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Avian aspergillosis Jacques Guillot, Guillaume Le Loc’h, Pascal Arné,

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1 Avian aspergillosis 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, 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… Aspergillus species have been recognised as a major cause of pulmonary disease in birds since the early 1800s and, as in humans, Aspergillus fumigatus is most frequent cause of disease. A retrospective analysis of penguin mortality at Edinburgh Zoo by Flach et al (1990) showed aspergillosis to be the most common cause of death in gentoo penguins (Pygoscelis papua), with chicks being the most susceptible. Annual mortality rates varied from below 20% to over 60% during the 25 year period, with 41% of birds overall being diagnosed with aspergillosis at post mortem. Most mortality occurred during the period July to September after chicks were moved to a crêche area, and it was theorised that climate may influence the prevalence.

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 ? Aspergillus species have been recognised as a major cause of pulmonary disease in birds since the early 1800s and, as in humans, Aspergillus fumigatus is most frequent cause of disease. A retrospective analysis of penguin mortality at Edinburgh Zoo by Flach et al (1990) showed aspergillosis to be the most common cause of death in gentoo penguins (Pygoscelis papua), with chicks being the most susceptible. Annual mortality rates varied from below 20% to over 60% during the 25 year period, with 41% of birds overall being diagnosed with aspergillosis at post mortem. Most mortality occurred during the period July to September after chicks were moved to a crêche area, and it was theorised that climate may influence the prevalence.

4 Susceptibility of birds
Birds are much more susceptible to aspergillosis than mammalian species • Environmental contamination by Aspergillus spp. Among all predisposing factors, the environmental contamination by Aspergillus fumigatus ust be taken into account. On this picture, you can see that the litter for this chicks is composed of straw Very frequently, the aliments are also contaminated

5 Susceptibility of birds
Birds are much more susceptible to aspergillosis than mammalian species • Environmental contamination by Aspergillus spp. • Avian anatomy and physiology lungs / air sacs 40-43°C In birds the anatomy of respiratory system is very different from that of mammals. There are no alveoles but tubular structures in connection with voluminous air sacs. These air-filled structures are very easily colonised by Aspergillus fumigatus. Posterior air sacs are most frequently affected. The internal temperature is very high in birds. This is also a very good condition for the development of a thermophilic fungal species such as Aspergillus fumigatus. Lack of epiglotis Lack of a diaphragme resulting in the inhability of a strong cough reflex

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 Predisposition of birds to aspergillosis may be attributed to some anatomical peculiarities, which preclude the mechanisms of ejection of inhaled fungal spores. The absence of resident macrophages within airway lumens and the dependence on heterophils (that use cationic proteins, hydrolase and lysosyme rather than catalase and myeloperoxidase) may also be responsible for the increased sensitivity of birds to aspergillosis (Klika et al., 1996; Harmon, 1998).

7 Epidemiology Aspergillus fumigatus / Aspergillus flavus, Aspergillus niger… humidity, dampness drying period … Overgrowth Specific virulence factors ? Epidemiology = in which circumstances aspergillosis occurs in birds ? Watering places

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 Kunkle & Rimler Early pulmonary lesions produced by non viable A. fumigatus and/or P. multocida lipopolysaccharide. 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
Kunkle & Rimler Early pulmonary lesions produced by non viable A. fumigatus and/or P. multocida lipopolysaccharide. Lair-Fulleringer et al. Poultry Science 2006

10 Epidemiology 10 healthy chicks, 23 isolates, 1 unique genotype
5 males 5 females 10 healthy chicks, 23 isolates, 1 unique genotype Twenty-three isolates were obtained from 6 healthy chicks, sacrified at their arrival in the confinement house. These isolates corresponded to the unique genotype C173D108 (Table 1). This genotype was also detected from two healthy turkeys, which were sacrified at day 37 and 44, respectively Lair-Fulleringer et al. J. Clin. Microbiol. 2003

11 9 healthy turkeys, 55 isolates, 17 genotypes
Epidemiology A total number of 55 A. fumigatus isolates were obtained from 9 healthy turkeys, which were sacrified during the breeding period. Lesions compatible with aspergillosis were never observed and each animal carried from one to 6 distinct A. fumigatus genotypes. A similar situation is described in cystic fibrosis patients who are supposed to be colonized rather than infected by A. fumigatus isolates . 9 healthy turkeys, 55 isolates, 17 genotypes Lair-Fulleringer et al. J. Clin. Microbiol. 2003

12 Epidemiology 2 carcass condemnations, 36 isolates, 2 genotypes
We finally examined 36 isolates from two animals (turkeys XVI and XVII) in which lesions of aspergillosis were detected at slaughter inspection. Histological examination confirmed the presence of hyaline fungal hyphae in lungs, abdominal air sac and kidneys. Typical conidial heads were also observed in the abdominal air sac. These turkeys were infected by their own distinct genotypes (C173D112 in turkey XVI and C169D102 in turkey XVII) (Table 1). Similarly, several investigations have demonstrated that only one or two genotypes are usually responsible for invasive aspergillosis in humans. The same genotype was detected in both healthy and infected birds suggesting that the absence of particular virulent genotypes for turkeys. Previous investigations have shown that no particular A. fumigatus genotype was associated with virulence in humans 2 carcass condemnations, 36 isolates, 2 genotypes Lair-Fulleringer et al. J. Clin. Microbiol. 2003

13 Epidemiology 362 air samples, 134 isolates 53 genotypes
A total number of 134 isolates representing 53 distinct genotypes were obtained from air samples. Thirty-three (33/53=62.3%) genotypes were observed only once. The remaining 20 genotypes were detected during several weeks with a maximum persistence of 8 weeks for genotype C175D76. Similar results were reported when the genetic diversity of A. fumigatus isolates from hospital environment was investigated 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… turkeys, quails, raptors, penguins, parrots, waterfowl • Stress • Other micro-organisms • Toxicosis • Therapeutics • Species • Avian strains • Individual susceptibility ? • Age • Sex Kunkle & Rimler Early pulmonary lesions produced by non viable A. fumigatus and/or P. multocida lipopolysaccharide. 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 ENVA Kaminski et al. ENVA

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

18 Diagnosis Clinical signs Epidemiology Radiology Endoscopy Hematology
Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Serology Biochemistry Electrophoresis Mycological culture PCR ? Cytology, histology Loss of definition or asymmetry of the air sacs

19 Juliet Joseph, Abu Dhabi Falcon Research Hospital
Diagnosis Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Biochemistry C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. Electrophoresis Juliet Joseph, Abu Dhabi Falcon Research Hospital Cytology, histology Mycological culture Serology PCR ?

20 Diagnosis Clinical signs Epidemiology Radiology Endoscopy Hematology
Falcons, parrots, wild birds in zoological or rehabilitation centers Clinical signs Epidemiology Radiology Endoscopy Hematology Biochemistry Supposed to be a very promising method… 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 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs.

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 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. Antibody titers not necessary correlated to clinical severity

23 Diagnosis Serology  detection of galactomannan
• agglutination (Pastorex Aspergillus®) • polyclonal sandwich ELISA C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. • 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 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. 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 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. Arca-Ruiba et al. Vet. Rec. 2006

26 Platelia® Aspergillus
Diagnosis Serology  detection of galactomannan Platelia® Aspergillus + - CIE 17 39 56 20 65 85 37 104 141 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. 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 C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. Some false positive results circulating GM from other fungi cross reactivity with bacterial components (food supplementation ?)

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

29 Diagnosis Clinical signs Epidemiology Radiology Endoscopy Hematology
Turkeys, chickens, quails, ducks, ostriches… Clinical signs Epidemiology Hematology Radiology Endoscopy Serology Biochemistry Electrophoresis Mycological culture Necropsy Cytology, histology C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs.

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

31 No treatment ! Treatment Turkeys, chickens, quails, ducks, ostriches…
C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs.

32 Prevention • Reduction of fungal contamination • Reduction of stress
• Chimioprevention ? • Vaccination C,D Yellow and green aspergillus colonies A1 and A2 on the airsac wall. Note the increased vascularity of the airsacs. 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 All birds are considered susceptible but the disease is particularly common in … • 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, 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, Kunkle, R.A. & Rimler, R.B. (1996). Pathology of acute aspergillosis in turkeys. Avian Diseases, 40, Lair-Fulleringer, S., Guillot, J., Desterque, C., Seguin, D., Warin, S., Chermette, R. & Bretagne, S Differentiation of Aspergillus fumigatus isolates from breeding turkeys and their environment by genotyping with microsatellite markers. Journal of Clinical Microbiology, 41, 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 Avian Diseases, 32, Peden, W.M. & Rhoades, K.R. (1992). Pathogenicity differences of multiple isolates of Aspergillus fumigatus in turkeys. Avian Diseases, 36, 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, Redig, P.T. (1993). Avian aspergillosis. In: Fowler ME (Ed) Zoo and wild animals medicine. WB. Saunders Company, Philadelphia, Richard, J.L. (1997). Aspergillosis. In: Diseases of poultry. Calmek B.W. (Ed), Mosby-Wolfe, London, Taylor, J.J. & Burroughs, E.J. (1973). Experimental avian aspergillosis. Mycopathologia Mycologia Applicata, 51, Tell, L.A. (2005). Aspergillosis in mammals and birds: impact in veterinary medicine. Medical Mycology, 43, S71-S73. All birds are considered susceptible but the disease is particularly common in …


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