Occurrence of Toxigenic Fungi in Occurrence of Toxigenic Fungi in Poultry feed & Molecular Poultry feed & Molecular

Detection of their Toxigenic ActivityDetection of their Toxigenic Activity

M.Sc. StudentRaed Najeeb Kadhim

SupervisorsSupervisorsProf.Dr. Mohammed H.Khudor Prof.Dr.Basil A.AbbasProf.Dr. Mohammed H.Khudor Prof.Dr.Basil A.Abbas

Mycotoxins• Secondary metabolites (chemicals) of a fungus that produce

toxic results in another organism.

• Lack of visible appearance of fungus does not negate presence

of mycotoxins. Toxins can remain in the organism after fungus

has been removed.

• Cytotoxic: disrupt cell structures such as membranes, and

processes such as protein, DNA, and RNA synthesis.

• Can be heat stable, not destroyed by canning or other

processes.

Some important Toxigenic Some important Toxigenic FungiFungi

Fusarium

Fusarium • The name of Fusarium comes from Latin fusus,

meaning a spindle• widely distributed on plants and in the soil.• found in rice, bean, soybean, and other crops• pathogenic to man and animals causing mycotic

keratitis, onychomycosis , cornea (keratomycosis) , (neutrpenia, i.e., very low neutrophils count) and hyalohyphomycosis, especially in burn victims and bone marrow transplant patients

• produce many toxins as fumonisins and trichothecenes

.

Alternaria

Alternaria

• Found in plants, soil, food, and indoor air environment• They are normal agents of decay and decomposition• Causing many infections to human and animals as alternariosis

, phaeohyphomycosis, onychomycosis, sinusitis, ulcerated cutaneous infections, and keratitis, as well as visceral infections and osteomyelitis , hypersensitivity reactions that sometimes lead to asthma and opportunistic infection in immunocompromised people such as AIDS patients

• Produce many toxins alternariol, monomethyl ether, tenuazonic acid, altertoxins and tentoxin.

Aspergillus

Aspergillus• Found in soil, plant debris, and indoor air environment• Cause infections in animals as well as in man, In birds , mycotic

abortion in the cattle and the sheep . Ingestion of high amounts of aflatoxin may induce lethal effects in poultry animals fed with grain contaminated with the toxin.

• Onychomycosis, sinusitis, cerebral aspergillosis, meningitis, endocarditis, myocarditis, pulmonary aspergillosis, osteomyelitis, otomycosis, endophthalmitis, cutaneous aspergillosis and hepatosplenic aspergillosis

• Produce many toxins as Aflatoxins B1, B2, ,G1,G2, M1. M1 , Gliotoxin, Cyclopiazonic acid, Sterigmatocystin, and Methoxy Sterigmatocystin

Penicillium

Penicillium• The name Penicillium comes from penicillus = brush, and this is

based on the brush-like appearance• commonly present wherever organic material is available , air and

dust of indoor environments, such as homes and public buildings• grow on seeds and other stored foods • Penicillium has been isolated from patients with keratitis

[ endophtalmitis, otomycosis, necrotizing esophagitis, pneumonia, endocarditis, peritonitis, and urinary tract infections. become a common opportunistic infection of HIV-infected individuals in southeast Asia

• produce penicillin, a molecule that is used as an antibiotic,• produce ochratoxin A toxin • .

Some Important Mycotoxins• Today 300 - 400 mycotoxins are known• Mycotoxins of human concern based on toxicity: Aflatoxin Deoxyniva-lenol (DON) or Vomitoxin Zearalenone Fumonisin T-2 toxin Ochratoxin A

Mycotoxins Chemical structure

• Aflatoxins

Tricothecenes

• Fumonisins

O

OOH

OHHHH

H

OH

OH

CH3

CH3

NH2

OHOH

OH

O

OCH3CH3

O OH

OOH

O

O OH

O

OH

O

O

O

O

H

H

O O

OCH3

Aflatoxins

• Produced by (Aspergillus. flavus, A. parasiticus and rare A. nomius)

• Types: aflatoxins B1 (AFB1) and B2 (AFB2), (AFG1) and G2 (AFG2).

• Aflatoxin B1 occurs most frequently and is most toxic and carcinogenic

• Many crops are affected Maize, Groundnut, Soybean, Cotton

Effects of Aflatoxins

• Human beings and Livestock– Cattle– Poultry

• Deleterious effects– Teratogenic– Immunosuppressive – Many times enhanced in the presence of hepatitis B

and C viruses. – Carcinogenic

Fungi Substrate Mycotoxin

Aspergillus flavus Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus parasiticus Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus nomius Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus ochraceus Barkey wheat Ochratoxin

Aspergillus carbonerius Grapes wine coffee Ochratoxin

Fusarium oxysporum Wheat barley maize Fumonisins

Fusarium sp. Wheat barley maize T-2 toxin

Penicillium verrucosum Wheat barley maize Ochratoxin

Claviceps purpurea Rye Ergot alkaloids

Stachybotrys Hay Satratoxins

MycotoxinsMycotoxinsFactors causing variation in effectsFactors causing variation in effects

• Species, breed• Age• Sex• Nutritional status• Other diseases• Other mycotoxins• Extent of exposure

Some mycotoxins are formed in the field, some in storage

o Storage conditions that favor production of mycotoxins:o Temperature (40 - 90o F ; 4 - 32o C)o Relative Humidity (> 70%)o Moisture (22-23% in grain)o Oxygen (1-2%)

Modes of Spore Transmission• Airborne, wind or indoor ventilation systems.• Attachment to insects of birds, thus transmitted

from plant to plant, or animal to animal, etc.• Via transportation mechanisms such as trucks, crop

machinery, etc.

Life Cycle of fungi

Route & mechanism of Intoxication of Mycotoxins

Ingestion/skin contact/ inhalation

Blood stream & lymphatic system

Inhibit protein synthesis

Damage macrophages system

Inhibit particle clearance of the lungs

Increase sensitivity to bacterial endotoxins

Mycotoxin Effects on Animals

• Feed refusal.• Impaired animal health, resulting in reduced production of

eggs, milk, weight gain, etc.• Metabolites are passed through the milk in cheese, dry milk,

and yogurt.• Disease.• Death in animals.• Mimic other herd health problems

Depressed immune system, Off-feed ,DiarrheaHemorrhaging , Abortions , Reproduction issues.

Quick Toxin ReviewOrgan System Affected Toxin(s)

Vascular AflatoxinDigestive Aflatoxin, T-2toxin,

VomitotoxinRespiratory Trichothecenes

Nervous Trichothecenes

Cutaneous Tricothecenes

Urinary Ochratoxin A, Citrinin

Reproductive Zearalenone, T-2 toxin

Immune Many

Effect of Mycotoxins on Domestic Animals

Effect of Mycotoxins on Poultry

Tolerance Levels for the Total Aflatoxin

Tolerance levels for the total aflatoxin(sum of aflatoxins B1, B2, G1 and G2) ranges from 1 to 35 μg/kg for foods, with an average of 10 g/kg; and from zero to 50 μg/kg for animal feed, with an average of 20 μg/kg . For AFM1 In milk, tolerance levels are between 0.05 and 0.5 μg/kg

Prevention

• Development of fungal resistant varieties of growing plants;• Control field infection by fungi of planting crops;• Making schedule for suitable pre-harvest, harvest and post-

harvest;• Lowering moisture content of plant seeds, after post

harvesting and during storage;• Store commodities at low temperature whenever possible;• Using fungicides and preservatives against fungal growth;• Control insect infestation in stored bulk grains with approved

insecticides.

Detection of Mycotoxins There are several methods:

•Chemical : TLC. ,GC. , GC-MS. , HPLC ., NH4 Sol.•Immunological: ELISA., Enzyme Inhibition Assays•Physical: UV Light .•Molecular: PCR

Method

Collection of samples of poultry feed

Culturing on PDA,MEA& SDA

Isolation

Identification

Morphological Microscopically Molecular physically Chemically

PCRUV

Cultured on CMA

NH4 Sol.

HPLCLight Microscope

Aims of study

• Detect the occurrence of mycoflora in poultry feed.

• Molecular –Base detection of mycotoxigenic fungi

• Investigate the types of mycotoxins in poultry feed

• Prevention and control of mycotoxins in poultry feed.

Thanks