Jacques Guillot, Guillaume Le Loch, Pascal Arné, Françoise Féménia, René Chermette Avianaspergillosis UMR INRA, AFSSA, ENVA, UPVM 956, Biologie Moléculaire

Jacques Guillot, Guillaume Le Loc’h, Pascal Arné,Françoise Féménia, René Chermette

AvianAvianaspergillosisaspergillosis

UMR INRA, AFSSA, ENVA, UPVM 956, Biologie Moléculaire et Immunologie Parasitaires et Fongiques, Ecole Nationale Vétérinaire d’Alfort, 94704 Maisons-Alfort Cedex, France

IntroductionIntroduction

Aspergillosis is considered as one of the most important infectious diseases in birdsAspergillosis is considered as one of the most important infectious diseases in birds

• • early 1800s: first observationsearly 1800s: first observations

scaup duck, jay, bullfinch, bustard and several swansscaup duck, jay, bullfinch, bustard and several swans

• • 1898: first cases in turkey poults 1898: first cases in turkey poults (Lignières & Petit in France)(Lignières & Petit in France)

• • Since then, cases in a very wide range of avian species…Since then, cases in a very wide range of avian species…

5 key questions…5 key questions…

Why are birds more susceptible than mammals ? Why are birds more susceptible than mammals ? Why are birds more susceptible than mammals ? Why are birds more susceptible than mammals ?

In which circumstances does aspergillosis occur in birds ? In which circumstances does aspergillosis occur in birds ? In which circumstances does aspergillosis occur in birds ? In which circumstances does aspergillosis occur in birds ?

Is it possible to make an early diagnosis in birds ? Is it possible to make an early diagnosis in birds ? Is it possible to make an early diagnosis in birds ? Is it possible to make an early diagnosis in birds ?

Is it possible to treat or prevent avian aspergillosis ? Is it possible to treat or prevent avian aspergillosis ? Is it possible to treat or prevent avian aspergillosis ? Is it possible to treat or prevent avian aspergillosis ?

What do the main clinical signs and lesions look like ? What do the main clinical signs and lesions look like ? What do the main clinical signs and lesions look like ? What do the main clinical signs and lesions look like ?

Susceptibility of birdsSusceptibility of birds

Birds are much more susceptible to aspergillosis than mammalian speciesBirds are much more susceptible to aspergillosis than mammalian species

• • Environmental contamination by Environmental contamination by AspergillusAspergillus spp. spp.



• • Avian anatomy and physiologyAvian anatomy and physiology

40-43°C40-43°C40-43°C40-43°C


lungslungs / / air sacsair sacs



• • Avian anatomy and physiologyAvian anatomy and physiology

• • Avian immunologyAvian immunology

Lack of resident macrophages in airways and air sacs

Heterophils without peroxydation


EpidemiologyEpidemiology

Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger…Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger…

OvergrowthOvergrowth

Specific virulence factors ?Specific virulence factors ?

humidity, dampnesshumidity, dampnessdrying period …drying period …


• • a 600 ma 600 m22 confinement building in the Center of France confinement building in the Center of France

• • a flock comprising 4500 turkeysa flock comprising 4500 turkeys females slaughtered at the age of 12 w and males at the age of 16 wfemales slaughtered at the age of 12 w and males at the age of 16 w

Genotyping of environmental Genotyping of environmental and clinical and clinical A. fumigatusA. fumigatus

isolatesisolates

(2 microsatellite markers)(2 microsatellite markers)Bart-Delabesse Bart-Delabesse et alet al. J. Clin. Microbiol. 1998. J. Clin. Microbiol. 1998

An epidemiological survey was conducted in FranceAn epidemiological survey was conducted in France


0

25

50

75

100

125

150

3 4 5 6 7 8 9 10 11 12 13 14 15 16

Aspergillus flavus Aspergillus fumigatus Other fungi

Lair-Fulleringer et al. Poultry Science 2006


5 males5 males 5 females5 females

10 healthy chicks, 23 isolates,1 unique genotype

Lair-Fulleringer et al. J. Clin. Microbiol. 2003


9 healthy turkeys, 55 isolates, 17 genotypes9 healthy turkeys, 55 isolates, 17 genotypes



2 carcass condemnations, 36 isolates, 2 genotypes



362 air samples, 134 isolates 53 genotypes

16 week-sampling period

Lair-Fulleringer et al. Poultry Science 2006


Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger…Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger…

OvergrowthOvergrowthSpecific virulence factors ?Specific virulence factors ?

• • SpeciesSpecies

• • Avian strainsAvian strains• • Individual susceptibility ?Individual susceptibility ?• • AgeAge• • SexSex

• • StressStress

• • Other micro-organismsOther micro-organisms

• • ToxicosisToxicosis

• • TherapeuticsTherapeutics

captive environmentcaptive environmenthandlinghandlingmigration…migration…

mycotoxins ?mycotoxins ?

corticosteroidscorticosteroids

turkeys, quails, turkeys, quails, raptors, penguins, raptors, penguins, parrots, waterfowlparrots, waterfowl

Clinical signs and lesionsClinical signs and lesions

Different avian speciesDifferent epidemiological situationsDifferent avian speciesDifferent epidemiological situations Many clinical signsMany clinical signs

Acute aspergillosisAcute aspergillosis = inappetance, depression, polydipsia, polyuria, dyspnoea, cyanosis= sometimes, sudden death without any signs

Acute aspergillosisAcute aspergillosis = inappetance, depression, polydipsia, polyuria, dyspnoea, cyanosis= sometimes, sudden death without any signs

Chronic aspergillosisChronic aspergillosis = signs are dependent on the area of invasion

change in voicerespiratory stridorexercice intoleranceataxia, torticollis, seizuresconjunctivitis, keratitisbeak malformation…

Chronic aspergillosisChronic aspergillosis = signs are dependent on the area of invasion

change in voicerespiratory stridorexercice intoleranceataxia, torticollis, seizuresconjunctivitis, keratitisbeak malformation…

fatal evolution


ENVA

Kaminski et al.

air sacculitis

Aspergillosisof eggs

ENVA


Chute

pneumonia

meningo-encephalitis

Séguin

arthritis

Séguin

uveitis

Séguin

DiagnosisDiagnosis

Falcons, parrots, wild birds in zoological or rehabilitation centers

Clinical signsClinical signs


RadiologyRadiology

EndoscopyEndoscopy

HematologyHematology

SerologySerology

BiochemistryBiochemistry

ElectrophoresisElectrophoresis

Mycological cultureMycological culture

PCR ?PCR ?

Cytology, histologyCytology, histology

DiagnosisDiagnosis



RadiologyRadiology

EndoscopyEndoscopy


SerologySerology

Juliet Joseph,

Abu Dhabi Falcon Research Hospital





PCR ?PCR ?



DiagnosisDiagnosis



RadiologyRadiology

EndoscopyEndoscopy

SerologySerology





PCR ?PCR ?


a promising approach ?

DiagnosisDiagnosis

SerologySerology

• • Immunologically, birds respond to Immunologically, birds respond to AspergillusAspergillus infection in infection in the same way as mammals and a type I response appears the same way as mammals and a type I response appears most beneficialmost beneficial

• • Birds also respond with specific antibody production Birds also respond with specific antibody production similar in its kinetics to mammalssimilar in its kinetics to mammals

• • Serological tests that may be used in birds include the Serological tests that may be used in birds include the detection of specific antibodies or fungal antigensdetection of specific antibodies or fungal antigens

DiagnosisDiagnosis

SerologySerology detection of specific antibodies

False negative results False negative results some birds may not be able to mount an appropriate responsesome infection locations result in limited antigenic stimulation

Antibody titers not necessary correlated to clinical severityAntibody titers not necessary correlated to clinical severity

High prevalence of seropositivity in captive penguinsLow prevalence of seropositivity in wild birds

DiagnosisDiagnosis

SerologySerology detection of galactomannan

• agglutination (Pastorex Aspergillus®)

• monoclonal sandwich ELISA (Platelia Aspergillus®)

• polyclonal sandwich ELISA

DiagnosisDiagnosis


Cray et al. ISHAM 2006 (poster P-0011)

839 serum samples (from suspected or confirmed cases)

Galactomannan in 50% of samples from penguinsin 25% of samples from other birds

Many chronic cases = negative for antibody but positive for galactomannanBut long term chronic cases = negative for both antigen and antibody !

DiagnosisDiagnosis


Arca-Ruiba et al. Vet. Rec. 2006

90 serum samples (from cases in falcons)

182 control serum samples (from healthy falcons)

Galactomannan in 12% of samples from infected falconsin 5% of samples from healthy birds

DiagnosisDiagnosis


Le Loch’ et al. ISHAM 2006 (poster))

Platelia® Aspergillus

+ -

CIE

+ 17 39 56

- 20 65 85

37 104 141

DiagnosisDiagnosis


Many false negative results Many false negative results variable release of GMvariable kinetics

according to avian species according to physiological status of each birdaccording to the level of immune complexing

Some false positive results Some false positive results circulating GM from other fungicross reactivity with bacterial components (food supplementation ?)

SerologySerology


DiagnosisDiagnosis



RadiologyRadiology

EndoscopyEndoscopy





PCR ?PCR ?


Hardy et al. AAV proc. 2003Dahlahausen et al. AAV proc. 2004

DiagnosisDiagnosis



Turkeys, chickens, quails, ducks, ostriches…


RadiologyRadiology

EndoscopyEndoscopy

SerologySerology




NecropsyNecropsy


TreatmentTreatment


Amphothericin BAmphothericin B

FlucytosineFlucytosine

KetoconazoleKetoconazole

ItraconazoleItraconazole

TerbinafineTerbinafineVoriconazole (Langhofer, AAV proc. 2004)Voriconazole (Langhofer, AAV proc. 2004)

Radiography, endoscopy or serology for the follow up

+ Supportive therapy + Surgical debridement of the lesions

TreatmentTreatment

No treatment !No treatment !

Turkeys, chickens, quails, ducks, ostriches…

PreventionPrevention

• Reduction of fungal contamination

• Chimioprevention ?

Vaccination with a heat-killed culture filtrate preparation has been reported Vaccination with a heat-killed culture filtrate preparation has been reported to reduce mortality in ducks and waterfowlto reduce mortality in ducks and waterfowl

• Reduction of stress

• Vaccination

Richard (1984) reduced mortalities by 50% in turkey poults vaccinated with Richard (1984) reduced mortalities by 50% in turkey poults vaccinated with germinated germinated A. fumigatusA. fumigatus conidia conidia

ConclusionsConclusions

« Avian aspergillosis »« Avian aspergillosis » = not a single entity but a complex of several diseases… = not a single entity but a complex of several diseases…

• • importance of prophylactic procedures importance of prophylactic procedures

• • poor prognosispoor prognosis

• • need for specific avian models of aspergillosisneed for specific avian models of aspergillosis

• • variable epidemiological situationsvariable epidemiological situations

• • several diagnostic tools requiredseveral diagnostic tools required

ReferencesReferences

Cray, C., Rodriguez, M. & Watson, T. (2006) Aspergillus serodiagnostics in avian species. 16th Congress of the International Society for Human and Animal Mycology (ISHAM), June 25-29th 2006, Paris (Poster).

Harmon, B. (1998). Avian heterophils in inflammation and disease resistance. Poultry Science, 77, 972-977.

Kearns, K.L. (2003). Avian aspergillosis. In: Recent advances in avian infectious diseases. Kearns KS, Loudis B (Eds). Ithaca, International Information Service.

Klika, E., Scheuermann, D.W., De Groodt-Lasseel, M.H.A., Bazantova, I. & Switka, A. (1996). Pulmonary macrophages in birds (barn owl, Tyto tyto alba), domestic fowl (Gallus gallus domestica), quail (Coturnix coturnix) and pigeon (Columbia livia). Anatomy Record, 246, 87-97.

Kunkle, R.A. & Rimler, R.B. (1996). Pathology of acute aspergillosis in turkeys. Avian Diseases, 40, 875-886.

Lair-Fulleringer, S., Guillot, J., Desterque, C., Seguin, D., Warin, S., Chermette, R. & Bretagne, S. 2003. Differentiation of Aspergillus fumigatus isolates from breeding turkeys and their environment by genotyping with microsatellite markers. Journal of Clinical Microbiology, 41, 1798-1800.

Le Loc’h, G., Arné, P., Bougerol, C., Risi, E., Péricard, J.M., Quinton, J.F., Bretagne, S. & Guillot, J. (2006) Detection of circulating serum galactomannan for the diagnosis of avian aspergillosis. 16th Congress of the International Society for Human and Animal Mycology (ISHAM), June 25-29th 2006, Paris (Poster).

Morris, M.P. & Fletcher, O.J. (1988). Disease prevalence in Georgia turkey flocks in 1986. Avian Diseases, 32, 404-406.

Peden, W.M. & Rhoades, K.R. (1992). Pathogenicity differences of multiple isolates of Aspergillus fumigatus in turkeys. Avian Diseases, 36, 537-542.

Redig, P.T., Post, G.S., Concannon, T.M. & Dunette, J. (1986). Development of an ELISA for the detection of aspergillosis in avian species. Proceedings of the Association Avian Veterinarians, 165-178.

Redig, P.T. (1993). Avian aspergillosis. In: Fowler ME (Ed) Zoo and wild animals medicine. WB. Saunders Company, Philadelphia, 178-181.

Richard, J.L. (1997). Aspergillosis. In: Diseases of poultry. Calmek B.W. (Ed), Mosby-Wolfe, London, 351-365.

Taylor, J.J. & Burroughs, E.J. (1973). Experimental avian aspergillosis. Mycopathologia Mycologia Applicata, 51, 131-141.

Tell, L.A. (2005). Aspergillosis in mammals and birds: impact in veterinary medicine. Medical Mycology, 43, S71-S73.

Documents

Jacques Guillot, Guillaume Le Loch, Pascal Arné, Françoise Féménia, René Chermette Avianaspergillosis UMR INRA, AFSSA, ENVA, UPVM 956, Biologie Moléculaire