2. Prof.Dr. Mohammed H.Khudor Prof.Dr.Basil A.Abbas
Occurrence of fungi in poultry feed with cultural and molecular detection of their aflatoxigenic activity
M.Sc. Student
Raed Najeeb Kadhim
Supervisors
Prof.Dr. Mohammed H.Khudor Prof.Dr.Basil A.Abbas

3. Mycotoxins
Low-molecular-weight natural products produced as secondary metabolites by fungi.
Lack of visible appearance of fungus does not negate presence of mycotoxins. Toxins can remain in the organism after fungus has been removed.
Mycotoxins greatly resist decomposition and even temperature treatments, such as cooking and freezing.
Resistant to breakdown in an animal’s digestive system.

4. Some important toxigenic fungi
Aspergillus
Toxigenic fungi
Penicillium
Fusarium
Alternaria

5. Aspergillus Found in soil, plant debris, and indoor air environment
Aspergillus flavus is The most important species which cause aspergillosis in animals as well as in man and in birds.
Aspergillus flavus causes mycotic abortion in cattle and sheep.Ingestion of high amounts of aflatoxin may induce lethal effects, also cause sinusitis, cerebral aspergillosis, meningitis, pulmonary aspergillosis, cutaneous aspergillosis and hepatosplenic aspergillosis.
Produces many toxins as aflatoxins (B1, B2, ,G1,G2, M1,  M2) Gliotoxin, Sterigmatocystin,  and Methoxy Sterigmatocystin.

6. Some important mycotoxins
Today mycotoxins are known
Common mycotoxins
Ochratoxin
Fumonisin
Aflatoxin
Deoxynivalenol
Zearalenone

7. Produced by Aspergillus. flavus, A.parasiticus and A. oryzae.
Aflatoxins
Produced by Aspergillus. flavus, A.parasiticus and A. oryzae.
There are types: aflatoxins B1 (AFB1) and B2(AFB2),G1(AFG1),G2(AFG2),M1(AFM1) andM2(AFM2).
Aflatoxin B1 occurs most frequently and is most toxic and carcinogenic.

8. Method

9. Collection of samples of poultry feed
Culturing on PDA,MEA& SDA
Isolation
Identification
A. flavus
Morphological by culture
Microscopically
Molecular
Cultural method (on CAM medium
Light
Microscope
PCR
Sequences analysis UV
NH4 Sol.

10. The Aim of Study 1 Study the occurrence of mycoflora in poultry feed.
Determination of aflatoxigenic A.flavus.
Compatible homology aflatoxigenic A.flavus strains with other strains in the gene bank.

11. Results 1- Morphological and microscopic identification

12. Aspergillus
Penicillium
Figure(1)

13. Rhizopus
Chladosporium
Figure(2)

14. Mucor
Alternaria
Figure(3)

15. Fusarium
Figure(4)

16. A.flavus
A.niger
Figure(5)

17. A.fumigatus
A.terreus
Figure(6)

18. A.flavipes
A.carbonarius
Figure(7)

19. A.ochraceus
A.candidus
Figure(8)

20. A.parasiticus
Figure(9)

21. Calculation of frequency and relative density of genera and Aspergillus spp.

22. Table(1):Range and average count cfu/g of recovered molds genera from poultry feed samples

23. Table(2):Frequency and relative density of recovered mold genera from poultry feed samples.

24. Table(3):Range and average count cfu/g of recovered Aspergillus spp
Table(3):Range and average count cfu/g of recovered Aspergillus spp. from poultry feed samples

25. Table(4):Frequency and relative density of recovered Aspergillus spp
Table(4):Frequency and relative density of recovered Aspergillus spp. from poultry feed samples

26. 2- Cultural detection a-UV light

27. Control
Control
Non aflatoxigenic
Aflatoxigenic
Figure(10)

28. b-Ammonia vapor

29. Control
Figure(11)

30. 3-Molecular by PCR

31. Figure(12): Agarose gel electrophoretic of PCR products obtained from DNA of fungal isolates showing amplicons for aflR primer. Lanes: M- 100bp standard, Lanes1–7: A. flavus (aflatoxin producer) amplicon corresponding to 798 bp.

32. Table(5):Detection of aflatoxigenic and nonaflatoxigenic A
Table(5):Detection of aflatoxigenic and nonaflatoxigenic A.flavus isolates from poultry feed by three methods 16

33. Sequencing analysis of PCR product

35. Conclusions
1-There were contamination with aflatoxin in poultry feed in farms and local markets in Basrah governorate. 2- There are several fungi in collected poultry feed . 3-Several identification and detection methods o aflatoxigenic Aspergillus flavus were used , the most specific , powerful and accurate methods were molecular methods (PCR and sequences analysis) .These two methods detect the specific gene and genetic sequences which produce the aflatoxin.

36. Conclusions
4- The sequences analysis revealed that the poultry feed were contaminated with aflatoxigenic A.flavus isolates , and these isolates were compatible(100% and 99%) with other A.flavus strains in gene bank.

37. Recommendations
1- Deep study should be done on mycotoxigenic fungi and the amount of mycotoxin in several farms storage and local markets in Basrah governorate. 2- Control and prevent factors such as moister and temperature which play important role in fungal growth and mycotoxin production by make them unsuitable .

38. Recommendations
3- Bioassay is very important measurement in this type of studies, so it is recommended to held more deep studies to estimate the effect of degradation residues on domestic animals especially blood parameters and status. 4- Application of biocontrol agent as a powerful control factor for aflatoxins especially the aflatoxin B1( AFB1).

39. CATCTATTCACACCCACACGGCTCTGCCCACCCCGAATGGTAGCAGTAGCGTCTCCGCCATCTTTTCTCATCAGAGTCCGCCGCCACCCGTGGAGACCCAGGGCCTTGGAGGAGATCTGGCTGGTCAGGAGCAAAGCACCCTGTCTTCCCTAACAGTCGATTCGGAATTCGGGGGCTCGTTGCAGTCAATGGAACACGGAAACCATGCCGATTTCTTGGCCGAGTCGACGGGGAGTCTTTTCGACGCGTTTTTGGAAGTAGGGACCCCCATGATCGACCCGTTCCTCGAGTCGGCCCCACTACCACCGTTTCAGGCGCGCTATTGCTGCTTTTCGCTAGCACTACAAACACTGACCCACCTCTTCCCCCACGCCCCGCTGGGCTGTCAACTACGGCTGACGGACGGTGAGGACAGTTCGTACAACCTGATGACGACTGATATGGTCATCTCGGGGAACAAGAGGGCTACCGATGCGGTCCGGAAGATCCTCGGGTGTTCGTGCGCGCAGGATGGCTACTTGCTGAGCATGGTCGTCCTTATCGTTCTCAAGGTGCTGGCATGGTATGCTGCGGCAGCAGGCACCCAGTGTACCTCAACGGCGGCGAGTGGAGAAACCAACAGTGGCAGCTGTAGCAACAGTCCCGCCACCGTGTCCAGTGGCTGTCTGACGGAAGAGCGCGTGCTGCACCTCCCTAGTATGGTGGGCGAGGATTGTGTGGATGAGGAAGACCAGCCGCGAGTGGCGGCACAGCTTGTTCTGAGCGAACTGCACTATTATGTTCTTTTATTTTTGTTGTTGCTCTTAGTCTGCGTTCTCTGTGTCTCTTATCTGTGGAGTGTATTATT

45. GGAGGGTTCCCTCGCGGGCTGGTCTTCTCATCCACACAATCCTCGCCCACCATACTAGGGAGGTGCAGCACGCGCTCTTCCGTCAGACAGCCACTGGACACGGTGGCGGGACTGTTGCTACAGCTGCCACTGTTGGTTTCTCCACTCGCCGCCGTTGAGGTACACTGGGTGCCTGCTGCCGCAGCATACCATGCCAGCACCTTGAGAACGATAAGGACGACCATGCTCAGCAAGTAGCCATCCTGCGCGCACGAACACCCGAGGATCTTCCGGACCGCATCGGTAGCCCTCTTGTTCCCCGAGATGACCATATCAGTCGTCATCAGGTTGTACGAACTGTCCTCACCGTCCGTCAGCCGTAGTTGACAGCCCAGCGGGGCGTGGGGGAAGAGGTGGGTCAGTGTTTGTAGTTCTATATAATCTTTATTTATATATTCTTTTATTTGTCTATATTGTAGATTTTTTTTATTTTTTATTCTTTCTTTTTTATAATATACTTTTTTTT

47. AGGGGGGGGGGGAATTGATCGCGGCTGGTCTTCTCATCCACACAATCCTCGCCCACTCATACTAGGGAGGTGCAGCACGCGCTCTTCCGTCAGACAGCCACTGGACACGGTGGCGGGACTGTTGCTACAGCTGCCACTGTTGGTTTCTCCACTCGCCGCCGTTGAGGTACACTGGGTGCCTGCTGCCGCAGCATACCATGCCAGCACCTTGAGAACGATAAGGACGACCATGCTCAGCAAGTAGCCATCCTGCGCGCACGAACACCCGAGGATCTTCCGGACCGCATCGGTAGCCCTCTTGTTCCCCGAGATGACCATATCAGTCGTCATCAGGTTGTACGAACTGTCCTCACCGTCCGTCAGCCGTAGTTGACAGCCCAGCGGGGCGTGGGGGAAGAGGTGGGTCATTTTTTGTTTTATTTGCGAATTTCTGTAATTTCGTTCCTGTTAAGGTCGTTTATTTTTTGTTGTACTATTTCTTTTCTTTTATTCTGATTTGTTTTTT