AVIAN ASPERGILLOSIS
Disease Name Aspergillosis. Syn. Pneumonomycosis, Bronchomycosis, Cytomycosis, Brooder pneumonia, and in part Pseudotuberculosis and Mycosis; in ostriches Chick fever and Yellow liver. Pneumonie du couveuse (Fr.).
Pathogens. Aspergillus fumigatus Fresenius, 1850-53. Syn. A. nigrescens Robin, 1853; A. aviarius Peck, 1891. Aspergillus flavus Link, 1809. (Schieblich, 1921). Aspergillus niger van Tieghem, 1867. (Hare, 1937). Aspergillus nidulans (Eidam) Wint.,1884. (Ainsworth & Rewell, 1949 ). Aspergillus terreus Thom, 1916. (Ainsworth & Austwick, 1955a).
Hosts. Domesticated and caged birds; fowl, turkey, duck, goose, pigeon, canary, budgerigar, parrot, ostrich. Captive wild birds, especially water birds; duck, geese, penguin, stork, flamingo, cormorant, parrot, hawk, owl, pheasant, peafowl. Free-living wild birds; wood-pigeon, seagull, rook, pheasant, sparrow, swan, jay, grouse, Manx shearwater, thrush.
Geographical Distribution. World-wide. Severe economic losses occur, chiefly in areas of intensive rearing.
The striking post-mortem picture presented by avian aspergillosis was no doubt responsible for its early recognition so that by the end of the nineteenth century some 60 contributions had been made on the disease of birds alone. At first there was little attempt to identify the "green mould" seen in the air-sac and bronchial lesions (Owen, 1832) and many of the early accounts are difficult to interpret for this reason. There was also much confusion between the pathogenic and saprophytic species of Aspergillus. Progress in the study of the disease was also slowed by controversy over the aetiological significance of the fungi found sporulating on the lesions and although the primary pathogenicity of most of these is now well established, the epidemiology and development of the disease still require further investigation.
There are extensive reviews of avian aspergillosis by Lesbouyries (1941), Urbain & Guillot (1938) and Verge (1927), in which may be found detailed accounts of the species of birds affected, and of the pathology, diagnosis, mycology and therapy of the disease.
Symptomatology
Because almost any organ of the avian body may be affected by aspergillosis, the symptoms are extremely varied. They may be respiratory, digestive or nervous and occur in birds of all ages.
Acute aspergillosis -This typically occurs in very young birds and may be associated with considerable losses. Watkins (1940), for instance, estimated that 10% of all deaths in chicks under 14 days old were caused by this disease. Typically there is loss of appetite, increased respiration, a rise in temperature, listlessness, foetid diarrhoea and rapid loss of condition. Convulsions sometimes occur and affected birds often die within 24-48 hours of the onset of symptoms. Epidemic outbreaks in young birds frequently have a very high death rate before control measures can be taken. Similar symptoms may occur in pullorum disease (Salmonella pullorum), from which this form of aspergillosis must be distinguished.
Chronic aspergillosis -The onset of this form is insidious and the affected birds may survive for long periods in a gradually declining state. The symptoms of the acute disease are often present in a milder form together with anaemia, yellowing of the faeces and the presence of a respiratory rattle. The chronic disease generally occurs sporadically and is the usual form in adult birds.
Pathology
Although the appearance of the lesions is almost diagnostic, it is necessary to eliminate tuberculosis and other granulomatous diseases. Differential diagnosis can be conclusive if the fungus is fruiting within the respiratory cavities of the bird but as this only occurs in the chronic stages of the disease, the demonstration of hyphae in the nodules is the most useful method. This is carried out by crushing the whole or a piece of a nodule under a coverslip on a slide in a drop of 20% potassium hydroxide or in lactophenol cotton blue. The hyphae can usually be seen projecting from the edges of the masses of caseous material and if the latter technique is used they will stain deeply.
Lungs - In the lungs the acute form typically appears as yellowish- white miliary nodules 1-3 mm. in diameter which are generally evenly distributed throughout the tissue. Each nodule is surrounded by a dark infiltrated zone, the remainder of the lung appearing normal. Chronic lesions either result from the coalescence of many nodules or are formed by the enlargement of single ones and may eventually become calcified. A peracute pneumonic form also occurs in which there is complete congestion of lung tissue without the formation of nodules.
Air-sacs -The serous membrane of the air-sacs becomes thickened in places by the development of small yellowish-white plaque-like lesions which enlarge rapidly, become raised in the centre, and then coalesce to form a complete membranous or caseous lining to the serosa up to 5 mm. thick.
Bronch and Trachea -These organs may become blocked either by lesions similar to those described in the air-sacs, or, in the pneumonic form, by mucoid discharge.
Wherever the surface of a lesion is exposed to the air, the conidiophores of A. fumigatus can develop, forming the characteristic greenish-blue or dark grey patches of sporing heads so often described by early observers.
Lesions in sites other than the respiratory organs are generally chronic:-
Alimentary canal - Plaque-like lesions may occur in the mouth, the gizzard and the intestines especially in turkeys (Lignires & Petit, 1898).
Liver, spleen, kidneys and ovaries - Nodular lesions have been found in all these organs sometimes associated with those in adjacent abdominal air-sacs (Emmel, 1929). Peritonitis is not uncommon and the disease in ostrich chicks has been termed "yellow liver" (Walker, 1915).
Nervous system -The occurrence of nervous symptoms in aspergillosis including convulsions and torticollis has been partly attributed by Henrici (1939) to the production of toxins by the growth of A. fumigatus in the body. There are however several reports of abscesses in the cerebrum and cerebellum containing the hyphae of A. fumigatus both in the presence (Raines et al.,1956) and absence (Jungherr & Gifford, 1944) of pulmonary and other lesions.
Eyes - Lesions have been noted in the eyes of baby chicks by Hudson (1947) and in turkeys by Moore (1953), the latter author finding the condition significantly more common in males than in females.
Bones and joints - Osteo-arthritis may be set up in the joints with subsequent lameness (Bizard & Pommay, 1887) and lesions in the long bones were observed very early by Heusinger (1821).
Skin -There is some doubt as to the reliability of the reports of skin infection (Lahaye, 1928), mostly because of the inability of the aspergilli to utilize keratin. Moulds can always be isolated from the skin of birds, where they are probably present as spores, and any sustained moist condition of the skin will lead to their germination.
Following castration - Babic (1930) reported infection with Aspergillus fumigatus at the site of surgical caponization, with spread to the liver and the air-sacs, and a similar incident was reported by Chute et al. (1955).
In the nodular lesions, there is a central necrotic zone containing a radiating mass of branching hyphae (2-3 micra diam.). These hyphae are generally clearly septate but may be either irregular in outline and appear to consist of swollen segments, or of normal elongated appearance and very sparsely septate. Numerous epithelioid and giant cells surround this centre and outside is a reaction zone of eosinophiles, lymphocytes and fibrous tissue (Lesbouyries, 1941). Völker (1924) claimed that the presence of eosinophiles was in itself sufficient to distinguish the infection from tuberculosis, which however may also be present in the same bird (Ainsworth & Rewell, 1949).
The development of the lesions has been followed in detail only by Nicolaus (1934). He found that lesions produced experimentally in the air-sacs of fowls were visible within eight hours of inoculation by the intratracheal route. At twelve hours they consisted of subepithelial nodules formed from pseudo-eosinophiles accumulating around the invading fungus hyphae. At three days he observed the temporary development of conidiophores within the central necrotic zone, the formation of daughter nodules and the appearance of histiocytes, epithelioid cells and giant cells. In the later stages a many-Iayered fibrous capsule was formed round the necrotic centre and the lesions reached a diameter of 25 mm. in twenty-two days.
Mycology
Diagnosis by direct examination of the lesions is not always successful and the isolation of the pathogen must always be undertaken to identify the species. The best method is to plate out small pieces of the lesions or affected tissues on to malt agar or Sabouraud glucose agar, containing antibacterial antibiotics. Mycelial growth is usually visible within twenty-four hours at 37°C. and the characteristic conidiophores are often produced within two days.
Aspergillus fumigatus is the commonest cause of avian aspergillosis. It was first described by Fresenius in 1850-53 from the lungs of a great bustard in the Frankfurt zoo but many of the earlier records of avian aspergillosis would appear to have been associated with this species although the descriptions are inadequate for certain identification. Other species of Aspergillus have also been incriminated (see list of pathogens) of which A. flavus and A. nidulans are probably the most frequent (Schieblich 1921; Ainsworth & Rewell, 1949). Yet further species reported from birds include A. glaucus (Spring, 1848), A.candidus (Rayel & Montagne, 1842) and A.dubius (Heusinger, 1821) but these are either well known saprophytes which were undoubtedly contaminants (e.g. A.glaucus series) or were doubtful identifications of pathogenic species.
The identification of the different species of Aspergillus has been greatly facilitated by the manual of Thorn & Raper (1945) and this can be referred to for keys and descriptions. The genus Aspergillus is characterized by the formation of a vesicle on the end of the upright conidiophore, followed by the appearance of peg-Iike structures over its surface. These are the sterigmata and they form chains of conidia from their distal ends, giving the columns of spores seen in culture. The different species are primarily distinguished on the structure of the sporing "heads" and on the colour of the spore mass. A few Aspergillus spp. form the "perfect" state in culture and are classed as Ascomycetes. In these, e.g. A. nidulans, the asci are formed inside closed fruiting bodies caIled cleistothecia and each contains up to eight ascospores. This type of spore probably plays little part in the epidemiology of aspergillosis although ascospores are generally more resistant: to adverse conditions than conidia.
There is some evidence that moulds other than Aspergillus spp. can cause similar lesions, but a great many of such reports appear to be due to the high rate of contamination of bird lungs by the spores of saprophytic fungi (Baker et al., 1934). Proof of infection by these fungi must depend on the isolation of a species with the same hyphal morphology as that seen in the lesions. Further work should show whether different species of Aspergillus cause distinct forms of the disease as for example in the association of caseating pneumonia with A. niger reported by Hare (1937).
Strain variation and Susceptibility.
Although Henrici (1939) states that "many strains of A.fumigatus isolated from air or vegetation show no pathogenicity" there seems little confirmation of this and a remarkable constancy in morphology and pathogenicity is a characteristic of this species. Occasional isolates are white, floccose and produce few spores, but these have not been reported from birds.
On the other hand considerable variation occurs in the susceptibility of different varieties of domestic birds to aspergillosis. Generali (1879) first showed that the finer bred races of pigeon succumbed to the disease more readily than others, and Asthana (1944) considered that the races of Rhode Island Red hens he studied were affected more seriously than Black Minorcas or White Leghorns. Brooksbank & Austwick (1955) showed how heavy losses in a hatchery occurred only in the weeks when eggs from inbreeding pens were hatching, whilst the alternating crossbred hatches escaped infection although in the same incubator. This outbreak also suggested that certain strains of Rhode Island Reds and White Leghorns were apparently more susceptible than others.
Epidemiology
There is no doubt that infection follows the inhalation of fungus spores from mouldy litter, grain, meal or other sources. The quantitative aspects of natural infection are quite unknown but the main habitat of A.fumigatus and other pathogenic species is in rotting plant and animal material especially damp hay, straw and grain which have heated up during storage. In this way A.fumigatus may become the predominant mould.
Although the source of infection for domestic fowls may often be traceable it is not so with aspergillosis in free-living and captive wild birds. Davis & McClung (1940) said that A.fumigatus developing in rotting seaweed was responsible for widespread deaths in herring gulls at Boston, U.S.A., whilst mouldy combine straw has been considered to have led to the deaths of pheasants. In captive water birds the litter and food are generally regarded as the inoculum source, but Ainsworth & Rewell (1949) found advanced cases in birds which had been captured only two days previously so that infection must have been present in the wild.
In connexion with the occurrence of aspergillosis in wild geese, Sladen & Austwick (1955) undertook a study of the fungus flora of the upper respiratory tracts of wild pink footed geese in lceland and Scotland. They were unable to detect the presence of A. fumigatus in throat swabs from these birds but found over a dozen other species of fungi which were thought to have been present as spores. The source of infection for free-living wild birds is still obscure.
Treatment and Control
The treatment of avian aspergillosis is influenced by the lack of clinical diagnosis, for the symptoms are not specific and generally the peak of mortality has been passed before any therapeutic measures can be instituted. Because of this reports of successful treatment or even spontaneous recovery are doubtful.
Various methods have been used in the direct treatment of individual birds. They include the inhalation of tar vapours (Otte, 1928), chlorine or sulphur dust, but so far no information is available concerning the use of iodide therapy which has been successfully employed in the experimental disease in rabbits and also in human cases.
By far the most effective method of control is by removing the source of infection, i.e. the mouldy litter or food; this should be burned and the premises adequately cleaned out, perhaps using a fungicidal solution. Another method of prevention is described by Urbain & Guillot (1938), who regularly fed the penguins in the Paris zoo with 5-6 mg. of potassium iodide daily for eight consecutive days in each month. Each dose being inserted into a fish. By this technique they found that the incidence of aspergillosis in these birds was greatly reduced. Good sanitation and careful control of feeding stuffs and litter remain the best methods of prevention in domestic birds.
Egg Infection
Aspergillus fumigatus can readily attack both fresh and incubating eggs once it has gained entry through the shell. Infection does not seem to occur in the oviduct but results from the external contamination of the shell after laying. Penetration occurs through the pores of the shell and the fungus grows well on the shell membranes, killing the embryo if present and occasionally penetrating into the white. Eventually conidiophores and spores are formed in the air-space and affected eggs can be detected by candling. On breaking open the air-space the greenish felt of hyphae and spores is seen.
The chief fungus reported from eggs in incubation is A.fumigatus but several others have been obtained from stored eggs. Of these probably Cladosporium herbarum is the most important in causing the "black spot" discoloration reported by Weston & Halnan (1927). Lucet (1897) demonstrated that clean eggs cannot be infected by direct application of the spores of A.fumigatus and that it was necessary to put a thin layer of butter or other grease on the shell before infection would take place. Hence he concluded that dirty eggs were more liable to infection than clean, and cited an outbreak of egg infection in ducks in which the nests were heavily contaminated with A.fumigatus.