Aspergillosis is caused by the filamentous fungus Aspergillus. Some Aspergillus species can cause serious disease in humans and animals through inhalation or ingestion. Aspergillus is found worldwide in soil and indoor environments. The most common pathogenic species are A. fumigatus and A. flavus, which can cause invasive infections in the lungs or other organs. Aspergillus has both pathogenic and beneficial uses industrially in producing enzymes and antibiotics. Certain Aspergillus species also produce dangerous mycotoxins like aflatoxins.

1. Aspergillosis
Presentor:
Bindu S
Division of Veterinary Microbiology

2. Aspergillus is a filamentous, most widespread fungal genus containing
both the pathogenic and beneficial species producing antibiotics,
antifungals and antitumor drugs

3. History
 In 1729 – Micheli first described Aspergillus
 In 1749- Reaumur first described Avian Aspergillosis in birds
 In 1842- Rayer and Montagene detected A. candidus in air sac
lesion of a bullfinch
 In 1863- Fresenius detected A.fumigatus in the lung of a great
bustard
 In 1961- Sargeant et al., identified Aspergillus flavus during
investigation of turkey-X disease
 In 1962- the name Aflatoxin was proposed
 In 1938- Datta reported association of aspergillus with bovine
hematuria in India
 In 1944- first isolated aspergillus from lungs, intestine, kidney, ovary ,
testes from poultry in India
 In 1996- Pal first reported guttural pouch mycosis in horses

4.  Some Aspergillus species cause serious disease in
humans and animals - it is pathogenic.
 Some Aspergillus species produce enzymes which
have important industrial applications.
 Aspergillus can produce mycotoxins – these are
often found in contaminated foodstuff and are
hazardous to the consumer.

5. Aspergillus species with Industrial Uses
 Genetically modified A. oryzae is used for the large scale
production of lipases used in biological washing powders.
 A.niger is used in the commercial production of citric acid, which
is widely used in the food industry.
 Fermentation of genetically modified A.oryzae is the major
source of recombinant chymogen which is used to curdle milk to
make hard cheeses.

6. Ecology
• Aspergillus is found predominately in the soil and also in air, water ,indoor
environment , vegetables and feed as saprophytes.
• Common contaminants of laboratory.
• It is a saprophytic fungi that breaks down carbon and nitrogen .
• In the conidia form however, it can become airborne in the atmosphere
and gain access to other hosts.
• The fungi can also act as opportunistic pathogen.
Unusual habitats are observed
(A. flavus, A.nidulans, A. niger , A.terreus) such as
microbial mat under fresh water or hypersaline water

7. In saprophytic phase , they are present
either as mycelium or a heavily
melanised survival structure called
sclerotia.

8. Pathogenic Aspergillus species
 The most common causing invasive disease are Aspergillus
fumigatus and Aspergillus flavus.
 The most common causing allergic disease are Aspergillus
fumigatus and Aspergillus clavatus.
which
- Allergic broncho pulmonary aspergillosis
- Invasive aspergillosis
- Aspergilloma
- Chronic pulmonary aspergillosis

9. Geographical distribution
 World wide distribution
 Most prevalent in warm regions
 Found everywhere from grasslands to mountainous terrain to high
temperature habitats.
 Highly aerobic fungus and therefore found in most oxygenated
environments.
 More common in cultivated fields compared to uncultivated fields..

10. Prevalence
 Majority of cases reported in mammalian species and in domestic animals
and pet animals only sporadic cases have been reported
 Rare in horses and donkeys.
 A.fumigatus has been found to be asssociated with diarrhoea in colts and
sometime equine abortions
 Ovine aspergillosis occur frequently
 Sometime generalized infections invading vascular and lymphatic system
as a extension of infection from respiratory tract to other organ of body

11. Etiology
 Kingdom:Fungi
 Phylum: Ascomycota
 Class: Eurotiomycetes
 Order: Eurotiales
 Family: Trichocomaceae
 Genus: Aspergillus
 Species: A.fumigatus , A.flavus, A.terreus, A.niger, A.nidulans, A.ustus

12. Morphology
 The mycelium is composed of septate hyphae with dichotomous
branching.
 In the tissues, only mycelium is observed
 In body cavities filled with air , the conidiophores with phialides are
found.
 Multiply by sexual and asexual spores. They possess fruiting bodies
called ascocarp (scletoium).
 Ascocarp contains number of sac like structure called ascus that is
filled with sexual spores- ascospores.
 In biseriate , a layer of cells called metullae cover the vesicle
 In uniseriate the structure of metullae is absent.

13. A.fumigatus A.flavus A.niger
Common cause of
aspergillosis
Usually a contaminant, may
cause disease
Usually a contaminant, may
cause disease
Conidiophores are long( 300-
500 micron) and have club
shaped vesicles that are 30-50
micron in diameter
Conidiophores are long ( 400-
800 micron)and are often
rough just beneath the
globose vesicle(25-45 micron)
Profuse conidiatian so that
circumferential conidia
obscure vesicle
Vesicle is uniseriate and are
covered by phialides / conidia
on only distal half
Metullae absent
Phialides arise
circumferentially and are
biseriate or sometimes
uniseriate
Biseriate, but heavy
conidiation usually obscures
metaulae and phialides
Conidia arise in chains Conidia are round, rough
form long chains
Conidia are spherical ,
3-5micron , and roughen
with maturity

14. Composition of cell wall
 A.flavus consists of glycoproeins, beta(1,3) glucan, beta(1,6) glucan.
 A.Fumigatus – beta(1,3) glucan, beta(1,6) glucan, alpha(1,3)glucan,
chitin, mannan, beta(1,5) galactofuranose, galactomannan and
galactomannoprotein

15. Transmission
• Inhalation or Ingestion
• Intramammary inoculation of spores
• In poultry farm by contaminated feed or litter.
• In moist environment, poor ventilation, humidity and long term storage of
feed in a farm
Impaired immunity/ stress due to administration
of antibiotic, vaccination metabolic bone disease,
hypovitaminosis A, over crowding, shipping,
starvation, inbreeding, toxicosis ,
reproductive activity , traumatic injuries
Predispo
-sing
factors

16. Antigenic characteristics
 Galactomannan (GM) is the major antigen of Aspergillus . It is a
part of cell wall along with chitin.
 It is released through the pores at the growing hyphal tips
during logarithmic growth phase of the fungi in highest amount
which helps in the detection of the antigen for diagnosis.
 GM is found in other fungi including Penicillium, Fusarium,
Alternaria, Histoplasma and yeasts including Cryptococcus which
can produce antigenic cross-reaction.
 The cell wall of Aspergillus contains 1, 3 β D glucan (BDG) which is
also secreted during late logarithmic growth phase of the fungi.

17. Virulence factors
Galactomannan (GM)
Melanin and Fks1
Protease
Siderophores
Calcium signalling pathway
Transcription factor
Ubiqutin encoding genes
Heat shock protein
Conidia
Autophagy related gene (ATG)

18. Immunity

19. Genome
 The genome of A.flavus contains eight chromosomes , genome size is 36.3
Mbp with 13,071 genes
 A 75 Kbp region in the chromosome consisting 29 gene clusters is
responsible for aflatoxin biosynthesis.
 The cluster is composed of – 1 gene encoding PKS
5 genes P450 mono-oxygenases
 Genes for global regulation MAPK, signal transduction, pathogenisity,
oxidative stress, fungal development and for several enzymes
 The genome of A.fumigatus is 29.4 Mbp containing 9630 genes
 Deep mRNA sequensing revealed 100’s of novel genes encoding small
proteins which are involved in colony growth and establishment of clinical
forms.

20. Pathogenesis
 Large number of conidia is required to establish infection
 Respiratory mucosa comprised of mucus, proteins, lipids, ions,
water and other ciliary secretions contribute to mucociliary clearance
 Conidial sialic acid act as ligand for adherence with alveolar
epithelial cells
 Lung injury act as major predisposing factor for invasive
aspergillosis
 Gliotoxin, fumagillin, helvolic acid produced by fungi damage the
respiratory mucosa
 In poultry , air sac is the primary target organ of fungi

21.  The conidial maturation begins which causes loss of hydrophobic
layer and exposure of the inner cell wall.
 The cell wall component act as ligand for the soluble and cell
associated Pattern recognition receptors.
 Most of conidia killed by ROS within alveolar macrophages
 The virulence factors of Aspergillus such as melanin, rodlets and
SOD can protect the conidia from ROS.
 Platelets also damage conidia by releasing seratonin.

22.  Germination depends on nutrient sensing and biosynthetic pathways.
 Under nutrient limited condition and other stress factor , the protein
folding capacity of the fungal EPR reaches its maximum limit and
UPR(unfolded protein response ) intiates .
 The enzymes elastase, aspartic proteinase, serine proteinase and
metalloprotease produced by Aspergillus.
 For survival & virulence of the fungi in vivo, there is requirement for
biosynthesis of uracil, folate and lysine.

23. Aspergillus utilize wide range of nitrogen sources
from environment
Degrade host proteins to obtain nutrients and
other AA
A. Fumigatus can also uptake the iron with the help
of siderophores.
Adaptation in alkaline pH and hypoxic condition
Gliotoxin, ribotoxins and haemolysin also help in
establishment of infection .
cpcA
locus
Pac C
SrbA

24. Fungi Host Disease
A.fumigatus, A.flavus,
A.niger, A.glaucus,
A.nidulans
poultry Avian aspergillosis
( Brooder pneumonia )
A. fumigatus Horse Guttural pouch mycosis
Nasal granuloma
Corneal ulcer
A. fumigatus Dogs Canine sinonasal aspergillosis
(Canine rhinitis)
Otitis externa
A. fumigatus Human Invasive aspergillosis
A. fumigatus Cattle, horse Sporadic abortion
A. fumigatus Cattle Mastitis
Pneumonia
Aspergillus spp. Calves
Horses
Mycotic gastritis
Keratomycosis

25. Toxins
Aflatoxins Different Aspergillus spp ,
A.flavus ( chief producer)
Ochratoxins A. ochraceus,
A. carbonarius
Citrinin A.terreus,
Penicillium citrinum
• Gliotoxin
• Ribotoxins
• Haemolysin
• Helvolic acid and fumagillin
• Fumitremorgin ( A,B,C)
• Tryptoquivaline A
A.fumigatus

26. Aflatoxins
 Aflatoxins are most potent toxic substances that occur naturally
 Aflatoxicosis due to ingestion of aflatoxins in contaminated feed.
 Aflatoxins are difuranocoumarin compounds produced by different species
of Aspergillus as secondary metabolite.
 It has six major types such as B1, B2, G1, G2, M1 and M2.
 Aflatoxin M1 and M2 are produced as metabolites of B1 and B2, and they
are commonly found in milk and to some extent in eggs respectively.,

27.  Aspergillus flavus is the chief producer which can affect many agricultural
crops .
 Other species such as A. fumigatus can produce aflatoxin B1 and G1.
 Aflatoxin, B1 (AFB1) is the most potent toxin and carcinogen than others in
human and animals including birds, fish and rodents.
 Maximum permissible limit of total aflatoxin and M1 (AFM1) is 20 ppb in
feed and 0.5 ppb in milk.
 Certain plant metabolite such as n-decyl aldehyde is inhibitory to aflatoxin
production.

28. Gliotoxin : It belongs to the family of epipolythiodioxopiperizines,
characterized by disulphide bridge across a piperizine ring which is
essential for their toxicity. It causes monocyte apoptosis , epithelial cell
damage.
Ochratoxins : it is potent nephrotoxic and carcinogenic.
A.ochraceus are mostly associated with dried and stored foods(cereals).
Whereas A .carbonarius is commonly found in fruits(grapes) that
mature in sunlight and at high temperature.
Citrinin: it is frequently found along with ochratoxin A in food stuff and
increases the toxicity synergistically.
Intoxication with both of the toxins together cause endemic
nephropathy. Citrinin is also embryocidal and foetotoxic.

29. Helvolic acid and fumagillin: Helvolic acid is a steroidal antibiotic. It can
inhibit the oxidative burst of macrophages and rupture of epithelial cells.
Fumagillin is an antitumour antibiotic which can inhibit angiogenesis,
endothelial cell proliferation and ciliary movement in the respiratory
epithelium.
Ribotoxins : Which specifically targets the sarcin/ricin domain of 28S rRNA
and inhibits the protein synthesis.
Mitogillin is highly cytotoxic causing cell death even in low concentration
Restrictocin
Mitogillin

30. Susceptibility to Disinfectants
o 35% Ethanol
o Copper 8- quinolinolate(0.4 mcg /ml)
o Very sensitive to radiation (500 & 100 Krad)
o Lower dose of radiation (1-5 Krad) can stimulate sporulation.

31. Avian Aspergillosis
 A.fumigatus and A.flavus is most commonly encountered.
 Most affected in turkeys but may affect chicken also.
 Acute outbreaks with high mortality and morbidity in young birds
 Chronic cases in adults affecting individual birds.
 Birds infected through inhalation of spores present in litter, feed or in the
soil of premises
 A.fumigatus has been reported to penetrate egg shell and infect embryos.
Acute
Chronic

32. Clinical signs and pathology
Acute form:
 Anorexia , sleepiness, gasping and sometime
convulsion.
 Occasionally invasion of brain and causes
paralysis or other CNS involvement
 Occular infection common usually unilateral
 Birds fail to grow and keep affected eyes
closed.
 Later on cheesy exudates in conjunctival sacs

33. Chronic form
• Often affects older birds (single individual/few birds in a flock )
• Mortality will be low
• Anorexia , gasping or coughing and rapid weight loss.

34. Necropsy finding
 Bright, greenish yellow caseous exudate is present in lungs or air sacs.
 Air sacs thickened , raised nodules of inflammatory exudates and mycelia
commonly present in lungs and in air sacs.
 Vary in size and in number and sometime causes button ulcers.

35. Histopathology
 The bronchi, bronchioles, and the alveoli to be filled with mucus , mycelia,
fibrin, and leucocytic and inflammatory cells.
 Foreign body giant cells may be seen
 Elements of the conidiophores may be found in the air passages but not in
the tissues.
hyphae

36. Bovine Aspergillosis
 Bovine mycotic abortion occurs third to eighth month of pregnanacy.
 Aborted fetus are rarely alive
 Retained placenta
 Placental lesions: yellow to grey cotyledons, thickened at periphery
 Intercotyledonry tissue appear leathery and is grey to tan in color.

37. Pathogenesis
 Spore inhalation
 Tissue and BV invasion
 Thrombus formation
 Granuloma formation
 Virulence factors: Gliotoxin, protease and elastase

38. Isolation and colony characteristics
 Aspergillus can be isolated in SDA with or without chloramphenicol
(0.05g/L)and other bacteriological media such as blood agar.
 Incubation at 37 C for 4-5 days , The spp which can’t tolerate this
temperature can be incubate at 25 C
 A. Fumigatus is thermophilic which is able to grow at 55-75 C

39. The colonies
are white
cotony which
becomes
granular with
green
coloration
after several
days
The colonies
are intially
white which
later become
cinnamon buff
coloured with
sugary texture
The colonies
are primarily
cotony and
later become
‘sugar texture’
with yellowish
green colour
The colonies
are white in
color in the
primary ,later
becomes black
due to
production of
black coloured
conidia
A.fumigatus A.terreus A.flavus A.niger

40. Diagnosis
 Clinical specimen collection
 Laboratory examination
- Direct examination
- Histopathology staining
- Isolation and identification
- Detection of antigen by serological test : AGPT, ELISA
- Moleculay biology: PCR-RFLP
Common
fungal
contaminant
in laboratory

41. Differential diagnosis
• From other respiratory tract infection causing organisms
• Mycotic bovine abortion
• IB, Pullorum disease in birds

42. Treatment
 No effective treatment exists .
 Voriconozole , Flucanozole, Itraconozole , Amphotericin B
 Nebulization , nasal or air sac flushing, surgical irrigation of abdominal
cavities
The nasal discharge usually resolves
within 7 – 14 days and rhinoscopy can
confirm the absence of fungal plaques.
If plaques are still present then a second
treatment can be followed

43. Control
• Dry, good quality litter and feed , hygiene .
• Thiabendazole and nystatin has been used in feed
• Avoid activities that involve close contact to soil or dust by wearing shoes,
protective clothing .
• Proper cleaning of skin injuries
• Antifungal medication if at high risk.

44. Reference
 Asthana RP. Aspergillosis in animals. Proc Indian Acad Sci Sec-
B. 1944:20:43-7.
 Veterinary mycology. In:Indranil Samanta.PP:32-44
 Clinical veterinary microbiology. In:P J Quinn et al.,PP:391-394