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

1. Jacques Guillot, Guillaume Le Loc’h, Pascal Arné, Françoise Féménia, René Chermette Avian aspergillosis UMR INRA, AFSSA, ENVA, UPVM 956, Biologie Moléculaire et Immunologie Parasitaires et Fongiques, Ecole Nationale Vétérinaire d’Alfort, 94704 Maisons-Alfort Cedex, France

2. Introduction Aspergillosis is considered as one of the most important infectious diseases in birds • early 1800s: first observations scaup duck, jay, bullfinch, bustard and several swans • 1898: first cases in turkey poults (Lignières & Petit in France) • Since then, cases in a very wide range of avian species…

3. 5 key questions… Why are birds more susceptible than mammals ? In which circumstances does aspergillosis occur in birds ? What do the main clinical signs and lesions look like ? Is it possible to make an early diagnosis in birds ? Is it possible to treat or prevent avian aspergillosis ?

4. Susceptibility of birds Birds are much more susceptible to aspergillosis than mammalian species • Environmental contamination by Aspergillus spp.

5. Susceptibility of birds lungs / air sacs Birds are much more susceptible to aspergillosis than mammalian species • Environmental contamination by Aspergillus spp. • Avian anatomy and physiology 40-43°C

6. Susceptibility of birds Birds are much more susceptible to aspergillosis than mammalian species • Environmental contamination by Aspergillus spp. • Avian anatomy and physiology • Avian immunology Lack of resident macrophages in airways and air sacs Heterophils without peroxydation

7. Epidemiology Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger… humidity, dampness drying period … Overgrowth Specific virulence factors ?

8. Epidemiology An epidemiological survey was conducted in France • a 600 m2 confinement building in the Center of France • a flock comprising 4500 turkeys females slaughtered at the age of 12 w and males at the age of 16 w Genotyping of environmental and clinical A. fumigatus isolates (2 microsatellite markers) Bart-Delabesse et al. J. Clin. Microbiol. 1998

9. Epidemiology Lair-Fulleringer et al. Poultry Science 2006

10. Epidemiology 5 females 5 males 10 healthy chicks, 23 isolates, 1 unique genotype Lair-Fulleringer et al. J. Clin. Microbiol. 2003

11. Epidemiology 9 healthy turkeys, 55 isolates, 17 genotypes Lair-Fulleringer et al. J. Clin. Microbiol. 2003

12. Epidemiology 2 carcass condemnations, 36 isolates, 2 genotypes Lair-Fulleringer et al. J. Clin. Microbiol. 2003

13. Epidemiology 362 air samples, 134 isolates 53 genotypes 16 week-sampling period Lair-Fulleringer et al. Poultry Science 2006

14. Epidemiology Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger… Overgrowth Specific virulence factors ? captive environment handling migration… • Stress • Other micro-organisms • Toxicosis • Therapeutics turkeys, quails, raptors, penguins, parrots, waterfowl • Species • Avian strains • Individual susceptibility ? • Age • Sex mycotoxins ? corticosteroids

15. Clinical signs and lesions Different avian species Different epidemiological situations Many clinical signs Acute aspergillosis = inappetance, depression, polydipsia, polyuria, dyspnoea, cyanosis = sometimes, sudden death without any signs fatal evolution Chronic aspergillosis = signs are dependent on the area of invasion change in voice respiratory stridor exercice intolerance ataxia, torticollis, seizures conjunctivitis, keratitis beak malformation…

16. Clinical signs and lesions Aspergillosis of eggs air sacculitis Kaminski et al. ENVA ENVA

17. Clinical signs and lesions meningo- encephalitis pneumonia Séguin Chute arthritis uveitis Séguin Séguin

18. Diagnosis Radiology Endoscopy Hematology Biochemistry Electrophoresis Cytology, histology Mycological culture Serology PCR ? Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology

19. Diagnosis Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Juliet Joseph, Abu Dhabi Falcon Research Hospital Biochemistry Electrophoresis Cytology, histology Mycological culture Serology PCR ?

20. Diagnosis Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Biochemistry Electrophoresis Cytology, histology Mycological culture Serology a promising approach ? PCR ?

21. Diagnosis Serology • Immunologically, birds respond to Aspergillus infection in the same way as mammals and a type I response appears most beneficial • Birds also respond with specific antibody production similar in its kinetics to mammals • Serological tests that may be used in birds include the detection of specific antibodies or fungal antigens

22. Diagnosis Serology  detection of specific antibodies High prevalence of seropositivity in captive penguins Low prevalence of seropositivity in wild birds False negative results some birds may not be able to mount an appropriate response some infection locations result in limited antigenic stimulation Antibody titers not necessary correlated to clinical severity

23. Diagnosis Serology  detection of galactomannan • agglutination (Pastorex Aspergillus®) • polyclonal sandwich ELISA • monoclonal sandwich ELISA (Platelia Aspergillus®)

24. Diagnosis Serology  detection of galactomannan 839 serum samples (from suspected or confirmed cases) Galactomannan in 50% of samples from penguins in 25% of samples from other birds Many chronic cases = negative for antibody but positive for galactomannan But long term chronic cases = negative for both antigen and antibody ! Cray et al. ISHAM 2006 (poster P-0011)

25. Diagnosis Serology  detection of galactomannan 90 serum samples (from cases in falcons) 182 control serum samples (from healthy falcons) Galactomannan in 12% of samples from infected falcons in 5% of samples from healthy birds Arca-Ruiba et al. Vet. Rec. 2006

26. Diagnosis Serology  detection of galactomannan Le Loch’ et al. ISHAM 2006 (poster))

27. Diagnosis Serology  detection of galactomannan Many false negative results variable release of GM variable kinetics according to avian species according to physiological status of each bird according to the level of immune complexing Some false positive results circulating GM from other fungi cross reactivity with bacterial components (food supplementation ?)

28. Diagnosis Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Biochemistry Electrophoresis Cytology, histology Mycological culture Serology Hardy et al. AAV proc. 2003 Dahlahausen et al. AAV proc. 2004 PCR ?

29. Diagnosis Radiology Endoscopy Hematology Biochemistry Electrophoresis Cytology, histology Mycological culture Serology Necropsy Turkeys, chickens, quails, ducks, ostriches… Clinical signs Epidemiology

30. Treatment Falcons, parrots, wild birds in zoological or rehabilitation centers Amphothericin B Flucytosine Ketoconazole Itraconazole Terbinafine Voriconazole (Langhofer, AAV proc. 2004) Radiography, endoscopy or serology for the follow up + Supportive therapy + Surgical debridement of the lesions

31. Treatment Turkeys, chickens, quails, ducks, ostriches… No treatment !

32. Prevention • Reduction of fungal contamination • Reduction of stress • Chimioprevention ? • Vaccination Richard (1984) reduced mortalities by 50% in turkey poults vaccinated with germinated A. fumigatus conidia Vaccination with a heat-killed culture filtrate preparation has been reported to reduce mortality in ducks and waterfowl

33. Conclusions « Avian aspergillosis » = not a single entity but a complex of several diseases… • need for specific avian models of aspergillosis • variable epidemiological situations • several diagnostic tools required • poor prognosis • importance of prophylactic procedures

34. References • 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.