Genotypic Characterization of Fungal Species Isolated From Broiler Breeder Chickens, Dead-In-Shell and Hatched Chicks

Document Type: Original Paper


1 Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt

2 Animal Health Research Institute, Beni-Suef Branch, Beni-Suef 62511, Egypt

3 Mycology Research Unit, Animal Health Research Institute, P.O. Box, 264, Dokki, Giza 12618, Egypt

4 Poultry Diseases Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 65211, Egypt


To investigate the prevalence of fungal agents in local Egyptian broiler breeder chicken’s premises, tracheal and cloacal swabs from chickens, feed, and water samples were collected. The targeted breed's dead-in-shell eggs and newly hatched chick’s samples were also tested. All fungal isolates were morphologically typed and the predominant fungal species were further subjected to molecular typing using PCR-RFLP and gene sequence analysis of the β-tubulin gene. Results revealed a high prevalence of fungal isolates in tracheal and cloacal swabs (39.3 - 48.1%) and feed and water samples (37.5% and 28.6%, respectively). Fungal isolation rates in dead-in-shell eggs of all breeds were high except in Dahaby breed. Aspergillus species including A. niger, A. flavus, and A. terreus were the predominantly isolated fungi from all collected samples. The ß-Tubulin genes PCR-RFLP of selected Aspergillus isolates showed a characteristic restriction pattern for each species; however, the method was unable to distinguish between strains. The β-tubulin gene phylogenetic and sequence analysis of selected A. flavus, and A. terreus from breeder chickens and their hatching chicks indicated their relatedness to isolates from bronchopulmonary Aspergillosis in humans in the Middle East. In conclusion, the Aspergillus species remains the most prevalent fungi in breeder chickens, their incubated eggs and hatched chicks indicating their widespread in hatcheries. The PCR-RFLP is an easy tool to discriminate between Aspergilli species, however, the β-tubulin sequence analysis more descriptive of potential sources of fungal contamination. Further epidemiological studies are needed to monitor avian and human Aspergilli in poultry houses with a special focus on antifungal drug-resistant strains.


Abd El-Galil Y, El-Kenawy AI, El-Gmiey SR&Abd El-Latif MM. 1995. Bacterial causes of lowering hatchability and early embryonic deaths in balady hatcheries in Dakahlia Governorate. Assiut Veterinary Medical Journal, 33:199-206.

Ahmed RSA. 2015. Characterization and differentiation of fungal and bacterial isolates associated with dead-in-shell embryos in breeder layers. Master thesis.Bacteriology, Mycology and Immunology Department, Beni-Suef University.

Akan M, Haziroğlu R, Ilhan Z, Sareyyüpoğlu B&Tunca R. 2002. A case of aspergillosis in a broiler breeder flock. Avian Disease, 46:497-501. doi: 10.1637/0005-2086(2002)046[0497:ACOAIA] 2.0.CO; 2

Altschul SF, Gertz EM, Agarwala R, Schaffer AA&Yu YK. 2009. PSI-BLAST pseudocounts and the minimum description length principle. Nucleic Acids Research, 37:815-824. doi: 10.1093/nar/gkn981

Arné P, Thierry S, Wang D, Deville M, Le Loc'h G, Desoutter A, Féménia F, Nieguitsila A, Huang W, Chermette R&Guillot J. 2011. Aspergillusfumigatus in Poultry.International Journal of Microbiology, 2011.doi: 10.1155/2011/746356

Balajee SA, Nickle D, Varga J&Marr KA. 2006. Molecular studies reveal frequent misidentification of Aspergillusfumigatus by morphotyping. Eukaryot Cell 5:1705-1712. doi: 10.1128%2FEC.00162-06

Beernaert LA, Pasmans F, Van Waeyenberghe L, Haesebrouck F&Martel A. 2010. Aspergillus infections in birds: a review. Avian Pathology, 39:325-331. doi:10.1080/03079457.2010.506210.

Brookman JL&Denning DW. 2000. Molecular genetics in Aspergillusfumigatus. CurrOpinMicrobiol 3:468-474. DOI:10.1016/S1369-5274(00)00124-7.

Cruickshank R, Duguid JP, Marmion BP&Swain RHA. 1975. Medical Microbiology, 12thEdition ed. London: Churchill. Livingstone.

Geiser DM, Dorner JW, Horn BW&Taylor JW. 2000. The phylogenetics of mycotoxin and sclerotium production in Aspergillusflavus and Aspergillusoryzae. Fungal Genetics and Biology, 31:169-179. doi: 10.1006/fgbi.2000.1215.

Geiser DM, Frisvad JC&Taylor JW. 1998. Evolutionary relationships in Aspergillus section Fumigati inferred from partial β-tubulin and hydrophobin DNA sequences. Mycologia, 90:831-845. doi: 10.2307/3761325.

Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P&Denning DW. 2007. Aspergillusflavus: human pathogen, allergen and mycotoxin producer. Microbiology, 153:1677-1692. doi: 10.1099/mic.0.2007/007641-0.

Imbert S, Normand AC, Ranque S, Costa JM, Guitard J, Accoceberry I, Bonnal C, Fekkar A, Bourgeois N, Houze S, Hennequin C, Piarroux R, Dannaoui E&Botterel F. 2018. Species identification and in vitro-antifungal susceptibility of Aspergillusterreus species complex clinical isolates: a French multicentre study. Antimicrobial agents and chemotherapy.doi: 10.1128/AAC.02315-17.

Jacobsen ID, Grosse K&Hube B. 2012.Embryonated chicken eggs as alternative infection model for pathogenic fungi. Methods in Molecular Biology, 845:487-496. doi: 10.1007/978-1-61779-539-8_34.

Jacobsen ID, Grosse K, Slesiona S, Hube B, Berndt A&Brock M. 2010. Embryonated eggs as an alternative infection model to investigate Aspergillusfumigatus virulence. Infection and Immunity, 78:2995-3006. doi: 10.1128/IAI.00268-10. 

Khairallah SH, Byrne KA&Tabbara KF. 1992. Fungal keratitis in Saudi Arabia. Documentaophthalmologica Advances in ophthalmology, 79:269-276. doi: 10.1007/BF00158257.

Krishnan S, Manavathu EK&Chandrasekar PH. 2005.A comparative study of fungicidal activities of voriconazole and amphotericin B against hyphae of Aspergillusfumigatus.Journal of Antimicrobial Chemotherapy, 55:914-920. doi: 10.1093/jac/dki100.

Kunkle RA. 2003.Aspergillosis. In: Saif YM, Editor. Diseases of Poultry, 11th Ed. Ames, Iowa, USA: Iowa State University press. Pages, 883-895.

Martin MP, Bouck KP, Helm J, Dykstra MJ, Wages DP&Barnes HJ. 2007. Disseminated Aspergillusflavus infection in broiler breeder pullets. Avian Disease, 51:626-631. doi: 10.1637/0005-2086(2007)51[626:DAFIIB]2.0.CO;2

May GS, Tsang ML, Smith H, Fidel S&Morris NR. 1987. Aspergillusnidulans beta-tubulin genes are unusually divergent. Gene, 55:231-243. doi:10.1016/0378-1119(87)90283-6.

Moustafa GZ. 1995. Effect of hatchery sanitation on the hatchability of breeder eggs. Master thesis.Veterinary Hygiene Department, Faculty of VeterinaryMedicine, Cairo University Egypt.

Neamatallah A, El-Leboudy A, Amer A&El-Shenawy N. 2009. Biosafety Against Fungal Contamination of Hen's Eggs and Mycotoxins Producing Species. Meteorology, Environment and Arid Land Agriculture Sciences, 20:63-73.

Pasqualotto AC. 2009. Differences in pathogenicity and clinical syndromes due to Aspergillusfumigatus and Aspergillusflavus.Medical mycology, 47:S261-270. doi: 10.1080/13693780802247702.

Peterson SW. 2008. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia, 100:205-226. doi: 10.1080/15572536.2008.11832477.

Reese S, Dalamani G&Kaspers B. 2006. The avian lung-associated immune system: a review. Veterinary research, 37: 311-324. doi: 10.1051/vetres:2006003.

Rippon JW. 1988. Medical Mycology-The Pathogenic Fungi and the Pathogenic Actinomycetes 3rdEdition. W.B. Saunders Co., Philadelphia, USA.

Smith AH&Rehberger TG. 2018. Bacteria and fungi in day-old turkeys vary among companies, collection periods, and breeder flocks. Poultry Science, 97:1400-1411. doi: 10.3382/ps/pex429.

Staab JF, Balajee SA&Marr KA. 2009. Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism. Journal of clinical microbiology, 47:2079-2083. doi: 10.1128/jcm.00551-09.

Stoute ST, Bickford AA, Walker RL&Charlton BR. 2009. Mycoticpododermatitis and mycotic pneumonia in commercial turkey poults in northern California.Journal of veterinary diagnostic investigation, 21:554-557. doi: 10.1177/104063870902100424.

Taj-Aldeen SJ, Hilal AA&Chong-Lopez A. 2003. Allergic Aspergillusflavusrhinosinusitis: a case report from Qatar. European archives of oto-rhino-laryngology, 260:331-335. doi: 10.1007/s00405-002-0547-x.

Tamura K, Stecher G, Peterson D, Filipski A&Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution, 30:2725-2729. doi: 10.1093/molbev/mst197.

Vaezi A, Fakhim H, Arastehfar A, Shokohi T, Hedayati MT, Khodavaisy S, Rezaei-Matehkolaei A, Badiee P, Hagen F, Lass-Florl C, Dannaoui E, Meis JF&Badali H. 2018. In vitro antifungal activity of amphotericin B and 11 comparators against Aspergillusterreus species complex. Mycoses, 61:134-142. doi: 10.1111/myc.12716.

Wang DY. 2012. Genetic diversity and antifungal susceptibility of Aspergillus spp. isolates from avian farms in Guangxi, China. Ph.D. thesis.Agricultural sciences, AgroParisTech, The National veterinary school of Alfort. Paris, France.

Zanganeh E, Zarrinfar H, Rezaeetalab F, Fata A, Tohidi M, Najafzadeh MJ, Alizadeh M&Seyedmousavi S. 2018. Predominance of non-fumigatusAspergillus species among patients suspected to pulmonary aspergillosis in a tropical and subtropical region of the Middle East. Microbial pathogenesis, 116:296-300. doi: 10.1016/j.micpath.2018.01.047