Occurrence of fungi in poultry feed with cultural and molecular detection of their aflatoxigenic

activityM.Sc. Student

Raed Najeeb Kadhim

SupervisorsProf.Dr. Mohammed H.Khudor Prof.Dr.Basil A.Abbas

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.

Some important toxigenic fungi

Aspergillus

Penicillium

Alternaria

Fusarium

Toxigenic fungi

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.

Some important mycotoxins

Today 300 - 400 mycotoxins are known

Common mycotoxins

Aflatoxin Deoxynivalenol ZearalenoneFumonisinOchratoxin

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.

Method

Collection of samples of poultry feed

Culturing on PDA,MEA& SDA

Isolation

Identification

Morphological by culture Microscopically Molecular

Cultural method (on

CAM medium

PCR UV NH4 Sol.Light Microscope

A. flavus

Sequences analysis

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.

Results1- Morphological and microscopic

identification

Aspergillus

PenicilliumFigure(1)

Rhizopus

Chladosporium Figure(2)

Mucor

Alternaria Figure(3)

Fusarium

Figure(4)

A.flavus

A.niger Figure(5)

A.fumigatus

A.terreus Figure(6)

A.flavipes

A.carbonarius Figure(7)

A.ochraceus

A.candidus Figure(8)

A.parasiticus

Figure(9)

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

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

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

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

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

2 -Cultural detectiona-UV light

Control

Figure(10)

Control Non aflatoxigenic

Aflatoxigenic

b-Ammonia vapor

Figure(11)

Control

3-Molecular by PCR

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 .

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

16

Sequencing analysis of PCR product

Conclusions1-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.

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.

Conclusions

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 .

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).

Recommendations

CATCTATTCACACCCACACGGCTCTGCCCACCCCGAATGGTAGCAGTAGCGTCTCCGCCATCTTTTCTCATCAGAGTCCGCCGCCACCCGTGGAGACCCAGGGCCTTGGAGGAGATCTGGCTGGTCAGGAGCAAAGCACCCTGTCTTCCCTAACAGTCGATTCGGAATTCGGGGGCTCGTTGCAGTCAATGGAACACGGAAACCATGCCGATTTCTTGGCCGAGTCGACGGGGAGTCTTTTCGACGCGTTTTTGGAAGTAGGGACCCCCATGATCGACCCGTTCCTCGAGTCGGCCCCACTACCACCGTTTCAGGCGCGCTATTGCTGCTTTTCGCTAGCACTACAAACACTGACCCACCTCTTCCCCCACGCCCCGCTGGGCTGTCAACTACGGCTGACGGACGGTGAGGACAGTTCGTACAACCTGATGACGACTGATATGGTCATCTCGGGGAACAAGAGGGCTACCGATGCGGTCCGGAAGATCCTCGGGTGTTCGTGCGCGCAGGATGGCTACTTGCTGAGCATGGTCGTCCTTATCGTTCTCAAGGTGCTGGCATGGTATGCTGCGGCAGCAGGCACCCAGTGTACCTCAACGGCGGCGAGTGGAGAAACCAACAGTGGCAGCTGTAGCAACAGTCCCGCCACCGTGTCCAGTGGCTGTCTGACGGAAGAGCGCGTGCTGCACCTCCCTAGTATGGTGGGCGAGGATTGTGTGGATGAGGAAGACCAGCCGCGAGTGGCGGCACAGCTTGTTCTGAGCGAACTGCACTATTATGTTCTTTTATTTTTGTTGTTGCTCTTAGTCTGCGTTCTCTGTGTCTCTTATCTGTGGAGTGTATTATT

ACGCCGTCATCGCGGCTGGTCTTCTCATCCACACAATCCTCGCCCACCATACTAGGGAGGTGCAGCACGCGCTCTTCCGTCAGACAGCCACTGGACACGGTGGCGGGACTGTTGCTACAGCTGCCACTGTTGGTTTCTCCACTCGCCGCCGTTGAGGTACACTGGGTGCCTGCTGCCGCAGCATACCATGCCAGCACCTTGAGAACGATAAGGACGACCATGCTCAGCAAGTAGCCATCCTGCGCGCACGAACACCCGAGGATCTTCCGGACCGCATCGGTAGCCCTCTTGTTCCCCGAGATGACCATATCAGTCGTCATCAGGTTGTACGAACTGTCCTCACCGTCCGTCAGCCGTAGTTGACAGCCCAGCGGGGCGTGGGGGAAGAGGTGGGTCAGTGTTTGTAGTGCTAGCGAAAAGCAGCAATAGCGCGCCTGAAACGGTGGTAGTGGGGCCGACTCGAGGAACGGGTCGATCATGGGGGTCCCTACTTCCAAAAACGCGTCGAAAAGACTCCCCGTCGACTCGGCCAAGAAATCGGCATGGTTTCCGTGTTCCATTGACTGCAACGAGCCCCCGAATTCCGAATCGACTGTTAGGGAAGACAGGGTGCTTTGCTCCTGACCAGCCAGATCTCCTCCAAGGCCCTGGGTCTCCACGGGTGGCGGCGGACTCTGATGAGAAAAGATGGCGGAGACGCTACTGCTACCATTCAGGGTGGGCAGAGCGTGTGGTGGTTGATTCGATTGACGATGAGATTGTGGATGCGGTTATATAACCCTCCCCTTCTCGGGCCGGGCGTTCTCGCTTCTGTCACATATTAATTTTCCGTTCTAACTAGCCTTC

CACTATCCACACACGCTCTGCCCACCCCCATGGTAGCAGTAGCGTCTCCGCCATCTTTTCTCATCAGAGTCCGCCGCCACCCGTGGAGACCCAGGGCCTTGGAGGAGATCTGGCTGGTCAGGAGCAAAGCACCCTGTCTTCCCTAACAGTCGATTCGGAATTCGGGGGCTCTTTGCAGTCAATGGAACACGGAAACCATGCCGATTTCTTGGCCGAGTCGACGGGGAGTCTTTTCGACGCGTTTTTGGAAGTAGGGACCCCCATGATCGACCCGTTCCTCGAGTCGGCCCCACTACCACCGTTTCAGGCGCGCTATTGCTGCTTTTCGCTAGCACTACAAACACTGACCCACCTCTTCCCCCACGCCCCGCTGGGCTGTCAACTACGGCTGACGGACGGTGAGGACAGTTCGTACAACCTGATGACGACTGATATGGTCATCTCGGGGAACAAGAGGGCTACCGATGCGGTCCGGAAGATCCTCGGGTGTTCGTGCGCGCAGGATGGCTACTTGCTGAGCATGGTCGTCCTTATCGTTCTCAAGGTGCTGGCATGGTATGCTGCGGCAGCAGGCACCCAGTGTACCTCAACGGCGGCGAGTGGAGAAACCAACAGTGGCAGCTGTAGCAACAGTCCCGCCACCGTGTCCAGTGGCTGTCTGACGGAAGAGCGCGTGCTGCACCTCCCTAGTATGATGGGCGAGGATTGTGTGGATGAGGAAGACCAGCCGCGAGTGGCGGCACAGCTTGTTCTGAGCGAACTGCACTAAAAACCCGTGCACCAAGCTAACTTTACT

GGAGGGTTCCCTCGCGGGCTGGTCTTCTCATCCACACAATCCTCGCCCACCATACTAGGGAGGTGCAGCACGCGCTCTTCCGTCAGACAGCCACTGGACACGGTGGCGGGACTGTTGCTACAGCTGCCACTGTTGGTTTCTCCACTCGCCGCCGTTGAGGTACACTGGGTGCCTGCTGCCGCAGCATACCATGCCAGCACCTTGAGAACGATAAGGACGACCATGCTCAGCAAGTAGCCATCCTGCGCGCACGAACACCCGAGGATCTTCCGGACCGCATCGGTAGCCCTCTTGTTCCCCGAGATGACCATATCAGTCGTCATCAGGTTGTACGAACTGTCCTCACCGTCCGTCAGCCGTAGTTGACAGCCCAGCGGGGCGTGGGGGAAGAGGTGGGTCAGTGTTTGTAGTTCTATATAATCTTTATTTATATATTCTTTTATTTGTCTATATTGTAGATTTTTTTTATTTTTTATTCTTTCTTTTTTATAATATACTTTTTTTT

AGGGGGGGGGGGAATTGATCGCGGCTGGTCTTCTCATCCACACAATCCTCGCCCACTCATACTAGGGAGGTGCAGCACGCGCTCTTCCGTCAGACAGCCACTGGACACGGTGGCGGGACTGTTGCTACAGCTGCCACTGTTGGTTTCTCCACTCGCCGCCGTTGAGGTACACTGGGTGCCTGCTGCCGCAGCATACCATGCCAGCACCTTGAGAACGATAAGGACGACCATGCTCAGCAAGTAGCCATCCTGCGCGCACGAACACCCGAGGATCTTCCGGACCGCATCGGTAGCCCTCTTGTTCCCCGAGATGACCATATCAGTCGTCATCAGGTTGTACGAACTGTCCTCACCGTCCGTCAGCCGTAGTTGACAGCCCAGCGGGGCGTGGGGGAAGAGGTGGGTCATTTTTTGTTTTATTTGCGAATTTCTGTAATTTCGTTCCTGTTAAGGTCGTTTATTTTTTGTTGTACTATTTCTTTTCTTTTATTCTGATTTGTTTTTT