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, M1) Gliotoxin, Sterigmatocystin, and Methoxy Sterigmatocystin.

Some important mycotoxins

Today 300 - 400 mycotoxins are known

Common mycotoxins

Aflatoxin

Deoxynivalenol

Zearalenone

Fumonisin

Ochratoxin

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.

AfB1 metabolism

• The main metabolizing organ for aflatoxin is the liver, but this can also occurs directly at the site of absorption, in the blood or in several extra-hepatic organs.

•Oral route is the main contamination means, inhalation may also occur as a result of people or animals being exposed to the grain’s dust.

•AfB1 is efficiently absorbed by duodenum.

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

Figure(10)

b-Ammonia vapor

Figure(11)

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