Identification of pathogenic Aspergillus isolates from captive birds in Australia

Identification of pathogenic Aspergillus isolates from captive birds in Australia Abstract Aspergillosis is a major cause of severe respiratory disease in birds. The prevalence of cryptic section Fumigati and other non–Aspergillus fumigatus species as causative agents is unknown. Species identity was determined in 30 isolates from affected birds from zoos, pet birds and poultry by PCR of the ITS1-5.8S-ITS2 and partial β-tubulin genes. The most prevalent isolate was A. fumigatus sens. str. in 87% (26) cases. Other Aspergillus species were identified in 13% (4) cases, including A. restrictus (1), A. flavus sens. str. (2), and A. nidulans-clade (1). This is the first report of A. restrictus causing avian disease. avian aspergillosis, avian mycoses, Australia Introduction Avian aspergillosis, first reported in the 1800s,1 is a significant cause of mortality among captive birds worldwide. It most commonly causes disease within the respiratory tract, and can progress to disseminated invasive aspergillosis. Two forms of lesions have been described; a granulomatous deep nodular form affecting non-aerated parenchyma, and a pyogranulomatous nonencapsulated superficial diffuse form involving lungs and serosae.2 Captive birds are highly susceptible to aspergillosis due to environmental factors (increased burden of fungal spores), host immune factors (immunosuppression from disease, stress, or therapeutics), and unique respiratory anatomy.1,2 Anatomical differences to mammals that enable unique airflow in birds include nonexpansile and unlobed lungs with no pleural cavity, looped parabronchi with bi-directional air flow capillaries instead of terminal ending alveoli, and cranial and caudal air sacs that extend the bronchial system beyond the lung to pneumatize bones and enable fresh airflow during inspiration and expiration.3 Infection occurs when innate defenses cannot eliminate spores in parabronchial air capillaries, resulting in fungal plaques that can penetrate surrounding tissues.2 Despite a range of antifungal therapeutic treatment options, infections are often fatal.2 The clinical importance of fungal molecular identification techniques has been demonstrated for human and nonavian animal species with aspergillosis, with cryptic section Fumigati species increasingly identified, prompting epidemiological studies.4–9 We previously identified isolates causing respiratory aspergillosis in cats and dogs and found that disease in dogs, like humans is predominantly caused by A. fumigatus sens. str.,8 while in cats, infections with cryptic species in section Fumigati including A. felis, A. udagawae, and A. thermomutatus are most prevalent.9 We performed environmental sampling for Aspergillus section Fumigati in homes of cats previously diagnosed with aspergillosis and in a recreational nature reserve and found that cryptic species reported to cause disease in humans or animals were common, comprising 25% of isolates overall.10 Aspergillus fumigatus sens. str. has been most commonly associated with avian infections based on phenotypic identification techniques, with A. flavus, A. niger, A. glaucus, and A. nidulans also reported.2 Thus, cryptic species may have been overlooked as a cause of avian aspergillosis. This study aimed to definitively identify fungal pathogens causing avian aspergillosis and to determine the prevalence of cryptic A. section Fumigati species. A secondary aim was to determine the form of aspergillosis present. Methods Australian avian veterinarians were contacted to recruit isolates from clinical cases of avian aspergillosis between 2013 and 2017. Aspergillus isolates cultured from lesions in captive birds consistent with aspergillosis, including fungal plaques or granulomas observed within the respiratory tract or other parenchyma during diagnostic investigation or at post mortem were included for study. Morphological identification of Aspergillus species was performed by microbiologists, based on consistent macroscopic and microscopic features including identification of smooth or rough walled conidia stemming from radiate or columnar conidial heads with globose or subclavate vesicles, stipe and a foot cell.11 Thirty isolates were received from Australian native and exotic avian species (Table 1), including 26 from zoos (New South Wales [n = 23, Taronga Conservation Society approved opportunistic sampling request R14B178], South Australia [n = 3]), one from private practice (Queensland) and three from commercial poultry producers (NSW). Histopathology post-mortem reports were available for review in 20 zoological cases. Table 1. Fungal molecular identification (internal transcribed spacer (ITS), partial beta-tubulin [BenA] gene), avian species and origin details for fungal isolates used in this study (n = 30), collected from 2013 to 2017. Molecular identification is based on homology percentage matches of ≥99% (ITS) and ≥99.5% (BenA) using NCBI-BLAST tool. Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  e, exotic; n, native. View Large Table 1. Fungal molecular identification (internal transcribed spacer (ITS), partial beta-tubulin [BenA] gene), avian species and origin details for fungal isolates used in this study (n = 30), collected from 2013 to 2017. Molecular identification is based on homology percentage matches of ≥99% (ITS) and ≥99.5% (BenA) using NCBI-BLAST tool. Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  e, exotic; n, native. View Large All isolates were subcultured onto malt extract agar and grown in the dark at 25°C for up to 7 days until sporulation occurred. DNA was extracted using the MoBio DNA isolation kit (QIAGEN MO BIO, Carlsbad, USA). Conventional polymerase chain reaction (PCR) was performed targeting the rDNA gene cluster, including the ITS1, 5.8S gene, and ITS2 regions using primers ITS1 and ITS4.12 Amplification of the partial β-tubulin (BenA) gene was performed using primers bt2a and bt2b.13 PCR product was sent to an external laboratory for purification and Sanger sequencing (Macrogen, Seoul, South Korea). Generated sequences were edited using BioEdit version 7.2.5.14 Species identification was determined using NCBI-BLAST tool and sequence homology of ≥99% (ITS) and ≥99.5% (BenA) with type strains sequences.15 If BenA homology was <99.5%, a separate alignment and phylogenetic analysis was performed in Mega 6.0616 with sequences of all section type-strains to confirm the closest type-strain match using the maximum likelihood method based on the Tamura-Nei model with gamma distribution and 1000 bootstrap replicates. Sequences were deposited in GenBank (MF540250-MF540309). Results Twenty-six of 30 (87%) isolates were identified as A. fumigatus sens. str., and the remaining four (13%) were other Aspergillus species, including A. restrictus (n = 1), A. flavus sens. str. (n = 2), and A. nidulans-clade (n = 1) (Table 1). The latter isolate had 99.1% BenA homology with A. nidulans sens. str. Phylogenetic analysis with section Nidulantes type-strains placed this isolate in the A. nidulans-clade, where it was most closely related to A. pachychristatus and A. latilabiatus. Affected avian species were 70% native and 30% exotic to Australia; passerines were most commonly affected (30%). Five A. fumigatus sens. str. isolates were from IUCN listed critically endangered native species (http://www.iucnredlist.org/search) including Plains Wanderer (Pedionomus torquatus) (3), Regent Honeyeater (Anthochaera Phrygia) (1), and Swift Parrot (Lathamus discolor) (1). Histopathology findings are summarized in supplementary Table 1. Seventeen of 20 cases had invasive aspergillosis, which was disseminated in 12 and focal in five (respiratory). There were three cases of noninvasive localized infection, involving conjunctiva, air sacs, and a coelomic cavity mass. All six respiratory cases (5 invasive, 1 noninvasive) involved the lower respiratory tract; localized to the air sacs (1), the lungs (4), or both air sacs and lungs (1). In four cases, a primary nonfungal disease process was identified including mycobacteriosis, egg yolk peritonitis, metastatic pancreatic adenocarcinoma and chronic liver disease. Discussion Aspergillus fumigatus sens. str. was the most common isolate amongst our study population (87%), similar to prevalence studies in humans, dogs, and the environment.4–8 While other Aspergillus species were identified in 13% of cases, no cryptic section Fumigati species were identified. This is in contrast to prevalence studies in humans (0.75–3.7% of cases)4–6 and reports on cats, in which invasive fungal rhinosinusitis is the most common form and is caused by cryptic species in section Fumigati.9 Although our sample size was small, we found a similar prevalence for A. flavus sens. str. (6%) and A. nidulans-clade (3%) as is reported in humans (A. flavus 8–13%; A. nidulans 0.5–2.5%).4–6 Our study is the first to our knowledge to report aspergillosis in birds due to A. restrictus. This case was in a Java Finch (Lonchura oryzivora) with chronic liver disease and disseminated invasive aspergillosis affecting air sacs, lung, and gizzard. Aspergillus restrictus has also been isolated from humans with disseminated infection following a heart valve replacement, although only phenotypic identification was performed.17,18 Our findings support an opportunistic pathogen role for this fungal species. This study demonstrates the value of molecular characterization of avian fungal isolates. Aspergillus species other than A. fumigatus sens. str. should also be considered as potential agents of this disease and may have important implications for patient management as seen in humans and other animals. Different Aspergillus species isolated from birds have been shown to have different susceptibilities to commonly used antifungal therapies, including itraconazole and amphotericin B.19 This information could prove invaluable in management of a disease that is already challenged by lack of knowledge in avian pharmacokinetics and late-stage clinical presentation.2 We investigated avian aspergillosis amongst captive birds including several critically endangered native Australian species. The pathogenesis of avian disease has been associated with an increased environmental load of filamentous fungi in captive/rehabilitation settings (5.7% of fungal isolates grown) compared to natural avian habitats (0.03%),20 with an A. fumigatus average air count nine times greater than natural habitats reported from rehabilitation centres.21 Furthermore, genotyping of clinical and environmental isolates has demonstrated that the rehabilitation setting is a source of clinical disease in captive birds, likely due to this increased fungal burden and the presence of existing disease and/or stress.21 This highlights the importance of environmental control strategies to reduce the risk of aspergillosis in captivity, and the importance of fungal load monitoring. Overall, the diversity of Aspergillus genus species causing infection in captive Australian birds was limited. Surveillance of wild birds may also be important for both human and animal health, as wild animal populations can be sentinels for infectious disease.22 Supplementary material Supplementary data are available at MMYCOL online. Acknowledgments The authors wish to thank veterinary pathologists Dr Lydia Tong, Dr Karrie Rose, and Dr Cheryl Sangster at Taronga Conservation Society, Mosman, New South Wales for performing histopathology, and veterinary clinicians Dr David McLelland and Dr Jenny McLelland at Adelaide Zoo, Adelaide, South Australia, and Dr Adrian Gallagher, Brisbane Bird Vet, Chermside, Queensland for providing samples. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Neumann. Aspergillosis in domesticated birds. J Comp Pathol . 2016; 155: 102– 104. CrossRef Search ADS PubMed  2. Beernaert LA, Pasmans F, Van Waeyenberghe L, Haesebrouck F, Martel A. Aspergillus infections in birds: a review. Avian Pathol . 2010; 39: 325– 331. Google Scholar CrossRef Search ADS PubMed  3. Dyce KM, Sack WO, Wensing CJG, eds. Textbook of Veterinary Anatomy , 3rd edn. Philadelphia: Saunders, 2002. 4. Negri CE, Gonçalves SS, Xafranski H et al.   Cryptic and rare Aspergillus species in Brazil: prevalence in clinical samples and in vitro susceptibility to triazoles. J Clin Microbiol . 2014; 52: 3633– 3640. Google Scholar CrossRef Search ADS PubMed  5. Balajee SA, Kano R, Baddley JW et al.   Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J Clin Microbiol . 2009; 47: 3138– 3141. Google Scholar CrossRef Search ADS PubMed  6. Alastruey-Izquierdo A, Mellado E, Peláez T et al.   Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study). Antimicrob Agents Chemother . 2013; 57: 3380– 3387. Google Scholar CrossRef Search ADS PubMed  7. Sabino R, Veríssimo C, Parada H et al.   Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources. Med Mycol . 2014; 52: 519– 529. Google Scholar CrossRef Search ADS PubMed  8. Talbot JJ, Johnson LR, Martin P et al.   What causes canine sino-nasal aspergillosis? A molecular approach to species identification. Vet J . 2014; 200: 17– 21. Google Scholar CrossRef Search ADS PubMed  9. Barrs VR, van Doorn TM, Houbraken J et al.   Aspergillus felis sp. nov., an emerging agent of invasive aspergillosis in humans, cats, and dogs. PLoS One . 2013; 8: e64871. Google Scholar CrossRef Search ADS PubMed  10. Talbot JJ, Houbraken J, Frisvad JC et al.   Discovery of Aspergillus frankstonensis sp. nov. during environmental sampling for animal and human fungal pathogens. PLoS One . 2017; 12: e0181660. Google Scholar CrossRef Search ADS PubMed  11. Samson RA, Houbraken J, Thrane U, Frisvad JC (eds). Food and Indoor Fungi . Utrecht: Centraalbureau voor Schimmelcultures, 2010. 12. White TJ, Bruns T, Lee SJWT. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols. A Guide to Methods and Applications . New York: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   13. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol . 1995; 61: 1323– 1330. Google Scholar PubMed  14. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids . 1999; 41: 95– 98. 15. Raja HA, Miller AN, Pearce CJ, Oberlies NH. Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod . 2017; 80: 756– 770. Google Scholar CrossRef Search ADS PubMed  16. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol . 2013; 30: 2725– 2729. Google Scholar CrossRef Search ADS PubMed  17. Pospísil K, Straka V, Otcenásek M et al.   Mycotic ulcerative aortitis after replacement of the aortic valve caused by the fungus Aspergillus restrictus. Vnitř Lék . 1984; 30: 292– 297. Google Scholar PubMed  18. Mencl K, Otčenàšek M, Špaček J, Řehulová E. Aspergillus restrictus and Candida parapsilosis - agents of endocarditis after heart valve replacements. Mykosen . 1985; 28: 127– 133. Google Scholar CrossRef Search ADS PubMed  19. Silvanose CD, Bailey TA, Di Somma A. Susceptibility of fungi isolated from the respiratory tract of falcons to amphotericin B, itraconazole and voriconazole. Vet Rec . 2006; 159: 282– 284. Google Scholar CrossRef Search ADS PubMed  20. Burco JD, Massey G, Byrne BA et al.   Monitoring of fungal loads in seabird rehabilitation centers with comparisons to natural seabird environments in Northern California. J Zoo Wildl Med . 2014; 45: 29– 40. Google Scholar CrossRef Search ADS PubMed  21. Burco JD, Etienne KA, Massey JG, Ziccardi MH, Balajee SA. Molecular sub-typing suggests that the environment of rehabilitation centers may be a potential source of Aspergillus fumigatus infecting rehabilitating seabirds. Med Mycol . 2012; 50: 91– 98. Google Scholar CrossRef Search ADS PubMed  22. Reif JS. Animal sentinels for environmental and public health. Public Health Rep . 2011; 126: 50– 57. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Identification of pathogenic Aspergillus isolates from captive birds in Australia

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Abstract

Abstract Aspergillosis is a major cause of severe respiratory disease in birds. The prevalence of cryptic section Fumigati and other non–Aspergillus fumigatus species as causative agents is unknown. Species identity was determined in 30 isolates from affected birds from zoos, pet birds and poultry by PCR of the ITS1-5.8S-ITS2 and partial β-tubulin genes. The most prevalent isolate was A. fumigatus sens. str. in 87% (26) cases. Other Aspergillus species were identified in 13% (4) cases, including A. restrictus (1), A. flavus sens. str. (2), and A. nidulans-clade (1). This is the first report of A. restrictus causing avian disease. avian aspergillosis, avian mycoses, Australia Introduction Avian aspergillosis, first reported in the 1800s,1 is a significant cause of mortality among captive birds worldwide. It most commonly causes disease within the respiratory tract, and can progress to disseminated invasive aspergillosis. Two forms of lesions have been described; a granulomatous deep nodular form affecting non-aerated parenchyma, and a pyogranulomatous nonencapsulated superficial diffuse form involving lungs and serosae.2 Captive birds are highly susceptible to aspergillosis due to environmental factors (increased burden of fungal spores), host immune factors (immunosuppression from disease, stress, or therapeutics), and unique respiratory anatomy.1,2 Anatomical differences to mammals that enable unique airflow in birds include nonexpansile and unlobed lungs with no pleural cavity, looped parabronchi with bi-directional air flow capillaries instead of terminal ending alveoli, and cranial and caudal air sacs that extend the bronchial system beyond the lung to pneumatize bones and enable fresh airflow during inspiration and expiration.3 Infection occurs when innate defenses cannot eliminate spores in parabronchial air capillaries, resulting in fungal plaques that can penetrate surrounding tissues.2 Despite a range of antifungal therapeutic treatment options, infections are often fatal.2 The clinical importance of fungal molecular identification techniques has been demonstrated for human and nonavian animal species with aspergillosis, with cryptic section Fumigati species increasingly identified, prompting epidemiological studies.4–9 We previously identified isolates causing respiratory aspergillosis in cats and dogs and found that disease in dogs, like humans is predominantly caused by A. fumigatus sens. str.,8 while in cats, infections with cryptic species in section Fumigati including A. felis, A. udagawae, and A. thermomutatus are most prevalent.9 We performed environmental sampling for Aspergillus section Fumigati in homes of cats previously diagnosed with aspergillosis and in a recreational nature reserve and found that cryptic species reported to cause disease in humans or animals were common, comprising 25% of isolates overall.10 Aspergillus fumigatus sens. str. has been most commonly associated with avian infections based on phenotypic identification techniques, with A. flavus, A. niger, A. glaucus, and A. nidulans also reported.2 Thus, cryptic species may have been overlooked as a cause of avian aspergillosis. This study aimed to definitively identify fungal pathogens causing avian aspergillosis and to determine the prevalence of cryptic A. section Fumigati species. A secondary aim was to determine the form of aspergillosis present. Methods Australian avian veterinarians were contacted to recruit isolates from clinical cases of avian aspergillosis between 2013 and 2017. Aspergillus isolates cultured from lesions in captive birds consistent with aspergillosis, including fungal plaques or granulomas observed within the respiratory tract or other parenchyma during diagnostic investigation or at post mortem were included for study. Morphological identification of Aspergillus species was performed by microbiologists, based on consistent macroscopic and microscopic features including identification of smooth or rough walled conidia stemming from radiate or columnar conidial heads with globose or subclavate vesicles, stipe and a foot cell.11 Thirty isolates were received from Australian native and exotic avian species (Table 1), including 26 from zoos (New South Wales [n = 23, Taronga Conservation Society approved opportunistic sampling request R14B178], South Australia [n = 3]), one from private practice (Queensland) and three from commercial poultry producers (NSW). Histopathology post-mortem reports were available for review in 20 zoological cases. Table 1. Fungal molecular identification (internal transcribed spacer (ITS), partial beta-tubulin [BenA] gene), avian species and origin details for fungal isolates used in this study (n = 30), collected from 2013 to 2017. Molecular identification is based on homology percentage matches of ≥99% (ITS) and ≥99.5% (BenA) using NCBI-BLAST tool. Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  e, exotic; n, native. View Large Table 1. Fungal molecular identification (internal transcribed spacer (ITS), partial beta-tubulin [BenA] gene), avian species and origin details for fungal isolates used in this study (n = 30), collected from 2013 to 2017. Molecular identification is based on homology percentage matches of ≥99% (ITS) and ≥99.5% (BenA) using NCBI-BLAST tool. Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  Fungal  Genbank accession  Avian species  Organ    species  number (ITS; BenA)  (native/exotic)  isolated from  Location  A. fumigatus sens.st.  MF540280; MF540250  Pacific Black Duck (n)  Lung  Zoo, NSW    MF540281; MF540251  Domestic Turkey (e)  Ocular  Zoo, NSW    MF540282; MF540252  Diamond Firetail Finch (n)  Lung  Zoo, NSW    MF540303; MF540273  Red-browed finch (n)  Liver  Zoo, NSW    MF540284; MF540254  Crimson Finch (n)  Lung  Zoo, NSW    MF540283; MF540253  Metallic Starling (n)  Lung  Zoo, NSW    MF540287; MF540257  Metallic Starling (n)  Liver  Zoo, NSW    MF540294; MF540264  Metallic Starling (n)  Lung  Zoo, NSW    MF540285; MF540255  Olive-backed Oriole (n)  Air sac  Zoo, NSW    MF540288; MF540258  Red Jungle Fowl (e)  Lung  Zoo, NSW    MF540296; MF540266  Red Jungle Fowl (e)  Abdominal mass  Zoo, NSW    MF540289; MF540259  Little Penguin (n)  Lung  Zoo, NSW    MF540292; MF540262  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540293; MF540263  Rufous Owl (n)  Ocular  Zoo, NSW    MF540295; MF540265  Regent Honeyeater (n)  Lung  Zoo, NSW    MF540297; MF540267  Scaly-breasted Lorikeet (n)  Pericardium  Zoo, NSW    MF540298; MF540268  Hardhead Duck (n)  Air sac  Zoo, NSW    MF540299; MF540269  Blue Fronted Amazon Parrot (e)  Lung  Veterinarian, QLD    MF540300; MF540270  Crimson Rosella (n)  Lung  Zoo, NSW    MF540301; MF540271  Plains Wanderer (n)  Lung  Zoo, NSW    MF540307; MF540277  Plains Wanderer (n)  Lung  Zoo, NSW    MF540309; MF540279  Plains Wanderer (n)  Air sac  Zoo, NSW    MF540305; MF540275  Swift Parrot (n)  Kidney  Zoo, SA    MF540306; MF540276  Sacred Ibis (n)  Lung  Zoo, NSW    MF540308; MF540278  Mandarin Duck (e)  Air sac  Zoo, SA    MF540302; MF540272  Black-faced Cockatoo (n)  Air sac  Zoo, NSW  A. restrictus  MF540286; MF540256  Java Finch (e)  Lung  Zoo, NSW  A. flavus sens. str.  MF540290; MF540260  Domestic Chicken (e)  Lung  Poultry producer, NSW    MF540291; MF540261  Domestic Chicken (e)  Lung  Poultry producer, NSW  A. nidulans-clade  MF540304; MF540274  Palm Cockatoo (n)  Tracheal mass  Zoo, SA  e, exotic; n, native. View Large All isolates were subcultured onto malt extract agar and grown in the dark at 25°C for up to 7 days until sporulation occurred. DNA was extracted using the MoBio DNA isolation kit (QIAGEN MO BIO, Carlsbad, USA). Conventional polymerase chain reaction (PCR) was performed targeting the rDNA gene cluster, including the ITS1, 5.8S gene, and ITS2 regions using primers ITS1 and ITS4.12 Amplification of the partial β-tubulin (BenA) gene was performed using primers bt2a and bt2b.13 PCR product was sent to an external laboratory for purification and Sanger sequencing (Macrogen, Seoul, South Korea). Generated sequences were edited using BioEdit version 7.2.5.14 Species identification was determined using NCBI-BLAST tool and sequence homology of ≥99% (ITS) and ≥99.5% (BenA) with type strains sequences.15 If BenA homology was <99.5%, a separate alignment and phylogenetic analysis was performed in Mega 6.0616 with sequences of all section type-strains to confirm the closest type-strain match using the maximum likelihood method based on the Tamura-Nei model with gamma distribution and 1000 bootstrap replicates. Sequences were deposited in GenBank (MF540250-MF540309). Results Twenty-six of 30 (87%) isolates were identified as A. fumigatus sens. str., and the remaining four (13%) were other Aspergillus species, including A. restrictus (n = 1), A. flavus sens. str. (n = 2), and A. nidulans-clade (n = 1) (Table 1). The latter isolate had 99.1% BenA homology with A. nidulans sens. str. Phylogenetic analysis with section Nidulantes type-strains placed this isolate in the A. nidulans-clade, where it was most closely related to A. pachychristatus and A. latilabiatus. Affected avian species were 70% native and 30% exotic to Australia; passerines were most commonly affected (30%). Five A. fumigatus sens. str. isolates were from IUCN listed critically endangered native species (http://www.iucnredlist.org/search) including Plains Wanderer (Pedionomus torquatus) (3), Regent Honeyeater (Anthochaera Phrygia) (1), and Swift Parrot (Lathamus discolor) (1). Histopathology findings are summarized in supplementary Table 1. Seventeen of 20 cases had invasive aspergillosis, which was disseminated in 12 and focal in five (respiratory). There were three cases of noninvasive localized infection, involving conjunctiva, air sacs, and a coelomic cavity mass. All six respiratory cases (5 invasive, 1 noninvasive) involved the lower respiratory tract; localized to the air sacs (1), the lungs (4), or both air sacs and lungs (1). In four cases, a primary nonfungal disease process was identified including mycobacteriosis, egg yolk peritonitis, metastatic pancreatic adenocarcinoma and chronic liver disease. Discussion Aspergillus fumigatus sens. str. was the most common isolate amongst our study population (87%), similar to prevalence studies in humans, dogs, and the environment.4–8 While other Aspergillus species were identified in 13% of cases, no cryptic section Fumigati species were identified. This is in contrast to prevalence studies in humans (0.75–3.7% of cases)4–6 and reports on cats, in which invasive fungal rhinosinusitis is the most common form and is caused by cryptic species in section Fumigati.9 Although our sample size was small, we found a similar prevalence for A. flavus sens. str. (6%) and A. nidulans-clade (3%) as is reported in humans (A. flavus 8–13%; A. nidulans 0.5–2.5%).4–6 Our study is the first to our knowledge to report aspergillosis in birds due to A. restrictus. This case was in a Java Finch (Lonchura oryzivora) with chronic liver disease and disseminated invasive aspergillosis affecting air sacs, lung, and gizzard. Aspergillus restrictus has also been isolated from humans with disseminated infection following a heart valve replacement, although only phenotypic identification was performed.17,18 Our findings support an opportunistic pathogen role for this fungal species. This study demonstrates the value of molecular characterization of avian fungal isolates. Aspergillus species other than A. fumigatus sens. str. should also be considered as potential agents of this disease and may have important implications for patient management as seen in humans and other animals. Different Aspergillus species isolated from birds have been shown to have different susceptibilities to commonly used antifungal therapies, including itraconazole and amphotericin B.19 This information could prove invaluable in management of a disease that is already challenged by lack of knowledge in avian pharmacokinetics and late-stage clinical presentation.2 We investigated avian aspergillosis amongst captive birds including several critically endangered native Australian species. The pathogenesis of avian disease has been associated with an increased environmental load of filamentous fungi in captive/rehabilitation settings (5.7% of fungal isolates grown) compared to natural avian habitats (0.03%),20 with an A. fumigatus average air count nine times greater than natural habitats reported from rehabilitation centres.21 Furthermore, genotyping of clinical and environmental isolates has demonstrated that the rehabilitation setting is a source of clinical disease in captive birds, likely due to this increased fungal burden and the presence of existing disease and/or stress.21 This highlights the importance of environmental control strategies to reduce the risk of aspergillosis in captivity, and the importance of fungal load monitoring. Overall, the diversity of Aspergillus genus species causing infection in captive Australian birds was limited. Surveillance of wild birds may also be important for both human and animal health, as wild animal populations can be sentinels for infectious disease.22 Supplementary material Supplementary data are available at MMYCOL online. Acknowledgments The authors wish to thank veterinary pathologists Dr Lydia Tong, Dr Karrie Rose, and Dr Cheryl Sangster at Taronga Conservation Society, Mosman, New South Wales for performing histopathology, and veterinary clinicians Dr David McLelland and Dr Jenny McLelland at Adelaide Zoo, Adelaide, South Australia, and Dr Adrian Gallagher, Brisbane Bird Vet, Chermside, Queensland for providing samples. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Neumann. Aspergillosis in domesticated birds. J Comp Pathol . 2016; 155: 102– 104. CrossRef Search ADS PubMed  2. Beernaert LA, Pasmans F, Van Waeyenberghe L, Haesebrouck F, Martel A. Aspergillus infections in birds: a review. Avian Pathol . 2010; 39: 325– 331. Google Scholar CrossRef Search ADS PubMed  3. Dyce KM, Sack WO, Wensing CJG, eds. Textbook of Veterinary Anatomy , 3rd edn. Philadelphia: Saunders, 2002. 4. Negri CE, Gonçalves SS, Xafranski H et al.   Cryptic and rare Aspergillus species in Brazil: prevalence in clinical samples and in vitro susceptibility to triazoles. J Clin Microbiol . 2014; 52: 3633– 3640. Google Scholar CrossRef Search ADS PubMed  5. Balajee SA, Kano R, Baddley JW et al.   Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J Clin Microbiol . 2009; 47: 3138– 3141. Google Scholar CrossRef Search ADS PubMed  6. Alastruey-Izquierdo A, Mellado E, Peláez T et al.   Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study). Antimicrob Agents Chemother . 2013; 57: 3380– 3387. Google Scholar CrossRef Search ADS PubMed  7. Sabino R, Veríssimo C, Parada H et al.   Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources. Med Mycol . 2014; 52: 519– 529. Google Scholar CrossRef Search ADS PubMed  8. Talbot JJ, Johnson LR, Martin P et al.   What causes canine sino-nasal aspergillosis? A molecular approach to species identification. Vet J . 2014; 200: 17– 21. Google Scholar CrossRef Search ADS PubMed  9. Barrs VR, van Doorn TM, Houbraken J et al.   Aspergillus felis sp. nov., an emerging agent of invasive aspergillosis in humans, cats, and dogs. PLoS One . 2013; 8: e64871. Google Scholar CrossRef Search ADS PubMed  10. Talbot JJ, Houbraken J, Frisvad JC et al.   Discovery of Aspergillus frankstonensis sp. nov. during environmental sampling for animal and human fungal pathogens. PLoS One . 2017; 12: e0181660. Google Scholar CrossRef Search ADS PubMed  11. Samson RA, Houbraken J, Thrane U, Frisvad JC (eds). Food and Indoor Fungi . Utrecht: Centraalbureau voor Schimmelcultures, 2010. 12. White TJ, Bruns T, Lee SJWT. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols. A Guide to Methods and Applications . New York: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   13. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol . 1995; 61: 1323– 1330. Google Scholar PubMed  14. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids . 1999; 41: 95– 98. 15. Raja HA, Miller AN, Pearce CJ, Oberlies NH. Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod . 2017; 80: 756– 770. Google Scholar CrossRef Search ADS PubMed  16. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol . 2013; 30: 2725– 2729. Google Scholar CrossRef Search ADS PubMed  17. Pospísil K, Straka V, Otcenásek M et al.   Mycotic ulcerative aortitis after replacement of the aortic valve caused by the fungus Aspergillus restrictus. Vnitř Lék . 1984; 30: 292– 297. Google Scholar PubMed  18. Mencl K, Otčenàšek M, Špaček J, Řehulová E. Aspergillus restrictus and Candida parapsilosis - agents of endocarditis after heart valve replacements. Mykosen . 1985; 28: 127– 133. Google Scholar CrossRef Search ADS PubMed  19. Silvanose CD, Bailey TA, Di Somma A. Susceptibility of fungi isolated from the respiratory tract of falcons to amphotericin B, itraconazole and voriconazole. Vet Rec . 2006; 159: 282– 284. Google Scholar CrossRef Search ADS PubMed  20. Burco JD, Massey G, Byrne BA et al.   Monitoring of fungal loads in seabird rehabilitation centers with comparisons to natural seabird environments in Northern California. J Zoo Wildl Med . 2014; 45: 29– 40. Google Scholar CrossRef Search ADS PubMed  21. Burco JD, Etienne KA, Massey JG, Ziccardi MH, Balajee SA. Molecular sub-typing suggests that the environment of rehabilitation centers may be a potential source of Aspergillus fumigatus infecting rehabilitating seabirds. Med Mycol . 2012; 50: 91– 98. Google Scholar CrossRef Search ADS PubMed  22. Reif JS. Animal sentinels for environmental and public health. Public Health Rep . 2011; 126: 50– 57. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Dec 8, 2017

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