers in the stomach intersection and diffuse, miliary, pseudodiphtheric, yellowish nodules in the esophageal mucosa was also observed. Other relevant findings were moderate infestation by Mallophaga ectoparasites, presence of mild pale greenish yellow viscous gastric content containing algae and unidentified nematode parasites, multiple organ congestion, and ingurgitation of large vessels.

Microscopically, lungs, air sacs, and serosa of blood vessels showed marked multifocal to coalescent granulomatous inflammation with multinucleated giant cells, hemorrhage, necrosis, and intralesional septate branched hyphae and conidiophore consistent with the genus Aspergillus (Fig. 2a and b), positively stained by PAS and GMS.

Fig. 2.

a Case 1. Lungs. Granuloma containing numerous septate branched hyphae (aspergilloma). Grocott methenamine silver. b Case 1. Lungs. Note septate branched hyphae (arrowhead) and conidiophores (black arrow) consistent with the genus Aspergillus. HE. c Case 3. Lungs. Note numerous PAS-positive yeast in a pulmonary granuloma. PAS. d.1 Case 3. Kidney. Note focal leukocytic (lymphocytes and histiocytes) interstitial infiltrate with intralesional protozoal structures. HE. d.2 Case 3. Kidney. Detailed view of protozoal structures consistent with Histomonas sp. and the leukocytic (lymphohistiocytic) response. HE

Case 2

The main gross findings were mild to moderate bilateral bruising and abrasion of the medial wing surface, thick and opaque multifocal to coalescing raised yellowish to greenish firm plaques varying from 1 to 5 mm in diameter in the anterior thoracic, posterior thoracic and abdominal air sacs (Fig. 1c), moderate to severe pulmonary congestion and edema, miliary yellowish nodules in stomach and esophageal submucosa, and presence of dark black viscous gastric content containing squid beaks and nematode parasites. The moderate autolytic state of the carcass prevented further evaluation of the remaining organs.

Microscopically, fragments of air sacs, lungs, and esophageal serosa showed marked multifocal granulomatous inflammation with multinucleated giant cells associated with necrosis and septate branched hyphae consistent with the genus Aspergillus, positively stained by PAS and GMS.

Case 3

Grossly, the main findings were bilateral mild to moderate abrasion, erythema and bruising of the medial wing surface, presence of multifocal amorphous pinkish nodular masses of approximately 4 cm in diameter adjacent to the heart and intercostal spaces, as well as diffuse firm yellowish nodules of caseous aspect on pleura (Fig. 1d), pulmonary parenchyma, stomach serosa, and intercostal spaces. A 6.5 × 3 cm light-tanned mass of irregular surface composed of coalescent nodules was present in the coelomic cavity, adjacent to the heart. Once incised, caseous content was observed. Pulmonary congestion and miliary yellowish nodules in stomach and esophageal submucosa associated with the presence of nematode parasites were also observed.

Microscopically, fragments of air sacs and lungs showed marked multifocal to coalescent granulomatous inflammation with multinucleated giant cells associated with necrosis and yeast structures (occasionally intracellular within giant cells) compatible with Candida and positively stained by PAS (Fig. 2c) and GMS. Numerous yeasts were also observed in the lumen of a cardiac artery. Additionally, mild to moderate focal lymphohistiocytic interstitial nephritis with protozoan structures consistent with Histomonas sp. were observed (Fig. 2d.1 and d.2).

Molecular findings

Positive amplification was observed for ITS 5.8S rRNA and D1/D2 regions in samples from all three Magellanic penguins. The ITS 5.8S rRNA sequences from the lung (case 1) and thoracic air sacs (case 2) shared 100% nucleotide (nt) identity to Aspergillus spp. (see Table 2). The 26S rRNA sequences of cases 1 and 2 also shared 100% nt identity to several Aspergillus spp. (see Table 2). Regarding case 3, ITS 5.8S rRNA was amplified from air sac and lung samples, while D1/D2 was amplified in an air sac sample. The retrieved ITS 5.8S rRNA sequence was identical (100% nucleotide identity) to Candida palmioleophila sequences from GenBank (e.g., MK394112 (type material) and LC317509). The same high identity (100%) was also observed between the obtained D1/D2 sequence and C. palmioleophila sequences previously described (e.g., KJ705005, KC111442, and JQ650231, Table 2). All the sequences were deposited in GenBank: the ITS 5.8S rRNA sequences of cases 1, 2, and 3 were submitted under accession numbers MN725773 (case 1), MN725772 (case 2), and MN724921 (case 3), while the 26S rRNA sequences of cases 1, 2, and 3 were deposited under accession numbers MN725079, MT256133, and MN725730, respectively.

Table 2.

Individual identification, type of tissue sample, amplified genetic region (nucleotide identities of the region internal transcribed spacer 1 (ITS 1) and 2 (ITS 2) and 5.8S rRNA gene (ITS 5.8S rRNA)), and D1 and D2 variable regions of the 26S rRNA gene obtained from Magellanic penguins after comparison with the closest sequences from GenBank

Id	Sample	Amplified genetic region
		ITS-1, ITS-2, 5.8S rRNA	26S rRNA
Case 1 SM98273	Lung	100% nt identity to Aspergillus spp. (e.g., MH141230, MH741436, KJ704773)	100% nt identity to Aspergillus spp. (e.g., MH878509, MH878496, MH877948)
Case 2 SM99821	Cranial thoracic air sac	100% nt identity to Aspergillus spp. (e.g., MH102084, MG662385, MH485381)	100% nt identity to Aspergillus spp. (e.g. CP047255, MH877419, MH876708, MH873944, FJ867931)
Case 3 SM94181	Unidentified air sac	100% nt identity to Candida palmioleophila (e.g., MK394112 (type material), LC317509)	100% nt to Candida palmioleophila (e.g., KJ705005, KC111442, JQ650231)
	Lung	100% nt identity to Candida palmioleophila (e.g., MK394112 (type material), LC317509)	Not amplified

Discussion

Respiratory mycoses were diagnosed in three out of 61 examined wild penguins that either were found dead or stranded alive and died during transport to a rehabilitation facility, in southern São Paulo state, Brazil; two of them caused by Aspergillus sp. and one by C. palmioleophila. Both agents, Aspergillus and Candida, are regarded as the most frequent genera of fungi isolated in birds [12, 33, 34]. Both are considered mainly opportunistic pathogens, able to cause diseases as a result of primary ailments or malnutrition [35], yet capable of acting as primary pathogens in avian species [12, 36]. The effort associated with Magellanic penguins’ migration from the breeding colonies in Argentina to southern Brazil and the poor body condition observed in the studied animals likely undermined their immunological system, contributing to the development of the observed mycoses. Similarly, in captive penguins with aspergillosis, host immune factors (immunosuppression due to underlying disease or stress associated with transport or captivity (for instance, inadequate exhibit/nest environment, and malnutrition caused by inadequate diet or dietary changes in captivity)), were identified as contributors for the disease [8, 9, 16, 18]. Furthermore, inadequate captive environmental conditions have been also described as contributors for aspergillosis in penguins under human care, including the presence of high spore loads, elevated amount of organic material, and deficient ventilation [12, 16]. On the other hand, the genus Candida is considered part of the avian mycobiota, commonly restricted to the upper digestive tract, mainly the crop [33]. Nevertheless, these agents can become pathogenic in face of disruption of physical, chemical, and/or immunological barriers [34].

Migrating penguins are strictly pelagic, staying on land solely during the breeding period, otherwise remaining in the continental shelf [37], and only found ashore when debilitated or dead. The most likely infection route in the two penguins diagnosed with aspergillosis was inhalation of Aspergillus conidia, as proposed in strictly aquatic free-ranging marine tetrapods (cetaceans) also affected by this agent [38, 39]. Certain characteristics of the highly efficient and specialized respiratory anatomy of birds may predispose this group to infection by Aspergillus spp. and other aerosolized pathogens: (1) absence of epiglottis and consequently more exposed lower respiratory tract, (2) absence of diaphragm, which prevents an efficient coughing reflex, (3) large air sac system with high oxygen tension, warm temperature, and less exposure to the immune system, and (4) limited distribution of pseudostratified ciliated columnar cells that collect foreign material [40]. Additionally, penguin trachea is bifurcated, which facilitates air turbulences, promoting local deposit of conidia and development of subsequent focal infections [11]. Inhalation possibly occurred while these individuals were still in the colony, where behavior (e.g., burrow-nesting) and environmental conditions (e.g., humidity and rainfall) have been suggested as possible influencing factors in the exposure to this ubiquitous agent [7]. The Candida infection source in case 3 is unknown; however, its previous isolation in asymptomatic Magellanic penguins under rehabilitation [24] suggests that this agent could be part of the species’ mycobiota. The penguins diagnosed with aspergillosis (cases 1 and 2) and candidiasis (case 3) in this study were juveniles, which is in accordance with previous reports of higher mycoses susceptibility in young birds, likely associated with an immature immune system [33, 41].

The genetic regions selected in this study —ITS-1 and ITS-2 and D1 and D2 variable regions of the 26S rRNA gene—are considered reliable fungal universal DNA barcodes [42]. The 100% identity observed between our yeast sequences and C. palmioleophila confirms its identification in the air sac and lung samples of case 3. In yeast, the percentages of nucleotide identity established as threshold for species identification are, respectively, 98.4% for ITS and 99.5% for D1 and D2 variable regions of the 26S rRNA gene [42]. Nevertheless, there are no threshold values for molds [43]. The ITS and D1-D2 sequences obtained in this study presented 100% identity to several species of Aspergillus.

The observed chronic aspergillosis lesions in the lung and air sacs (cases 1 and 2) were similar to those previously described in penguins [12, 16] and are consistent with chronic aspergillosis [44]. The air sacs, especially the posterior thoracic and abdominal, are considered primary sites of infection in birds due to their respiratory physiology [45], eventually affecting the lungs and other tissues.

Candida palmioleophila was amplified in air sac and lung samples of one Magellanic penguin (case 3). In the past, this yeast species was often mistaken with other Candida species—C. famata and C. guilliermondii [46]. Confirmed reports of C. palmioleophila include opportunistic infections in humans, leading to endogenous endophthalmitis and intravenous catheter-associated fungemia [47, 48]. In birds, C. palmioleophila has been described in scarlet ibis (Eudocimus ruber) and in turkeys (Meleagris gallopavo), without associated lesions [34, 49]. Despite being described in several bird species, to the authors’ knowledge, there is a single report of Candida sp. in penguins: in Magellanic penguins undergoing rehabilitation in Brazil that did not present any associated lesions [24]. The candidiasis lesions observed in the air sacs and lung of case 3 were similar to C. albicans and C. tropicalis granulomatous nodular lesions found in the lung and air sacs of two fowls [50]. Although respiratory candidiasis is considered rare in birds [36], C. albicans has been cultured from respiratory lesions of several bird orders in captivity [19, 51]. Additionally, Candida spp. have been associated with alimentary (the most common infection site), reproductive, dermal, ocular, and systemic lesions [12, 33]. Based on our findings, the respiratory mycoses found in this study contributed to the animals’ death. Additionally, this animal presented nephritis with intralesional structures consistent with Histomonas sp. similar to those described by Sentíes-Cué et al. [52]. This protozoan was previously described in renal tissue of birds [53], also associated with inflammation [52]. To the authors’ knowledge, this is the first description of histomoniasis in penguins worldwide.

To the authors’ knowledge, this is the first report of aspergillosis in free-ranging Magellanic penguins and of respiratory candidiasis in penguins worldwide. Interestingly, antibodies against Aspergillus sp. were found in 15.1% (61/405) of free-ranging adult Magellanic penguins from colonies in Argentina [7], reinforcing the hypothesis that the animals in our study had contact with the agent while in the colony instead of during migration/wintering. Aside from Magellanic penguins, exposure to Aspergillus has been described in free-ranging nesting blue penguins (100%, 8/8) from New Zealand and in free-ranging yellow-eyed penguins (60%, 90/150) from New Zealand and two Subantarctic islands [54]. On the other hand, southern rockhopper penguin (Eudyptes chrysocome, n = 9) from a Subantarctic island and Adélie penguin (Pygoscelis adeliae, n = 5) from an Antarctic colony tested seronegative [54], while no Aspergillus exposure was observed in free-ranging Humboldt penguins (n = 32) from Peru [55].

Reports of postmortem gross and microscopic diagnosis of aspergillosis in free-ranging penguins are limited. Hocken [56] described macroscopic lesions compatible with respiratory mycosis, possibly aspergillosis, in seven of 213 (3%) necropsied malnourished blue penguins from New Zealand. Obendorf and McColl [57] microscopically diagnosed aspergillosis in lung and air sac samples from two free-ranging adult little penguins from Australia (of an overall number of 48 that were collected during an episode of unusual mortality – 4.2%). Finally, Gill and Darby [20] reported histopathologic lesions consistent with aspergillosis in a yellow-eyed penguin chick found dead in New Zealand during a mass mortality event of unknown etiology that killed over half of the colony’s adult population. Interestingly, some of the adult carcasses (n = 9) studied by Gill and Darby [20] were Aspergillus-seropositive but failed to present lesions consistent with this mycosis [54].

This study diagnosed mycoses in three out of 61 free-ranging Magellanic penguins, including the first two cases of aspergillosis in wild penguins from the Atlantic Ocean and the first case of respiratory candidiasis in penguins worldwide. The most likely predisposing factor for these mycoses in the studied animals was immunosuppression, associated with malnutrition and the effort invested in this species’ winter migration. The young age of these animals may have also been a predisposing factor. Based on our results, Aspergillus sp. and C. palmioleophila should be considered morbidity agents in free-ranging Magellanic penguins.

Author contribution

ACE/ADB/CS: conceptualization, methodology and analysis, writing-original draft. RZR/PENS/MAG/PCSC: methodology and analysis and research. CS/JLCD: writing -review and editing, supervision, and analysis.

Funding

This study was financed by the Coordination and Improvement of Higher Level or Education Personnel (CAPES), Brazilian National Council for Scientific and Technological Development (CNPq) (grant numbers 304999-18, 165364/2018-1, and 141868/2019-8), São Paulo Research Foundation (FAPESP) (grant numbers 2018/20956-0 and 2018/25069-7), and Projeto de monitoramento de Praias da Bacia de Santos (PMP-BS), the former required by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) of the Brazilian Ministry of Environment for the environmental licensing process of the oil production and transport by Petrobras.

Data availability

All data generated or analyzed during this study are included in the manuscript.

Code availability

Not applicable.

Declarations

Ethics approval

The samples used in this study were collected as part of the Santos Basin Beach Monitoring Project (Projeto de monitoramento de Praias da Bacia de Santos - PMP-BS), licensed by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) of the Brazilian Ministry of Environment under ABIO N° 640/2015, and in full compliance with specific federal permits issued by the Brazilian Ministry of Environment and approved by the Biodiversity Information and Authorization System (SISBIO 59150-4). All procedures were performed according to the Ethical Committee in Animal Research of the School of Veterinary Medicine and Animal Sciences, University of São Paulo (process number 1753110716).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests