J. Vet. Med. B 48, 551±554 (2001) Ó 2001 Blackwell Wissenschafts-Verlag, Berlin ISSN 0931±1793

Short Communication Departmento de AnatomõÂa y AnatomõÂa PatoloÂgica Comparadas, Edi®cio de Sanidad Animal, Facultad de Veterinaria, Campus Universitario de Rabanales, Universidad de CoÂrdoba, CoÂrdoba, Spain

Systemic Aspergillosis in an Oiled Magallanic Penguin (Spheniscus magellanicus) L. CARRASCO1,4, J. S. LIMA JR2, D. C. HALFEN2, F. J. SALGUERO1, P. SAÂNCHEZ-CORDOÂN1 and G. BECKER3 Addresses of authors: 1Departmento de AnatomõÂa y AnatomõÂa PatoloÂgica Comparadas, Edi®cio de Sanidad Animal, Facultad de Veterinaria, Campus Universitario de Rabanales, Universidad de CoÂrdoba, 14014 CoÂrdoba, Spain; 2Laboratorio Regional de DiagnoÂstico, Facultad de VeterinariaUFPel, Brasil; 3Centro de RecuperacioÂn de Animales Marinos (CRAM), Rio Grande, Brasil; 4 Corresponding author: E-mail: [email protected] With 2 ®gures (Received for publication August 2, 2000)

Summary This report describes a case of fatal aspergillosis caused by A. fumigatus during the recovery of an oiled Magallanic penguin. The possible role of aspergillosis as a possible complication responsible for the mortality of penguins surviving the ®rst days of treatment for oil is emphasized.

Introduction Aspergillosis, de®ned as any infectious condition caused by members of the fungal genus Aspergillus, has been considered the major disease and cause of mortality of captive 1 penguins (SaeÈz, 1971; Khan et al., 1977; Flach et al., 1990). However, there are few reports in which this disease has been diagnosed in free live or recently captured penguins (Hawkey et al., 1984). In captive penguins, the occurrence of aspergillosis has been related to different infectious diseases and nephropathy (Flach et al., 1990), and it has been demonstrated that aspergillosis is clearly age-dependent in captive penguins, occurring only sporadically in adults (Flach et al., 1990). In free-living animals, aspergillosis has been described in a penguin during convalescence from trauma (Hawkey et al., 1984). Magallanic penguins live on the southern coasts of South America, in the Paci®c and Atlantic oceans, on the Falkland Islands and on Tierra de Fuego (Harrison, 1989; Williams, 1995). They migrate to northern territories such as the Brazilian and Peruvian coasts to feed on ®sh (mostly anchovies). During migration, penguins are exposed to adverse environmental conditions, such as the pollution of the ocean by oil and derivatives. The effects of oil pollution at sea are evident in many ways, perhaps most dramatically illustrated by emaciated oil-soaked birds on beaches (Randall et al., 1980). Oil pollution has been considered an important cause of mortality in free-living penguins as well as other aquatic birds (Jehl, 1975; Randall et al., 1980). U. S. Copyright Clearance Center Code Statement: 0931-1793/2001/4807±0551 $15.00/0

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Oil pollution causes signi®cant physiological aberrations in haemoglobin, the endocrine system, sodium potassium pumps and the intestinal tract by affecting oxygen 2 transport, electrolite balance and nutrient absorption (Peakall et al., 1981). Affected animals usually show dehydration, hypotermia, skin irritation, eye ulcers and conjunctivitis (Williams, 1995; Miller and Welte, 1998), and approximately 32 % of oiled penguins die before or during the cleaning treatment (Randall et al., 1980). This report describes a case of fatal aspergillosis during the recovery of an oiled Magallanic penguin.

Materials and Methods A Magallanic penguin (Spheniscus magallanicus) of approximately 2.3 kg, 65 % of mean adult 3 weight (Stoskopf and Kennedy-Stoskopf, 1986), was found on the coast of Brazil with oiled feathers, poor body condition, hypothermia and apathy and was therefore sent to the Centro de RecuperacioÂn de Animales Marinos [(CRAM) Rio Grande, Brazil]. After examination, the penguin was treated with active coal and, on the next day, it was washed with water at 45°C with neutral detergent to remove oil from feathers. During the period of its stay at CRAM, the bird became weak and died 2 weeks after being caught. In the necropsy, several soft and greyish white nodules ranging from 1 to 3 mm in diameter were found to be scattered throughout the lung, liver and kidney. The serous membranes of the air sacs and stomach were thickened and opaque due to ®brinocaseous deposits. Tissue samples from lung, liver, kidneys and air sacs were rinsed in 20 % KOH for 20±30 min at room temperature. Microscope analysis revealed hyphae entwined around the parenchyma of the studied organs. For the isolation and characterization of the fungi, tissue samples of these organs were cultured in Sabouraud's dextrose agar, pH 5.7, and incubated for 72 h at 28°C. For the histopathological study, tissue samples from lung, air sacs, heart, liver, spleen, kidneys, glandular stomach and intestine were ®xed in 10 % buffered formalin and embedded in paraf®n wax. Sections (3±4 lm) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Grocott's methanamine silver (GMS).

Results Growth of green to grey-green colonies was obtained from all the studied organs. Hyphae were septate, with a conidia of 2±3 lm diameter, with condiophores 300 lm in length and vesicles 20±30 lm in diameter with a single series of phialides (6±8 lm in length). Morphologic characteristics identi®ed the fungus as A. fumigatus. Histopathologically the nodules observed in lung, liver and kidneys corresponded to acute aspergillosis. It was characterized by widespread necrosis in which numerous PASand GMS-positive septate hyphae, 3±5 lm across, with parallel walls and dichotomous branches, were seen growing in a radial pattern (Fig. 1). Within and around the necrotic areas heterophils and some mononuclear cells had accumulated. On the serosa of the glandular stomach and air sacs a ®brin deposit containing heterophils and hyphae with similar characteristics to those previously described was observed. In the lung and serosa of the gastric stomach invasion of blood vessels and consequent development of thrombosing vasculitis were frequently observed (Fig. 2). In the rest of the organs studied no signi®cant histopathological lesions were observed.

Discussion As in other birds, aspergillosis usually causes respiratory lesions in penguins, with involvement of lung and air sacs (Khan et al., 1977; Hawkey et al., 1984; Carrasco et al., 1993; Jensen et al., 1997), although these lesions are usually accompanied by lesions in the

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Fig. 1. Acute aspergillosis in the lung, characterized by necrosis and numerous hyphae growing in a radial pattern surrounded by cell debris, heterophils and mononuclear cells. Stained by Grocott's methanamine silver method. Magni®cation ´200.

Fig. 2. Serosa of the glandular stomach. A. fumigatus hyphae in the wall and in the lumen (arrows) of a thrombosed blood vessel are visible. Stained using PAS. Magni®cation ´400.

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liver (Khan et al., 1977; Hawkey et al., 1984). These ®ndings are considered as indicative of a systemic process, which in our case was clear as, in addition to respiratory lesions, lesions in liver, kidney and stomach serosa, as well as invasion of blood vessels, were observed. Invasion of blood vessels may be considered as evidence of the haematogenous spread of the fungus (Jensen et al., 1997). In the pathogenesis of aspergillosis in penguins, considered to be the major cause of mortality of these animals in captivity (SaeÈz, 1971; Khan et al., 1977; Flach et al., 1990), the possible role of stress caused by captivity or environmental change has been emphasized, as well as that of the transfer of the birds to new areas with very different climates (Khan et al., 1977; Hawkey et al., 1984; Flach et al., 1990). For this reason, in the recovery of oiled penguins, aspergillosis may be considered a possible complication, and it is possible that this disease is responsible for the mortality of surviving penguins during the ®rst days of treatment, in which the majority of oiled penguins die (Randall et al., 1980). On the other hand, mortality of oiled penguins has been also associated with low weight, up to 64 % of the mean adult weight (Randall et al., 1980), as was found in our case. For this reason, efforts should be made to reduce stress in the recovery of oiled penguins and strict hygiene should also be encouraged at all times, because aspergillosisfree penguins may become chronically infected, as indicated by the lesions observed in recently captive (Hawkey et al., 1984) or imported animals (Khan et al., 1977), while the stress situation may have been responsible for the fatal course of the disease.

Acknowledgements This paper was supported by grants from Plan Andaluz de InvestigacioÂn (AGR-137).

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