ASPERGILLOSIS: GROSS AND

MICROSCOPIC LESIONS AND

DIAGNOSTIC APPROACH.

Bhupendra Khati 1
R-2020-VPP-05M
Department of Veterinary Pathology
 INTRODUCTION
• Aspergillosis is one of the important fungal infection, which is caused by

Aspergillus fumigatus and less commonly by other Aspergillus species. (Richard

1991, Barnes and Denning, 1993).

• Aspergillus fumigatus is a ubiquitous saprophyte and opportunistic fungal

pathogen (Plopper and Adams, 1987).

• In birds, the disease is usually confined to the lower pulmonary system with

granulomatous lesions in air sacs and lungs.

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• Chicks become infected during hatching or during first or two days in the

brooder house, so called brooder pneumonia.

• All the domestic birds like poultry, turkey quails and duck, as well as wild

birds are susceptible to aspergillosis infection.

• Other synonyms are fungal or mycotic pneumonia, and pneumonomycosis.

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ETIOLOGY
• The two major species of fungus Aspergillus which cause aspergillosis in

poultry are,

• Aspergillus fumigatus

• Aspergillus flavus

• Other species include A. terreus, A. glaucus, A. niger.

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• These organisms are common soil saprophytes, occurring in decaying vegetative

matter and feed grains.

• They grow on organic matter in warm humid environments.

• Fungal hyphae are 4 -12 μm in diameter and bear conidiopores producing conidia

(spores) 2 - 6 μm in diameter that are easily spread in air.

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TRANSMISSION
• Infections are acquired from environmental exposure.

• Infection is by inhalation of spores that usually originate from infected eggs.

• Contamination of the equipment's may result in hatchery infection.

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TRANSMISSION

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TRANSMISSION
• Contaminated feed or poultry
house litter also produce
infection.

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PATHOGENESIS

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CLINICAL SIGNS
1. Acute form

• Common in young birds (with high morbidity and mortality)

• Clinical signs include dyspnoea, anorexia, cyanosis, polydypsia, foul smell

diarrhoea and emaciation.

• Occasionally the birds may die suddenly without showing any clinical signs (Per-

acute).

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CLINICAL SIGNS
2. Chronic form
• More common in older birds.

• Clinical signs include inappetence, emaciation, dyspnoea, gasping, increased thirst, fever,
diarrhoea and signs of nervous involvement.
• Ocular changes include ophthalmitis, blepharospasm, photophobia, and Mycotic keratitis
(periorbital and eyelid swelling with cheesy yellow exudates in the conjunctival sac as well
as necrotic granulomatous dermatitis.
• Neurological signs include paralysis, ataxia, tremor, torticollis, lameness, convulsions,
opisthotonos condition.

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LESIONS
• Extensive involvement of the respiratory tract can occur before

development of clinical signs.

• The primary location of lesions is the lungs and air sacs although

other organs may be involved.

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 GROSS LESIONS
1st Journal (Girma et al., 2016)
• Pulmonary lesions are characterized by multiple hard
creams to yellow coloured, circumscribe plaques a
few mm to several cm in diameter (lungs surface,
inside the lungs, scattered in ventral surface of • Multiple caseous
sternum and air passages)
• The plaques also found in the syrinx, air sacs, liver
and intestines.
• Lung parenchyma was consolidated and single or
multiple necrotic areas are visible on cut surfaces of
lungs.
3rd Journal (Nururrozi 2nd Journal (Sultana et al., 2015)
et al., 2020)
• Yellow caseous • Pulmonary lesions, characterized by
exudate covering multiple hard creams to yellow
the air sac. coloured, circumscribe plaques a few
mm to several cm in diameter seen
throughout the lungs surface, inside
nodules or plaques, the lungs, scattered in ventral surface
severely thickened of sternum and air passages.
air sac . • The plaques also found in the syrinx,
• Nodules and air sacs, liver and intestines.
necrosis in over • Lung parenchyma was consolidated
half of the lung and single or multiple necrotic areas
area, parenchymal are visible on cut surfaces of lungs.
consolidation lung.

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 GROSS LESIONS
1st Journal (Girma et al., 2016) 3rd Journal 2nd Journal
(Nururrozi et (Sultana et al.,
al., 2020) 2015)

• Mycotic pododermatitis along with pulmonary aspergillosis in turkeys.

• In footpads, keratinized epidermal disruption, encrustations and acute
inflammation were noted.
• Epidermal cysts associated with A. fumigatus have been described in the comb of
a silky bantam chicken. It leads to periorbital swelling, swollen and adhered
eyelids with turbid discharge, cloudy cornea and cheesy yellow exudates within
the conjunctival sac. Osteo-arthritis and granulomatous osteoarthritis of the hip
joints with necrosis of the femur head was observed in turkey.
• Abscesses in the cerebellum and cerebrum. It may occur with or without
pulmonary and other lesions.
• Granuloma formation in the brain and lungs of layer chicken.

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Figure. a. Lung shows the presence of cream colour nodules in plural surface, air sacs (arrow).
b. Creamy to yellow colour nodules shows throughout the lung (arrow).

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Figure. Thickening and turbidity of the walls of Figure. Multiple caseous nodule or plaques
the thoracic air sac (3 days post infection(dpi)). and severe thickening of the air sacs (14 dpi).

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Figure. Nodules and necrosis in the lungs (5
dpi).

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MICROSCOPICAL LESIONS
 Air sacs

 Thickening due to massive infiltration of heterophils, multinucleated giant cells

and other types of leukocytes.

 Germinating conidia were seen in the membrane interstitium, and

lymphohistiocytic perivasculitisin less severely affected area.
 Granulomas composing central necrotic cellular debris and heterophils with peripheral

palisade of epitheliod macrophages and aggregates of lymphocytes.

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 MICROSCOPIC LESIONS
1st Journal (Girma et al., 2016)
• The microscopical examination showed
congestion of pulmonary and perialveolar
blood vessel and perivascular edema.
• The normal architecture of the lung and air
sacs were replaced by disseminated
granulomatous foci. The center of the
granulomatous foci contained caseous
necrosis and necrotic cellular debris
surrounded by rims of heterophils,
lymphocytes, macrophages and
multinucleated giant cells.
• A few, more severe, densification and
inflammatory lesions were focally present on
the pleura and the underlying pulmonary
lobules.
3rd Journal (Nururrozi et al., 2nd Journal (Sultana et al., 2015)
2020)
Inflammation, hyphae, and • Congestion of pulmonary and
spores found in air sac and perialveolar blood vessel and
lung. perivascular edema.
• The normal architecture of the lung and
Granulomatous air sacs were replaced by disseminated
inflammation in lung, air granulomatous foci.
sac. • Center of the granulomatous foci
contained caseous necrosis and necrotic
cellular debris surrounded by rims of
heterophils, lymphocytes, macrophages
and multinucleated giant cells.
• A few, more severe, densification and
inflammatory lesions were focally
present on the pleura and the underlying
pulmonary lobules.
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Figure. Showing congestion of pulmonary, Figure. Areas of caseous necrosis (black arrow) and
perialveolar blood vessel and diffuse edema of cellular debris (red arrow) in lung. H&E stain, 10×.
pulmonary tissues (arrow) in Lung. H&E stain, 10×.

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Figure. Aspergillosis showing granuloma Figure. Diffuse densification of the pleural
formation with caseated center (arrow) in lung. parenchyma by congestion and an
H&E stain, 10×. inflammatory cellular infiltration (arrows)
in lung. H&E stain, 10×.

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Figure. Local inflammation surrounded with Figure. Granulomatous inflammation
hyphae and spores in air sacs at 3 dpi. surrounded hyphae, submucosal edema, and
infiltration of inflammatory cells at 5 dpi.

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Figure. Bronchopneumonia with spores and
septate hyphae at 5 dpi.

Figure. Granulomatous inflammation of the lungs with

severe necrotic areas, heterophils surrounded by
macrophages, and connective tissue.

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DIAGNOSTIC APPROACH
 Signs of aspergillosis are non-specific so making diagnosis difficult with signs
alone.
 Since, clinical diagnosis is difficult, cases of aspergillosis in birds are often
diagnosed based on post-mortem findings of white caseous nodules in the
lungs or air sacs of affected birds.
 In aspergillosis, individual test does not provide reliable diagnosis.
 So, confirmatory diagnosis usually relay on disease history, clinical
presentation, post-mortem findings, haematology, biochemistry, serology,
radiographic changes, endoscopy, and culture of the fungus (Jones and Orosz,
2000).

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DISEASE HISTORY

• The history of the bird can reveal a stressful event and some under lining

environmental factors and immune suppressive condition or treatment.

• It may also reveal chronic debilitating, voice change or exercise intolerance.

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CLINICAL REPRESENTATION

 Dyspnoea, anorexia, cyanosis, polydypsia, foul smell diarrhea and emaciation.

 In chronic case Ocular changes like ophthalmitis, blepharospasm,

photophobia, and mycotic keratitis.

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POST-MORTEM FINDINGS

 Lesions typically involve the respiratory system; chronic lesions usually

involve the entire respiratory system, but acute cases have lesions in the lungs
and air sacs.
 Chickens with aspergillosis typically have white or yellow plaques or
nodules/granulomas and a mold-like lesion, or a general cloudiness, in the air
sacs and lungs of affected birds.
 But, definitive diagnosis is based on the isolation of Aspergillus species by
culture or by the detection of the organism during histological examination.

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HISTOPATHOLOGY

 The tissue samples (lungs, trachea, pharynx and thoracic air sacs as well as
other organs) fixed in 10% neutral buffered formalin are processed and
embedded in paraffin blocks and stain with haematoxylin and eosin (HE)
method. Aspergillus hyphae stained poorly in H and E stained sections.
 Differential stains such as Periodic acid-Schiff (PAS), Bauer’s and Gridley’s
stains differentiate and easily identify the hyphae and mycelia.
 Special stains for fungus Grocott’s and Gomori Methenamine Silver stain
should be employed to detect the presence of fungal hyphae.

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Fig. Fungal hyphae with dichotomous branching diagnostic of
aspergillus species are depicted (arrows), Histology (H & E
stain).
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Fig. Gomori methenamine silver stain of the patient's skin
biopsy showing fungal hyphae with septations

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MICROBIOLOGY

 Due to the ubiquity of Aspergillus conidia, isolation of the fungi without

presence of lesions is not diagnostic.
 Sources of samples for culture include deep tracheal swabs, culture of sinus
aspirates, choanal swabs, and material obtained from endoscopic evaluation of
the coelomic cavity.
 Identification can be made by preparing a wet smear. For this, a nodule is
dissected out and crushed on a slide beneath a cover slip in a drop of 20%
potassium hydroxide and lactophenol cotton blue. (The lactophenol cotton blue
stains the fungal hyphae).
 For proper identification of the species, the pathogenic organism must be
isolated by culturing it on differential media (Sabouraud’s glucose agar)
incubated at 37°C for 24 hours. Species of Aspergillus can be identified by
observing the characteristic conidial head and colony.

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Fig. Aspergillus spp on Lactophenol Cotton Blue (LPCB)
mount.

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HAEMATOLOGY

 Typically, birds that mount an appropriate immune response show a heterophilic leucocytosis,
monocytosis, lymphopenia, hyperproteinemia, and a nonregenerative anemia. The total white
blood cell count (WBC) is usually higher than 20,000/µL and may range as high as or higher
than 100,000/µL.
 The following characteristics may be indicative of aspergillosis:
 Elevated liver values (aspartate aminotransferase and lactate dehydrogenase)
 Elevated creatine kinase
 Increased uric acid
 Electrolyte abnormalities
 Decreased albumin: globulin ratio (<0.5)
 Moderate to severe leukocytosis with heterophilia (25,000–100,000 cells/mcL)
 Hyperglobulinemia (beta and gamma) Hypoalbuminemia Hypoglycemia

Repeated blood work can be used to evaluate disease progression and treatment success.
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RADIOLOGY
 Radiographic signs consistent with aspergillosis include:

 A prominent parabronchial pattern; loss of definition or asymmetry of the air

sacs; hyperinflation of the abdominal air sacs; focal soft tissue opacities within
the oropharynx, periorbital sinuses, trachea, syrinx, lungs, and air sacs;
thickening of the air sac membranes; outlining of air sac membranes by fluid
accumulation within the coelomic cavity; and hepatomegaly.

 Unfortunately, when radiographic signs are easily identifiable, the prognosis is

very poor.
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SEROLOGY

• Several serological assays have been developed to assist in the diagnosis of

aspergillosis. These include the agar gel immunodiffusion (AGID) test, the
indirect enzyme-linked immunosorbent assay (ELISA) for antibody, and the
ELISA for antigen. These tests are used frequently; however, the results
should be interpreted in conjunction with the clinical history and other
diagnostic tests.

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THE AGAR GEL IMMUNODIFFUSION (AGID)

• The AGID technique measures precipitating antibodies to A. fumigatus, and although

sensitive for long-standing disease, may not be able to diagnose aspergillosis in its early
stages.

• Most likely, the antibodies measured may represent immunoglobulin (Ig) G levels.

• In a study on the immunologic response of aspergillosis in pigeons (Columba livia), the birds
were immunized by weekly injections of A. fumigatus. Using an IgM- and IgG-specific A.
fumigatus ELISA assay, it was found that the IgM levels were detectable early and reached
their maximum levels by the second week. In contrast, the IgG levels started to increase only
in the second week and reached their maximum titre at 63 days (ninth week).

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DIFFERENTIAL DIAGNOSES 
• Differential diagnoses include:

• Infectious bronchitis

• Newcastle disease

• Infectious laryngotracheitis

• Mycobacteriosis

• Colibacillosis

• Other mycoses (eg, ochroconosis, zygomycosis)

• Oncogenic tumors (eg, Marek disease , avian leukosis )

• Nutritional encephalomalacia

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7579.1000382
 Nururrozi, A., Yanuartono, Y., Widyarini, S., Ramandani, D., & Indarjulianto, S. (2020).
Clinical and pathological features of aspergillosis due to Aspergillus fumigatus in broilers.
Veterinary World, 13(12), 2787–2792. https://doi.org/10.14202/vetworld.2020.2787-2792
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