Toxigenicity and phylogeny of Aspergillus section Flavi in poultry feed in Iran

Document Type : Original Articles


1 Feed Hygienist, Institute of Agricultural Education and Extension, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

2 Department of Mycology, Pasteur Institute of Iran, Tehran, Iran


Background and Purpose: This study was conducted to evaluate the presence of aflatoxigenic strains and level of aflatoxin in poultry feed. Aflatoxigenic strains were investigated in corn and soybean meal as the ingredients of poultry feed, as well as in two types of commercial feed, namely pellet and mash. The gene sequencing was performed to identify the species of Aspergillus section Flavi.
Materials and Methods: All samples were randomly collected from feed storage silos located in Iran in 2018. The samples were cultured on specialized media for 2 weeks at 28ºC. Identification of Aspergillus section Flavi isolates was based on macro- and microscopic morphological criteria and molecular analysis. The thin-layer chromatography (TLC) was applied to confirm the aflatoxigenic isolates. In addition, the level of aflatoxin B1 (AFB1) produced by these isolates was determined by high-performance liquid chromatography. The strains were subjected to sequence analysis, and Bt2 PCR products were purified by the QIAquick PCR purification kit. At the final stage, the phylogenetic tree was built.
Results: Among 54 isolates identified as Aspergillus section Flavi, 20 (37%) isolates were found to produce aflatoxin at a range of 11.28±1.18 to 2239.92±92.26 μg/g fungal dry weight. The aflatoxigenic isolates had the frequencies of 45%, 40%, 10%, and 5% in the corn, pellet, soybean meal, and mash samples, respectively. Furthermore, the mean concentrations of AFB1 were significantly higher in the corn samples (707.04±39.05) than that of other poultry feed samples (p <0.05). A total of 34 (63%) isolates were detected as non-aflatoxigenic on the yeast extract-sucrose broth in TLC analysis. The toxigenic isolates produced the highest (2232.62±55.49) and lowest (11.28±1.18) levels of AFB1 in the corn samples, compared to other feedstuffs. Furthermore, the mean level of AFB1 in mash product was 554.09±10.36 μg/g, compared to a mean level of 229.22±11.09 μg/g in pellets. The isolates were randomly selected, sequenced, and then analyzed. Subsequently, the phylogenetic tree of Aspergillus section Flavi was plotted.
Conclusion: The process of converting raw ingredients to compound poultry feed is more hazardous when there is not enough time and temperature provided to eliminate aflatoxigenic isolates. Therefore, Aspergillus section Flavi in poultry feed can pose a threat to the poultry industry and poultry products, thereby affecting the health status of humans. Unprocessed/processed materials, such as corns and pelleted feed, need further monitoring, especially when conditions are not optimal for destroying the fungus.


1. Alam MS, Islam MR, Banu MS. Aboundance of fungal flora in relation to moisture content and storage period in different types of poultry feed ingredient. Pak J Biol Sci. 2001; 41:194-8.
2. Khosravi AR, Shokri H, Zaboli F. Grain-borne mycoflora and fumonisin B1 from fresh-harvested and stored rice in Northern Iran. Jundishapur J Microbiol. 2013; 6(5):e6414.
3. Kpodo K, Thrane U, Hald B. Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins. Int J Food Microbiol. 2000;61(2-3):147-57.
4. Nowrozi H, Farhadi A, Kachuei R, Ghaemmaghami SS. Two important fusarium in esophageal cancer, fusarium verticillioides and fusarium proliferatum: mycoflora isolation and molecular characterization in Iranian feed and corn. J Res Med Dent Sci. 2018; 6(6):239.
5. Pinotti L, Ottoboni M, Giromini C, Dell’Orto V, Cheli F. Mycotoxin contamination in the EU feed supply chain: A focus on cereal byproducts. Toxins. 2016; 8(2):45.
6. Magnoli C, Hallak C, Astoreca A, Ponsone L, Chiacchiera S, Palacio G, et al. Surveillance of toxigenic fungi and ochratoxin A in feedstuffs from Córdoba province, Argentina. Vet Res Commun. 2005; 29(5):431-45.
7. Azarakhsh Y, Sabokbar A, Bayat M. Incidence of the most common toxigenic Aspergillus species in broiler feeds in Kermanshah province, West of Iran. Global Vet. 2011; 6(1):73-7.
8. Khanafari A, Soudi H, Miraboulfathi M. Biocontrol of Aspergillus flavus and aflatoxin B1 production in corn. J Environ Health Sci Eng. 2007; 4(3):163-8.
9. Ghaemmaghami SS, Nowroozi H. Toxigenic fungal contamination for assessment of poultry feeds: Mashed vs. Pellet. Iran J Toxicol. 2018; 12(5):5-10.
10. Coulombe Jr RA. Biological action of mycotoxins. J Dairy Sci. 1993; 76(3):880-91.
11. Jalili M. A review on aflatoxins reduction in food. Iran J Health Saf Environ. 2016; 3(1):445-59.
12. Bryden WL. Food and feed, mycotoxins and the perpetual pentagram in a changing animal production environment. Anim Product Sci. 2012; 52(7):383-97.
13. Medina A, Rodriguez A, Magan N. Effect of climate change on Aspergillus flavus and aflatoxin B1 production. Front Microbiol. 2014; 5:348.
14. Labuda R, Tancinova D. Fungi recovered from Slovakian poultry feed mixtures and their toxinogenity. Ann Agric Environ Med. 2006; 13(2):193-200.
15. Hong SB, Go SJ, Shin HD, Frisvad JC, Samson RA. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia. 2005; 97(6):1316-29.
16. Gibbons JG, Salichos L, Slot JC, Rinker DC, McGary KL, King JG, et al. The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Curr Biol. 2012; 22(15):1403-9.
17. Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CH, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014; 78:141-73.
18. Greco MV, Franchi ML, Rico Golba SL, Pardo AG, Pose GN. Mycotoxins and mycotoxigenic fungi in poultry feed for food-producing animals. Sci World J. 2014; 2014:968215.
19. Oliveira GR, Ribeiro JM, Fraga ME, Cavaglieri LR, Direito GM, Keller KM, et al. Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil. Mycopathologia. 2006; 162(5):355-62.
20. Samson RA, Hoekstra ES, Frisvad JC. Introduction to foodand airborne fungi. Centraalbureau voor Schimmelcultures, Utrecht. Fungi Mycot Fruit Cereals. 2004; 151:1-389.
21. Atehnkeng J, Ojiambo PS, Donner M, Ikotun T, Sikora RA, Cotty PJ, et al. Distribution and toxigenicity of Aspergillus species isolated from maize kernels from three agro-ecological zones in Nigeria. Int J Food Microbiol. 2008; 122(1-2):74-84.
22. Razzaghi-Abyaneh M, Shams-Ghahfarokhi M, Allameh A, Kazeroon-Shiri A, Ranjbar-Bahadori S, Mirzahoseini H, et al. A survey on distribution of Aspergillus section Flavi in corn field soils in Iran: population patterns based on aflatoxins, cyclopiazonic acid and sclerotia production. Mycopathologia. 2006; 161(3):183-92.
23. Razzaghi-Abyaneh M, Shams-Ghahfarokhi M, Rezaee MB, Jaimand K, Alinezhad S, Saberi R, et al. Chemical composition and antiaflatoxigenic activity of Carum carvi L., Thymus vulgaris and Citrus aurantifolia essential oils. Food Control. 2009; 20(11):1018-24.
24. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7):1870-4.
25. Keller KM, Queiroz BD, Keller LA, Ribeiro JM, Cavaglieri LR,
Pereyra ML, et al. The mycobiota and toxicity of equine feeds. Vet Res Commun. 2007; 31(8):1037-45.
26. Kheirkhah M, Chadeganipour M, Dehghan P, Mohammadi R. Identification of morphological and molecular Aspergillus species isolated from patients based on beta-tubulin gene sequencing. Yafteh. 2017; 19(1):87-97.
27. Klich MA. Aspergillus flavus: the major producer of aflatoxin. Mol Plant Pathol. 2007; 8(6):713-22.
28. Accensi F, Abarca M, Cabanes F. Occurrence of Aspergillus species in mixed feeds and component raw materials and their ability to produce ochratoxin A. Food Microbiol. 2004; 21(5):623-7.
29. Probst C, Bandyopadhyay R, Price LE, Cotty PJ. Identification of atoxigenic Aspergillus flavus isolates to reduce aflatoxin contamination of maize in Kenya. Plant Dis. 2011; 95(2):212-8.
30. Ezekiel C, Atehnkeng J, Odebode A, Bandyopadhyay R. Distribution of aflatoxigenic Aspergillus section Flavi in commercial poultry feed in Nigeria. Int J Food Microbiol. 2014; 189:18-25.
31. Monge MdP, Dalcero AM, Magnoli CE, Chiacchiera SM. Natural co-occurrence of fungi and mycotoxins in poultry feeds from Entre Ríos, Argentina. Food Addit Contam Part B Surveill. 2013; 6(3):168-74.
32. Yiannikouris A, Jouany JP. Mycotoxins in feeds and their fate in animals: a review. Anim Res. 2002; 51(2):81-99.
33. Lee KR, Yang SM, Cho SM, Kim M, Hong SY, Chung SH. Aflatoxin B1 detoxification by Aspergillus oryzae from meju, a traditional Korean fermented soybean starter. J Microbiol Biotechnol. 2017; 27(1):57-66.
34. Prencipe S, Siciliano I, Garibaldi A, Gullino ML, Spadaro DC. Aspergillus section Flavi from chestnuts: biological, molecular and chemical characterization. Piacenza: National meeting italian society for plant pathology; 2017.
35. Ariyo AL, Anthony MH, Lami MH. Survey of mycotoxigenic fungi in concentrated poultry feed in Niger State, Nigeria. J Food Res. 2013; 2(2):128-35.
36. Fraga ME, Curvello F, Gatti MJ, Cavaglieri LR, Dalcero AM, da Rocha Rosa CA. Potential aflatoxin and ochratoxin A production by Aspergillus species in poultry feed processing. Vet Res Commun. 2007; 31(3):343-53.
37. Ghaemmaghami SS, Modirsaneii M, Khosravi AR, Razzaghi-Abyaneh M. Study on mycoflora of poultry feed ingredients and finished feed in Iran. Iran J Microbiol. 2016; 8(1):47-54.
38. Rodrigues I, Naehrer K. A three-year survey on the worldwide occurrence of mycotoxins in feedstuffs and feed. Toxins. 2012; 4(9):663-75.