Avian Aspergillosis

Allison German, BVSc, MSc, MRCVS

E-mail: AllisonGerman@yahoo.com

Langford School of Veterinary Science, Bristol University from January 2001

Table of Contents

1  Aspergillus spp. and Avian Aspergillosis: An Overview

1.1 The agent and the environment

Aspergillus fumigatus is an opportunistic, ubiquitous soil saprophyte. It is a mycelium-forming true fungus, classified as an Ascomycete, capable of asexual reproduction. The fungus may be found as a contaminant of animal feeds, straw or rotting plant or animal matter, with growth promoted by poor hygiene. Inadequate ventilation and dusty environments increase the risk of exposure (Oglesbee, 1997). An example of the importance of poultry litter as a source and predisposing cause of Aspergillus spp. is given by Huton (1966). Forbes (1990) describes an increase in incidence in aspergillosis in a raptor collection due to the use of contaminated shredded wood bark.

1.2 Risk factors

Factors predisposing to an increased risk of aspergillosis include stress (shipping, quarantine, capture, overcrowding); concurrent disease (especially prolonged illnesses resulting in exhaustion of the immune system or immunosuppressive conditions); trauma (injury, smoke inhalation); immunosuppressive therapeutics (corticosteroids); poor husbandry (environmental contamination, overcrowding); poor diet (malnutrition, hypovitaminosis A); prolonged antibiotic therapy (tetracyclines) (Oglesbee, 1997).

1.3 Prevalence and susceptibility

Avian aspergillosis was first described in the early eighteenth century; the first case recorded in England being in 1832, by Owen, in a flamingo (cited by Campbell, 1973). The diversity of avian species affected by aspergillosis is illustrated by Rao and Acharjyo (1988), both in their review of literature and their retrospective study. Over a 15 year period at Nandankanan Zoo, 300 out of 1500 birds, representing 11 species were diagnosed with the mycosis on post mortem. The authors noted a particular problem with peracute disease in chicks.

The causal agent of avian aspergillosis is usually Aspergillus fumigatus Fresenius, with occasional reports of A. flavus and A. nidulans (Beneke, 1980). A. terreus has also been reported in a captured pigeon (Pal, 1992). Individual strains differ markedly in pathogenicity. Other mycotic agents in birds include Candida spp., Dactylaria gallopava, Sporothrix (Sporotrichum) schenckii, Absidia ramosa, Rhinosporidium seeberi (now reclassified as a protozoan), Microsporum spp. and Trichophyton spp. (Beneke, 1980). There have also been reports of Mucor spp. (puffins), Geotrichum candidum (pelican) (Albers, 1975, cited by Stoskopf and Kennedy-Stoskopf, 1986), blastomycosis (Humboldt penguin) and actinomycosis (black-footed penguin) (Heidenreich and Hinz, 1978, cited by Stoskopf and Kennedy-Stoskopf, 1986).

Cases of aspergillosis in free-living birds are rare, being mostly linked to poor weather conditions and climate. In a compilation of reports of mycoses in India over the period 1944-1975, in addition to cases in domestic species, pharyngeal isolates of Aspergillus spp. were made from sparrows, pigeons and a parrot (Monga, 1972, cited by Monga and Mohapatra, 1980). The disease can also be seen secondarily to lead poisoning in wild birds (Locke et al, 1969)

Penguins represent a species in captivity particularly susceptible to aspergillosis (Ainsworth and Rewell, 1949), as illustrated by an epidemic at London Zoological Gardens, where 19 out of 24 King penguins died (17 due to aspergillosis) following importation. Campbell (1973) substantiated this observation, with a reported 100% mortality due to acute aspergillosis in a group of Humboldt penguins, imported to the Royal Zoological Gardens, Dublin, from South America.

Other susceptible species are outlined in Table 1.

Raptors Goshawks (Accipiter gentilis)
Gyrfalcons (Falco rusticolus)
Immature red-tailed hawks (Buteo jamaicensis)
Golden eagles (Aquila chrysaetos)
Rough-legged hawks (Buteo lagopus)
Bald eagles (Haliaeetus leucocephalus) with lead poisoning
Psittacines Blue-fronted amazon (Amazona aestiva aestiva)
African grey (Psittacus erithacus)
Hill mynah (Gracula religiosa)
Cockatoo
Others Eider ducks
Trumpeter swans
Penguins

Table 1: Avian species highly susceptible to aspergillosis (Adapted from Redig, 1993)

1.4 Pathology

There are two recognised manifestations of the disease, the clinical outcome being dependent on the extent of imbalance between the host respiratory immune defences and the ability of the organism to germinate and produce an invasive mycelium (Oglesbee, 1997). Acute cases are seen following inhalation of a high dose of spores, due to heavy environmental contamination (Redig, 1993). The fungus rapidly colonizes the lungs and the host mounts a granulomatous response. Severe terminal dyspnoea is the main clinical sign, though listlessness, pyrexia, diarrhoea and convulsions may be observed. Treatment is usually ineffective and diagnosis is on post mortem. The histological appearance includes miliary fungal granulomas throughout the lungs, with multinuclear cell infiltrates (Oglesbee, 1997). Lesions are also often present in the anterior thoracic air sacs (Buxton and Fraser, 1977). Severe air sacculitis has been shown to develop in the air sac interstitium within 24 hours of experimental inoculation in turkeys. Subpleural pneumonia and pleuritis, from haematogenous spread, also developed within 24 hours (Kunkle and Rimler, 1996). The chronic form is the most common manifestation, resulting from inhalation of a normal spore load by a stressed or immunosuppressed adult bird. Primary colonisation of the air sacs and syrinx occurs (areas of high oxygen tension and poor perfusion), with spread of spores throughout the respiratory system following sporulation. Luminal fungal plaques and necrotic debris may obstruct the airways. Sporulation can be observed grossly as dark fruiting bodies. Owls tend to form well-encapsulated granulomas and thus can tolerate the fungus better (Oglesbee, 1997; Ainsworth and Rewell, 1949).

Haematogenous spread may result in disseminated lesions, involving pneumatic bone, peritoneum, internal organs or the CNS (Redig, 1993). Localised lesions at the point of entry may develop. The disease progression is slow and onset insidious, hence diagnosis prior to extensive lesions is rare (Oglesbee, 1997).

1.5 Clinical signs

Acute aspergillosis usually presents fairly non-specifically, with inappetance, depression, polydipsia, polyuria, dyspnoea, cyanosis, or sudden death without prior clinical signs (Oglesbee, 1997; Reidarson and McBain, 1992, Baumgartner, 1988). The chronic syndrome shows symptoms dependent on the area of invasion. Clinical signs often result from obstruction of the airways by the mycelium and through systemic effects following toxin release (Redig, 1986). Hypersensitivity responses in certain individuals may exacerbate the lesions. A change in voice can be pathognomonic, progressing to dyspnoea, tachypnoea and exercise intolerance. Upper respiratory tract involvement may present as mucoid to mucopurulent nasal discharge, beak malformation and nasal or periorbital swelling. Non-specific signs, such as weight loss, diarrhoea and lethargy are often seen, sometimes as the only indication of disease (Baumgartner, 1988). Hepatic involvement may cause biliverdinuria and hepatomegaly; CNS involvement ataxia, torticollis and seizures or hind limb paresis/paralysis (Oglesbee, 1997).

1.6 Avian aspergillosis: Epidemiology and case reports

1.6.1 General

Analysis of data over the period 1969-1976 by Sheridan (1980), revealed an annual incidence of 4% for avian aspergillosis in Ireland. Higher mortalities were seen in young birds. The disease was a sporadic problem in pheasant rearing, incidence varying between 0 and 10%, depending on environmental contamination of bedding. He postulated that penguins, such as those involved in the Dublin outbreak (Campbell, 1973) may harbour A. fumigatus as a saprophyte in their lungs, with resulting systemic imbalance allowing colonisation and disease development.

1.6.1.1. Ostriches

20% mortality due to aspergillosis was described in ostrich chicks over a three month period by Perelman and Kuttin (1991). No respiratory signs were observed, just lethargy and anorexia. On post mortem, granulomatous pneumonia and fungal osteomyelitis was observed. The source of infection was environmental contamination. Individual cases of mortality, following mis-diagnosis and prolonged antibiotic therapy, have also been reported in adult birds (Marks et al 1994; Fitzgerald and Moisan 1995)

1.6.1.2. Waterfowl

Syringeal aspergilloma, causing death by asphyxiation, was reported in three Canada geese (Branta canadensis) by Stroud and Duncan (1982) following routine post mortem work at a wildlife refuge. Abnormal heterophils were observed in a king shag (Phalacocorax albivenier) which died of aspergillosis, despite miconazole therapy, four weeks after its arrival into captivity (Hawkey et al, 1984). These heterophils have since been reported by the authors in other avian species suffering non-mycological disease. A juvenile red-crowned crane (Grus japonensis) that died with respiratory signs was found to have a pulmonary aspergilloma on post mortem (Stroud and Duncan, 1983). Examination of the Bursa of Fabricius revealed active lymphoid tissue, consistent with the granulomatous response to the A. flavus infection.

A serological survey by Astorga et al (1994) in the Guadalquivir Marshlands, Spain, showed 1.1% of 712 wild waterfowl to have anti-Aspergillus fumigatus antibodies, as determined by agar gel immunodiffusion, using commercial A. fumigatus somatic antigen. Species represented included grey heron, mallard, pochard, coot and green-winged teal.

1.6.1.3. Zoological collections

Captive birds tend to be more susceptible to infection. A report of 78 cases of aspergillosis in birds from the London Zoological Gardens was detailed by Ainsworth and Rewell (1949). Diagnosis was made at post mortem over a two year period. An outbreak of aspergillosis in a zoological park aviary, where six passerines died with Aspergillus spp. infection, was investigated by Dykstra et al (1997). Stressors identified included high temperatures due to failure of the air conditioning system, nearby building work, high visitor throughput and nesting activity. The environmental spore load was investigated using a gravitometric (drop plate) method, swabs of surfaces and cultures of food, soil and nest material. A high load of fungal spores was not detected. Subsequent comparison of air sampling methods showed a volumetric impaction method to be more sensitive and allow quantitation.

1.6.2 Free-living birds

1.6.2.1. Psittacines

High mortality rates in Philippine Red Vented Cockatoos revealed 74% to be due to aspergillosis (Burr, 1981). Only close confined birds, rather than those in flight aviaries, were affected, with 65% of confirmed cases occurring at the start of or during the monsoon period. No source of infection could be found in the captive situation. Investigation of the wild population found a high percentage of deaths occurring at the start of the monsoon. Cultures demonstrated 89% of nest material to be positive for A. fumigatus, compared to 4% for other local psittacine species. The choice of nesting areas by the cockatoos as hollow rotten trees containing humus material, compared to dry shallow trees, selected by other psittacines, was implicated.

1.6.2.2. Gulls

Threlfall (1967) reviewed disease in herring gulls (Larus argentatus Pontopp) and reported four cases of aspergillosis. In view of the susceptibility of young gulls to the disease when being raised in captivity, he theorised that the mycosis might be endemic in the wild, becoming unmasked by the stress resulting from captivity. An outbreak of aspergillosis in young captive herring gulls, selected for pesticide study, resulted in a 23% mortality rate (Friend and Trainer, 1969). Thiamine deficiency was identified and treated after characteristic clinical signs in the first few weeks. It was concluded that the gulls were carriers of the fungal spores, developing overt disease following the stress of captivity and nutritional deficiency, rather than developing acute disease from primary exposure. Corroboration of the above theories was proved by Brand et al (1988) who investigated disease in wild birds at a wildlife refuge. 31% of birds examined were affected, 92% of these being gulls, and 96% of the herring gulls being sub-adult. Young gulls were significantly over-represented, with aspergillosis being epizootic (affecting many animals in the same region at the same time) during late summer, early autumn, when the influx of sub-adult gulls is high.

1.6.2.3. Mallards

A 13% mortality in mallards (Anas platyrhynchos) overwintering on a seepage ditch was found to be due to aspergillosis (Pearson, 1969). A potential source of infection was mouldy corn, though infection could not be reproduced when the feed was given to experimental mallards. Another die-off in mallards due to acute aspergillosis was reported by Bair et al (1988), with the likely cause again implicated as a combination of severe weather and poor quality corn. 86% of the birds were in poor body condition. Two epornithics (an epidemic in a population of birds) were reported by Adrian and colleagues (1978) in October 1975 (n=270) and 1976 (n=117) in Denver, Colorado. The 1975 outbreak was preceded by heavy snow; both outbreaks lasted less than seven days. The pathology indicated acute disease (miliary granulomas in lungs; air sac plaques), with a few birds having alimentary involvement. Aspergillus spp. were identified from cultures. 70% of birds were juveniles. Cultures from nearby ensilage pits, where the birds could feed, were positive for Aspergillus spp.

1.6.2.4. Die-offs in other species

Mortality of 1000-1500 common crows (Corvus brachyrhynchos) due to aspergillosis was reported in a Game Management Area in Nebraska by Zinkl and colleagues (1977). A potential causal factor was dry weather, resulting in compromise of the muco-ciliary respiratory epithelium. An 18% prevalence of aspergillosis was recorded in common loons (Gavia immer) collected form the Florida coastal regions, by White et al (1976), over a five year period. Surprisingly, there was a statistically higher prevalence in non-oiled loons, as compared to oiled loons, during an oil spill.

1.6.2.5. Raptors

Wild goshawks (Accipiter gentilis atricapillus) were trapped and blood sampled on their migration through Minnesota during the period 1972-73 (Redig et al, 1980). Tracheal swab and culture on SDA revealed 53% positive birds in 1972, compared to 7% in 1973. A. fumigatus was isolated from all cases; A. terreus and A. niger found in mixed cultures in a few cases. Chronic aspergillosis was diagnosed in 67% of captured birds in 1972, compared to 12% in 1973. The authors suggested this provided evidence of an epizootic of aspergillosis in goshawks during 1972. Predisposing factors were likely to be an increase in goshawk numbers, alongside a decrease in the prey populations, coinciding with the stress of migration.

A case of generalised disseminated aspergillosis was reported in an Abyssinian tawny eagle (Aquila rapax raptor) shortly after capture from the wild (Fatunmbi and Bankole, 1984). Diagnosis was made on post mortem, culture, microscopic and histopathological examination. A great horned owl (Bubo virginianus) died at a rehabilitation centre after treatment for emaciation and dehydration. On post mortem, miliary granulomas were observed in the lungs and air sacs, which produced a fungal growth consistent with Aspergillus spp. on culture on Sabouraud agar (Clark et al, 1987). Aspergillosis has also been reported in wild eagles (Coon and Locke, 1968; Horner, 1989), characterised by an alteration in feeding behaviour.

1.6.3 Psittacines

1.6.3.1. Captive psittacines

In a retrospective study by McMillan and Petrak (1989), 45 cases of psittacine aspergillosis were reviewed. They found a high predilection for disease in young, newly imported birds, with Blue-fronted Amazons (Amazona aestiva) being over-represented, often having aspergillosis in conjunction with pox virus infection. Poor nutrition (seed diet) was implicated as a predisposing factor, particularly the development of hypovitaminosis A. Weight loss, emaciation and respiratory distress were the most common clinical signs. Neurological cases were also recognised. Most cases died shortly after presentation, although the nature of the disease was found to be chronic on post mortem. Histopathological results correlated with semi-invasive disease; 66% of cases had granulomatous pneumonia, 53% air sacculitis. Non-specific radiographic findings consistent with pneumonia and consolidating air sacculitis were observed in ten birds.

African greys appear to be particularly prone to development of aspergillosis. The species predilection may be due to an immune deficiency problem or related to initial capture technique (Rosskopf and Woerpel, 1996). Ritchie (1990) also reported a species predilection in African grey parrots, Amazon parrots and cockatoos. Oglesbee (1991) highlighted a case of chronic aspergillosis in an African Grey, that followed an incident of a house fire. The case was successfully treated with amphotericin B, fluid therapy, oxygen, antibiotics and dexamethasone, with ketoconazole as longer term therapy.

A batch of mouldy parrot seed was implicated as the source of an outbreak of aspergillosis in parrots by Simpson and Euden in 1991. Nineteen birds were affected over a three month period. A. fumigatus was cultured from eight out of ten independent seed samples. The pathology was characterised by pulmonary oedema, possibly attributable to mycotoxin production

Fourteen case reports of aspergillosis in psittacine chicks, five diagnosed antemortem, were described by Van der Heyden (1993). Predisposing factors included nest box contamination despite good hygiene, prolonged antibiotic therapy with or without corticosteroids, stress arising from metabolic bone disease and concurrent viral disease, with six cases having no identifiable risk factors. 58% of cases presented with respiratory compromise; 60% with abdominal enlargement. A. fumigatus was cultured from all cases. The acute form tended to predominate in chicks, with septicaemia rather than granuloma formation being the main expression of disease. Ascites was common, either due to peritonitis or cardiovascular impairment. The primary lesion could potentially have been pulmonary, though entry of the fungus via the gastrointestinal tract or a skin lesion was also theorised. Treatment with oxygen, antibiotics, frusemide, flunixin meglumine, amphotericin B and supportive care were successful if the case was diagnosed early enough.

1.6.3.2. Quarantine of psittacines

Kaplan et al (1975) highlighted the risk of aspergillosis in quarantine. 45 psittacines that died out of 655 imported to the United States for study into psittacosis were found to have macroscopic lesions suggestive of aspergillosis on post mortem. 35 of these cases were confirmed on histopathology, 32 on culture. 16 cases were due to A. oryzae, 13 to A. fumigatus and 3 due to a combined infection. Causal factors implicated were the stress of capture and transport (23%), cortisone acetate treatment (8.6%, one third of the cortisone treatment group) and prolonged antibiotic therapy (chlortetracycline) (17%). Cultures of air and food were negative, thus the source of infection remained unidentified. Aspergillosis has also been reported as concurrent to psittacosis (Dolphin and Olsen, 1977). A frequent diagnosis of aspergillosis, concurrent to Newcastle disease was made by Dharma and Sudana (1983) at an Indonesian quarantine station, the two diseases being the most usual diagnosis in cockatoos at the laboratory.

Imported psittacines in a Switzerland quarantine station were reported to suffer up to 70% mortality due to aspergillosis, diagnosed on post mortem (Baumgartner, 1988). Anti-A. fumigatus IgG titres were found to increase, indicating the need for prophylactic therapy. A high incidence (12.9%) of acute aspergillosis was observed in psittacine birds in quarantine in Japan by Tsai and colleagues (1992). Lesions involved the nasal cavity, lungs and air sacs, causing exudative rhinitis and invasive granulomatous lesions. Cutaneous aspergillosis in association with pox virus was also observed. The highest prevalence was seen in Amazons, followed by African greys, lovebirds and parakeets. Severe cases showed angioinvasion. The likely predisposing factor to infection was shipping stress.

1.6.4 Sphenisciformes

A report concerning medical problems in wild-caught penguins during the period 1972-1977 at the Baltimore Zoo (Sladen et al, 1979) described 63% mortality in Black-footed penguin (Spheniscus demersus) chicks, 11% caused primarily by aspergillosis. Only one juvenile died of primary aspergillosis, a second suffered the disease with concurrent malaria infection. The rockhopper (Eudyptes crestatus) colony lost 13 out of 14 individuals. Three birds suffered aspergillosis, of which only one case was primary. The authors described previously high mortalities due to the disease during the period 1958-65 in Humboldt (Spheniscus humboldti) and Adelie (Pygoscelis adeliae) penguins (100% and 53% of mortalities respectively). Reduction in prevalence was attributed to husbandry changes.

An outbreak of Aspergillus fumigatus in imported penguins at the Zoological Park, New Delhi, was reported by Khan et al (1977). Seven out of eight birds died within 17 days of arrival, showing signs of dullness and anorexia. Post mortem, microscopy and culture of available tissues confirmed the diagnosis in three of the penguins, involving the lungs and liver. The diagnosis was extrapolated to the other birds. Attempts to obtain a culture of the organism from the environment were unsuccessful, the authors surmising that the infection was acquired in the country of origin or during transit.

Nakeeb et al (1981) reported anorexia, lethargy and respiratory distress in recently imported Humboldt penguins at the Aquarium of Niagara Falls, New York. Two birds died within 39 days of arrival. Treatment with amphotericin B was initiated in the remaining two, following A. fumigatus cultures from the nasopharynx of both birds. One bird died subsequently, following cessation of therapy after drug-induced anorexia. The last bird survived, receiving a 34 day course of intraperitoneal amphotericin B. Multifocal granulomatous pneumonia, with Aspergillus fumigatus present in some nodules, was diagnosed on post mortem, culture and histopathological examination.

A retrospective analysis of penguin mortality at Edinburgh Zoo by Flach et al (1990) showed aspergillosis to be the most common cause of death in gentoo penguins (Pygoscelis papua), with chicks being the most susceptible. Annual mortality rates varied from below 20% to over 60% during the 25 year period, with 41% of birds overall being diagnosed with aspergillosis at post mortem. Most mortality occurred during the period July to September after chicks were moved to a crêche area, and it was theorised that climate may influence the prevalence. Vaccination with a killed suspension of Aspergillus fumigatus did not reduce mortality, and ketoconazole treatment of cases was ineffective.

Reidarson and McBain (1992) theorised that it is likely that all captive penguins are infected to some extent. However, only those malnourished, stressed by moult, egg-laying or chick rearing, or suffering concurrent disease, are likely to develop clinical disease. They advised to test (by ELISA) any penguins with suggestive clinical signs or haematologic changes.

1.7  Diagnosis

1.7.1  General

Most acute cases are often only diagnosed at post mortem examination. Following a presumptive diagnosis, based on clinical signs, species susceptibility, stressors and physical examination, treatment should be initiated. The definitive diagnosis should be made once the individual is stabilised, allowing determination of the intensity and duration of therapy. Knowledge of the serological profile of the flock aids diagnosis and monitoring of response to therapy (Redig, 1993).

A thorough history should be taken, including evaluation of the environment, alongside physical signs. Aspergillosis should be considered in cases that are deteriorating despite antibiotic therapy (Oglesbee, 1997). The blood profile may show a leukocytosis, 20,000-100,000 cells per microlitre; heterophilia with left shift, monocytosis and lymphopenia. In chronic infections, a non-regenerative anaemia, hyperproteinaemia and hyperglobulinaemia may be seen. Increases in aspartate aminotransferase and bile acids may occur with hepatic involvement (Oglesbee, 1997; Reidarson and McBain, 1992; Redig, 1993).

1.7.2 Radiography

Radiographical changes seen in advanced disease include a parabronchial pattern, loss of definition of airsacs, asymmetry due to consolidation or hyperinflation, or focal densities. Hepatomegaly or renomegaly may be visible with involvement of these systems (Oglesbee, 1997; Baumgartner, 1988). Clavicular and thoracic air sac involvement is often seen in penguins (Reidarson and McBain, 1992); abdominal and caudal thoracic air sacs tend to be more commonly affected in raptors (Redig, 1993). Contrast radiography and CAT scans have also been reported as diagnostic aids (Redig, 1986).

1.7.3 More invasive techniques

An aseptic, deep tracheal swab, examined microscopically for hyphae and spores or cultured, is useful, particularly in raptors (Baumgartner, 1988; Redig, 1981). Endoscopy allows tracheal examination for the presence of syringeal granulomas. These should be biopsied for microscopic evaluation and culture. Abdominal and posterior thoracic airsac laparoscopic examination may show diffuse cloudiness or plaques. Again, these should be biopsied. Plaques may be white, yellow or green-grey (Oglesbee, 1997; Baumgartner, 1988). Airsac examination should be bilateral, and may be used to monitor progress, particularly in raptors, who tolerate the procedure well (Redig, 1993). Exploratory laparotomy, though more invasive, allows assessment of the extent of systemic involvement, and enables surgical debridement of lesions (Redig, 1986).

1.7.4 Direct examination, culture and histopathology

Microscopically, on bronchoalveolar lavage (BAL), fragments of fungus are seen with thick hyphae and parallel walls. With endoscopy, fruiting bodies may be seen in the air sacs or syrinx (Oglesbee, 1997). Culture can be achieved using Sabouraud dextrose agar (SDA) or blood agar at room temperature. 4% malt agar with chloramphenicol at 28°C to look at contaminants and 37°C to inhibit bacteria, is used by most human laboratories (and at 45°C for BAL to inhibit yeast growth) (G.S. Shankland, personal communication). White colonies appear at 18-24 hours and turn green (if sporulation occurs) after 48 hours. However, many colonies do not sporulate well. Growth of between 1-4 colonies is diagnostic (Redig, 1993), and staining with lactophenol cotton blue or new methylene blue can aid visualisation (Oglesbee, 1997). Care should be taken with interpretation, as Aspergillus species are common laboratory contaminants (Buxton and Fraser, 1977). On histopathology, Aspergillus spp. have slender, tubular septate hyphae, with parallel-sided walls, dichotomous 45° branching and spherical spores. Haematoxylin-eosin, periodic acid-Schiff or Grocott stain may be used to aid visualisation. (Oglesbee, 1997).

Post mortem diagnosis of aspergillosis tends to be that most commonly reported. Carrasco et al (1998) reported the histopathological findings in an Amazon parakeet (Amazona aestiva). They diagnosed pulmonary aspergillosis, with concomitant zygomycosis, through the use of indirect immunohistochemical techniques. The latter agent was found to be disseminated from a primary pulmonary lesion, causing a thrombosing vasculitis with localisation in the myocardium, kidneys and air sacs. They used the case to highlight the risks taken when making aetiopathological diagnosis in avian mycoses.

1.7.5 Serology (also see Avian Immunology and Serological Assays)

Serological diagnosis was originally based on precipitin formation in agar gel, though the development of enzyme-linked immunosorbent assays (ELISA) has allowed earlier diagnosis (within 7 days of exposure) and monitoring of response to treatment (Redig, 1993). The benefits of immunodiffusion techniques lie in their simplicity, low cost and precision. Agar gel double diffusion (AGDD), and counter-immunoelectrophoresis (CIE) are the most commonly used. Precipitins form when the optimal concentration of antigen and antibody are reached (Beard, 1980). Additional techniques include collodium agglutination, immunoelectrophoresis, radioimmunoassay (RIA) and indirect haemagglutination (IHA). IHA is relatively simple, cheap and commercially available and has been adapted for use with avian sera (Baumgartner, 1988). Antigen detection can be made through latex particle agglutination (LA) or ELISA.

Redig (1981) compared the ability of diagnostic tests to identify aspergillosis in raptors (wild and captive). He found that no single test was completely efficient. AGDD was particularly poor, implying a sub-optimal antibody response in this species. Tracheal swabbing and endoscopy were the most sensitive. When clinical signs indicated invasive disease, laparoscopy with endoscopy and radiography were most useful.

1.8 Treatment

Treatment is often unsuccessful and prognosis poor, especially in well-colonised infections with poor blood supply and a granulomatous host response. The best treatment involves surgical debridement of the lesions, followed by topical therapy in conjunction with aggressive systemic antifungals. Amphotericin B, 5-fluorocytosine, ketoconazole, miconazole, enilconazole, itraconazole, rifampicin and dimethyldithiocarbamate have all been reported in treatment of aspergillosis (Redig, 1993). Topical therapy includes nebulisation, nasal and airsac flushing and surgical irrigation of abdominal cavity (Oglesbee, 1997). Supportive therapy is given as required (Redig, 1993). Immunostimulation, via the administration of levamisole hydrochloride, could possibly augment therapy (Redig, 1986).

Amphotericin B is fungicidal and used in first line treatment by intratracheal or intravenous injection or by nebulisation. Pharmacodynamics indicate a shorter half life in birds, requiring higher doses for effective therapy (Redig, 1986). Toxic side effects include nephrotoxicity and crystal deposition in soft tissues around sinuses (Oglesbee, 1997). However, nephrotoxicity has not been reported in avian species (Redig, 1986), just an occasional transitory ataxia, following intravenous administration in raptors. Local irritation at the administration site, vomiting and depression have also been observed (Ritchie and Harrison, 1994). Sladen et al (1979) reported experimental infection of chickens by direct airsac inoculation with Aspergillus fumigatus, used to investigate the use of amphotericin B treatment by nebulisation (100mg in 250ml 5% dextrose). The results showed improved success with early treatment. Two cases of successful treatment in penguins with clinical signs of dyspnoea and weight loss were described.

Oral ketoconazole has been used, although it is now superseded by itraconazole, which has a greater activity against Aspergillus spp. Tablets require administration with a fatty meal, but an oral syrup has been developed to improve absorption. Itraconazole has been reported to cause anorexia in African Greys (Quesenberry et al, 1991). Therapeutic concentrations take time to develop, hence amphotericin B should be used initially. Treatment should be maintained for several months following resolution of clinical signs (Oglesbee, 1997; Flammer, 1994).

Kaufman and Paul-Murphy (1989) described the treatment of a nasal granuloma, involving A. flavus, in an African Grey parrot (Psittacus erithacus) using enilconazole in nebulised and topical preparations following initial therapy with amphotericin B and ketoconazole. An outbreak of aspergillosis in captive tufted puffins (Lunda cirrhata), reported by Monroe et al (1994), detailed a variety of treatment protocols, with success being achieved using itraconazole, 40 mg/Kg bid and nebulised enilconazole, 1:50 dilution for 45 minutes daily, a dose much greater than that previously used in avian species. The use of clotrimazole, administered via nebulisation, as an adjunct to therapy has been reported by Joseph et al (1994) in raptors and psittacines. Although this was not a controlled study, successful therapeutic regimes were achieved with minimal side effects. Gass (1979) reported success in halting an outbreak of acute aspergillosis in imported Humboldt penguins using miconazole intravenously. Furley and Greenwood (1982) also described success (74%) in treating aspergillosis in raptors using intramuscular miconazole. However, complete return to fitness did not occur due to residual granulomata mechanically compromising respiratory function. However, Lawrence (1983) reported the drug ineffective in psittacines. A new azole drug with activity against Aspergillus spp., voriconazole, is currently being evaluated (Pfizer).

Reports of treatment in penguins include the use of itraconazole at 8.3mg/Kg/d for 49 days to treat pulmonary aspergillosis (A.niger) in a gentoo penguin (Pygoscelis papua). Increasing the dose to 17mg/Kg/d caused anorexia. The bird showed a negative titre on ELISA. Treatment was stopped with apparent recovery, but the bird died three weeks later, with cerebral aspergillosis on post mortem (Hines et al, 1990). Ocular aspergillosis in a king penguin (Aptenodytes patagonica) was successfully treated with itraconazole at 8mg/Kg/d for 29 days (Hines et al, 1990). This penguin had an IgG titre consistent with Aspergillus exposure on ELISA. The authors also compared itraconazole serum levels in two uninfected gentoo penguins, treated with 10mg and 20mg/Kg/d respectively. They found that it took 6 days for serum levels to reach steady state. Both dose regimes produced serum levels above the minimal inhibitory concentration. Individual variation in response to the drug were noted; a loading dose of 2-4 times the maintenance dose was advised.

1.9 Prevention

Environmental hygiene and ventilation should be maximised and stress reduced. Prophylactic treatment should be considered in high risk species (Table 1) at times of stress, with serological-based monitoring for the flock and incoming birds. Periods of stress to consider are during injury or disease; certain developmental stages (e.g. post-fledgling in goshawks and gyrfalcons); wild capture and psittacine quarantine (Redig, 1993).

The use of a commercial biologic in the treatment and prophylaxis of aspergillosis in penguins and other avian species was reported by Stodard (1990), suggesting its use was effective in reducing mortality in susceptible species. Vaccination with a heat-killed culture filtrate preparation has been reported to reduce mortality in eiders and waterfowl (Donnelly et al, cited by Redig, 1993). Also, reports of the effectiveness of a vaccine in preventing outbreaks in susceptible populations was mentioned by Ritchie (1990). However, no convincing trials have been reported.

June 12, 2000