Avian Aspergillosis: Clinical Signs, Histopathology & Diagnosis

by Dr. Tara Nath Gaire

Department of Veterinary Medicine and Public Health
Institute of Agriculture and Animal Science (IAAS), Rampur, Chitwan, Nepal.

Complete Article: Avian Aspergillosis by Dr. Tara Nath Gaire

Contents

Abstract … Introduction … Etiology … Growth … Toxins … Epidemiology … Transmission … Pathogenesis … Clinical Signs, Gross Lesions, Histopathology and Diagnosis (below) … Treatments and Acquired Immunity … Prevention and Control … References Cited

Clinical Signs

Clinical manifestations depend on the infective dose, the spore distribution, pre-existing diseases, and the immune response of the host (Dahlhausen et al., 2004). Avian aspergillosis is often classified as acute or chronic. Acute aspergillosis is thought to be the result of inhaling an overwhelming number of spores, while chronic aspergillosis is generally associated with immune suppression (Vanderheyden, 1993). Acute aspergillosis may include a variety of nonspecific clinical signs: anorexia, lethargy, ruffled feathers, respiratory signs, polydipsia, polyuria, stunting, or sudden death. In chicks, contaminated in ovo or during hatching, the disease, commonly known as brooder pneumonia, is highly fatal in the first ten days of life and results in a major respiratory distress. Two outbreaks of omphalitis where the primary cause was A. fumigatus have been investigated in young turkeys.In poultry farms, mortality rate may rise slightly or increases suddenly, peaks during a few days, and then returns to initial state Respiratory signs include dyspnoea, gasping, hyperpnoea with panting, nonproductive coughing, wheezing, cyanosis , and sometimes nasal discharge .In the chronic form, dyspnoea, depression, dehydration, and emaciation are described. Nervous system involvement causes ataxia, tremor, opisthotonos, lateral recumbency, torticollis, seizures, convulsions, lameness, and hind limb paresis .Occurrence of nervous and ophthalmic troubles one week after an acute episode of aspergillosis has been reported in a turkey flock. Cloudiness of the eye with severe conjunctivitis and turbid discharge were associated with paralysis in broiler breeders.

Although aspergillosis is predominantly a disease of the respiratory tract, any organ can be infected. Nasal aspergillosis causes exudative rhinitis (Tsai et al., 1992), possibly accompanied by malformation of the nostrils, beak and cere (Bauck et al., 1992). Mycotic keratitis can cause blepharospasm, photophobia, periorbital swelling, turbid discharge, swollen and adhered eyelids, cloudy cornea and cheesy yellow exudates within the conjunctival sac (Beckman et al., 1994; Hoppes et al., 2000). Neurological signs can be caused by Aspergillus spp. (Jensen et al., 1997). Epidermal cysts associated with A. fumigatus have been described in the comb of a silky bantam chicken (Suedmeyer et al., 2002). Aspergillus blepharitis and dermatitis involving the eyelids and the head have been described in a peregrine falcon–gyrfalcon hybrid (Falco peregrinus x Falco rusticolus) (Abrams et al., 2001). Right ventricular dilatation (cor pulmonale) due to pulmonary hypertension, with or without ascites, and congestion of the lungs caused by ventricular failure occasionally occurs (Julian and Goryo, 1990; Höfle et al., 2001). Birds suffering from aspergillosis do not always show respiratory problems.

Gross Lesions

The severity and the degree of development of the disease determine both morphology and extension of macroscopic lesions. Extensive involvement of the respiratory tract can occur before clinical signs are apparent. Typically, lesions consist of white to yellowish granulomas ranging from miliary (mm in diameter) to large roughly spherical granulomatous nodules (>2cm) involving serosae and parenchyma of one or multiple organs. Single or multiple necrotic areas are visible on cut surfaces. The primary location of lesions is the air sacs and lungs although oesophagus, proventriculus, gizzard, small intestine, liver, kidney, spleen, skin, trachea, peritoneum, brain, eye, muscle, or heart may be involved. Lung parenchyma is either consolidated or has focal granulomas of varied size (Figure 1). When coalescing in air sacs, these masses form cheesy caseous plaques covering the thickened membranes and even obstructing the entire lumina where fungal sporulation may occur as evidenced by a grey-greenish velvet.

Figure 1: Numerous nodules in the lung of poultry with acute aspergillosis

Nonulcerative or mildly ulcerative keratitis was reported in a turkey flock . Broiler pullets presented periorbital and eyelid swelling with cheesy yellow exudates in the conjunctival sac . Circumscribed white to greyish areas were observed in the cerebellum of broiler breeders and turkeys . Ribs of ostriches , sternum of broiler breeders , yolk sacs of poults and hip joints of turkeys constituted unusual locations of aspergillosis lesions.

The lesions depend considerably on the site of infection. Either localisation or generalisation may be observed. Individual lesions may be observed, for example, in the syrinx or in a single air sac. The lungs are most frequently involved. Pulmonary lesions vary from miliary nodules up to larger nodules.In some cases there may be localised hepatization, and in others grossly visible mycelial masses may be present in the air passages and bronchi. There may be generalised involvement of the air sacs. Occasionally a circular disc-shaped necrotic mass with a concave surface, loosely attached to which there is a circular more or less flat or convex plaque, may be observed. Various manifestations of the disease have been described. Lange (1914) recorded nodules in the lungs and the thoracic and abdominal cavities of chickens, ducks, geese, and pigeons. These nodules varied from pinhead or millet seed size up to the size of a pea. They were yellow in colour, of an elastically soft cartilaginous consistency, and homogeneously caseous. Individual nodules were noted on the intestinal serosa and in the parenchyma of the liver in a goose.

The nodules in the lungs of a turkey observed by Schlegel (1915) were pinhead to lentil size and were surrounded by an infiltrated or hemorrhagic corona with considerable hepatization. There were also grayish yellow, fibrin purulent disc- or plate-shaped masses of exudates 2-5mm thick on the pleura. Inflammation and detritus masses were present in the bronchi. The anterior thoracic, maxillary, and cervical air sacs contained yellow caseous flat discs and masses consisting of inflammatory exudates and mycelia. The left lower thoracic, upper posterior thoracic, and left abdominal air sacs were greatly distended. The walls of these air sacs were thickened and covered with a fur like growth of mold. Adjacent to the air sacs there were lentil-sized, knob-shaped, concentrically layered, turbid yellow, solid nodules composed of fibrin and mycelia. There were about 200cc of reddish turbid fluid in the abdominal cavity.

There were no circumscribed yellowish foci in the outbreak in chicks reported by Savage and Isa (1933). There was a diffuse grayish yellow infiltration in the lungs, with about one-third of each lung involved. Mortality was 90% in this outbreak.In pneumomycosis in a flamingo described by Mohler and Buckley (1904), the lungs were filled with nodules, and the mucosa of the bronchi was covered with membranous masses that consisted primarily of the fungous mycelium.

Archibald (1913) found gray round colonies of the fungus in the bronchioles in an ostrich, whereas in a case described by Jowett (1913) the lungs were covered with miliary foci. Lahaye (1928) states that Aspergillus glaucus may be the cause of a disease of the skin in pigeons, particularly in young birds. Any part of the body may e affected with yellow scaly spots. The feathers in the affected areas are dry and easily broken.

Durant and Tucker (1935) observed yellowish white nodules up to 5 x 8mm in the lungs of wild turkey poults being reared in captivity. The hyphae of the fungus also penetrated the tissue of the lung, and there was involvement of the adjacent air sacs.In canaries observed by De Jong (1912) there were small whitish yellow, crusty coatings on the tongue, palate, and aditus laryngis and in the trachea and syrinx. Caseous foci in the lungs and caseous coatings on the pleura and peritoneum were also observed.

Histopathology

The histological picture as described by Nieberle (1923) consists of focal pneumonia, multiple necrosis, and nodular formations which resemble tubercles. The diffuse pneumonic foci are indicative of fibrinous or catarrhal pneumonia. The alveoli, bronchioles, and bronchi are filled with mucus, stained fibrin, nuclear fragments, detritus, leucocytic and inflammatory cells, and mycelia. The mycelium penetrates the walls, and the surrounding pulmonary parenchyma shows an exudative cellular inflammation or necrosis. The tubercle-like nodules show in the center a radiating turf of hyphae surrounded by a reactive inflammatory wall which resembles granulation tissue. Foreign body giant cells are frequently observed. The fruiting organs (conidiophores, sterigmata, conidia) occur more frequently in the air sacs.

In turkey cases of ophthalmitis described by Moore (1953), the primary involvement was in the viterous humor and the adjoining tissues. In one turkey he observed the presence of mycelia in the crystalline lens. Haematoxylin-eosin stain is often augmented with periodic acid-Schiff, Grocott, and Gomori’s methenamine silver stains in order to display fungal elements in embedded tissue sections. The fluorescent optical brightener blankophor proves to be a valuable tool for demonstrating Aspergillus sp. hyphae .Based on histopathological difference a deep nodular form and a superficial diffuse form of aspergillosis. A well-organised granulomatous reaction develops in nonaerated parenchyma whereas a superficial diffuse form, containing fungal elements and a nonencapsulated pyogranulomatous reaction, predominates in serosae and lungs .Organised granulomas are clearly encapsulated by an outer thick fibrous layer whereas pyogranulomas lack clear borders. The first feature corresponding to late granuloma may also be an important sign of the chronic form of aspergillosis, especially in adults Pyogranulomas organization presents a centre with variable amounts of septate, dichotomously branching hyphae surrounded by a palisade of radially arranged foreign body giant cells, macrophages, heterophils, and lymphocytes. Phagocytized fungal elements are regularly observed in the eosinophilic cytoplasm of multinucleated cells. Lymphocytes may infiltrate the margins of the granuloma Progressive inflammation in lungs resulting in small granuloma coalescence induces more extensive lesions and may lead to parabronchial obliteration with necrotic eosinophilic material containing erythrocytes, degenerated heterophils, and exfoliated epithelial cells . Numerous conidiophores and free spores appear in granulomas that open to the air spaces of the respiratory tract. Typical granulomatous reactions associated or not with fungal elements have been observed in brain conjunctiva, liver, spleen, gizzard, small intestine, hip joints, and trachea.

Diagnosis

The signs of aspergillosis are non-specific, making diagnosis difficult (Dahlhausen et al., 2004). Moreover, no single test provides certainty. Diagnosis usually relies upon an accumulation of evidence from the history, clinical presentation, haematology and biochemistry, serology, radiographic changes, endoscopy and culture of the fungus (Jones and Orosz, 2000).The history of the bird can reveal a stressful event and/or some underlying environmental factors and/or an immunosuppressive condition or treatment (Jenkins, 1991). It may also reveal chronic debilitation, voice change or exercise intolerance (Oglesbee, 1997).The clinical signs depend on which form of aspergillosis the bird develops and which organs are involved (Jones and Orosz, 2000). Hence, aspergillosis should be included in the differential diagnosis of respiratory tract and systemic diseases (Jenkins, 1991; Jones and Orosz, 2000).

Results of haematology and plasma biochemistry can be considered indicative rather than diagnostic (Jones and Orosz, 2000). Leukocytosis of 20,000 to more than 100,000 white blood cells per microlitre (Jenkins, 1991; Oglesbee, 1997), heterophilia with a left shift (degenerative shift), monocytosis and lymphopenia have been described in aspergillosis cases (Forbes, 1992). In addition, non-regenerative anaemia, increased total protein and globulin fraction can be observed (Vanderheyden, 1993; Reidarson and McBain, 1995; Jones and Orosz, 2000). Acute infections often present an increase in ?-globulins, while chronic infections show an increase in ?-globulin and/or ?-globulin fractions . However, immunosuppressed birds may have hypoproteinaemia (Ivey, 2000; Cray et al., 2009a) and white blood cells may be in the normal range (Flammer and Orosz, 2008). Overall, changes in protein electrophoresis are non-specific, but can be useful to estimate disease progression and the response to therapy (Ivey, 2000; Cray et al.,2009a).

Serological tests have been developed to confirm an early and more definite diagnosis of aspergillosis (Peden and Rhoades, 1992). However, in the acute stage, antibody production trails behind antigen exposure by 10 to 14 days (Brown and Redig, 1994); and if the bird is immunosuppressed, the low antibody production results in false negative results (Redig, 1994). In these cases, detection of circulating Aspergillus antigen in the serum may be more helpful (Cray et al., 2006). In chronic cases in which antigen levels may be low, detection of antibodies may be useful (Jones and Orosz, 2000). A number of serological test methods have been applied in birds, including counter-immunoelectrophoresis, agar gel immunodiffusion and enzyme-linked immunosorbent assays—but, in general, negative serological tests do not rule out aspergillosis, and positive tests are only considered diagnostic when backed up by other evidence (Peden and Rhoades, 1992; Brown and Redig, 1994; Redig, 1994; Redig et al., 1997; Le Loch et al.,2005; Arca-Ruibal et al., 2006; Cray et al., 2006, 2009a,b).

Although radiographs may not be helpful, lateral and ventrodorsal views can be taken in a bird suspected of having aspergillosis. For those that are unlikely to survive anaesthesia, standing or perching lateral views as well as dorsoventral views are helpful (Jones and Orosz, 2000). The radiographic changes of pneumonia and consolidating airsacculitis are non-specific (McMillan and Petrak, 1989). Consistent with radiography, computed tomography scans reveal the extent of lesions—but the diagnosis of aspergillosis still requires identification by biopsy, smear or culture (Phalen, 2000).

Endoscopy is invasive and requires anaesthesia, but it allows the extent of the lesions to be seen as well as the progress of infection during treatment (Redig, 1994; Jones and Orosz, 2000). This technique enables evaluation of the entire respiratory tract. Tracheal endoscopy is useful for showing a single lesion, such as a thick white discharge or plaque occluding the trachea or syrinx (Jenkins, 1991; Marks et al., 1994; Redig, 1994). The lower respiratory tract is best evaluated by laparoscopy (Jones and Orosz, 2000). Endoscopy of the abdominal air sac can show a diffuse cloudiness or white or yellow plaques. In the event of sporulation, plaques are covered with green–gray pigmented mould. Samples for culture and cytology should be taken directly with biopsy forceps or via air sac lavage (Jenkins, 1991; Taylor, 1993; Oglesbee, 1997).On necropsy, the yellow, green or white granulomatous foci can be noted in chronic aspergillosis patients (Jenkins, 1991; Vanderheyden, 1993). Acute aspergillosis causes numerous miliary granulomatous foci (McMillan and Petrak,1989; Jenkins, 1991).

Definitive diagnosis requires demonstration of the presence of the organism by cytology or histopathology and its identification by culture (Dahlhausen et al., 2004). It is important to mention that isolation of the fungus alone does not confirm the infection because Aspergillus organisms are ubiquitous and can be contaminants (Jensen et al., 1997; Flammer and Orosz, 2008). However, an abundant culture from any organ should be regarded as diagnostic. On the contrary, a negative culture does not rule out aspergillosis (Redig, 1994; Jensen et al., 1997).

Histopathological lesions can be suggestive, but because in vivo hyphae of hyaline filamentous fungi are very similar and their in situ manifestations are not pathognomonic, this technique does not allow fungal species identification (Kaufman et al., 1997; Cray et al., 2009a). Thus the aetiological diagnosis should ideally be confirmed by immunohistochemistry, although few reports on immunohistochemical techniques using monoclonal or polyclonal antibodies in birds with aspergillosis are available (Carrasco et al., 1993; Jensen et al., 1997; Beytut et al., 2004, Beytut, 2007).A few reports of different polymerase chain reaction assays (including real-time polymerase chain reactions) tested on heparinized whole blood, tracheal washings, air sac fluids, respiratory tract granulomas, or (biopsy) tissue samples from birds support the value of this assay in diagnosing avian aspergillosis (Dahlhausen et al., 2004; Cray et al., 2009a). However, further research is necessary before such assays can be included in the work-up of the avian practitioner.

Treatments and Acquired Immunity

Prevention and Control

References Cited

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