Recombinant DNA technology (Immunological screening)
seminar
1. Occurrence of Toxigenic Fungi inOccurrence of Toxigenic Fungi in
Poultry feed & MolecularPoultry feed & Molecular
Detection of their ToxigenicDetection of their Toxigenic
ActivityActivity
M.Sc. Student
Raed Najeeb Kadhim
SupervisorsSupervisors
Prof.Dr. Mohammed H.Khudor Prof.Dr.Basil A.AbbasProf.Dr. Mohammed H.Khudor Prof.Dr.Basil A.Abbas
2. 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.
5. 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
.
7. 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.
9. 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
11. 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
12. 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
13. Mycotoxins Chemical structure
• Aflatoxins
Tricothecenes
• Fumonisins
O
O
OH
OH
HHH
H
O
H
OH
CH3
CH3
NH2
OHOH
OH
O
O
CH3CH3
O OH
OOH
O
O OH
O
OH
O
O
O
O
H
H
O
O
O
CH3
14. 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
15. 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
17. MycotoxinsMycotoxins
Factors causing variation in effectsFactors causing variation in effects
• Species, breed
• Age
• Sex
• Nutritional status
• Other diseases
• Other mycotoxins
• Extent of exposure
18. Some mycotoxins are formed in the field, some
in storage
oStorage 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%)
19. 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.
21. Route & mechanism of Intoxication of Mycotoxins
Ingestion/skin contact/ inhalationIngestion/skin contact/ inhalation
Blood stream & lymphatic systemBlood stream & lymphatic system
Inhibit protein synthesisInhibit protein synthesis
Damage macrophages systemDamage macrophages system
Inhibit particle clearance of the lungsInhibit particle clearance of the lungs
Increase sensitivity to bacterial endotoxinsIncrease sensitivity to bacterial endotoxins
22. 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 ,Diarrhea
Hemorrhaging , Abortions , Reproduction issues.
23. Quick Toxin Review
Organ System Affected Toxin(s)
Vascular Aflatoxin
Digestive Aflatoxin, T-2toxin,
Vomitotoxin
Respiratory Trichothecenes
Nervous Trichothecenes
Cutaneous Tricothecenes
Urinary Ochratoxin A, Citrinin
Reproductive Zearalenone, T-2 toxin
Immune Many
26. 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
27.
28. 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.
29. 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
30. Method
Collection of samples of poultry feed
Culturing on
PDA,MEA& SDA
Isolation
Identification
Morphological Microscopically Molecular physically Chemically
PCR
UV
Cultured
on CMA
NH4
Sol.
HPLC
Light
Microscope
31. 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.
