Detection of Aflatoxigenic Aspergillus flavus in Poultry Feed

Cultural and molecular detection of
aflatoxigenic activity in Aspergillus flavus
isolated from poultry feed
A thesis submitted to the council of College of Veterinary Medicine-
University of Basrah in partial fulfillment of the requirements for the
Master Science Degree in Veterinary Medicine / Microbiology
By:
Raed Najeeb Kadhim Alkhersan
B.Sc. (2002)
Supervised by:
Prof.Dr. Mohammed H.Khudor Prof. Dr.Basil A. Abbas
2016 AD 1437 AH
Republic of Iraq
Ministry of Higher Education and Scientific Research
University of Basrah
College of Veterinary Medicine

ِ‫م‬‫ِي‬‫ح‬َّ‫ر‬‫ل‬‫ا‬ ِ‫ن‬َ‫م‬ْ‫ح‬َّ‫ر‬‫ل‬‫ا‬ ِ‫ه‬َّ‫ل‬‫ل‬‫ا‬ ِ‫م‬ْ‫س‬ِ‫ب‬
‫َا‬‫م‬َ‫و‬ ‫ِّي‬‫ب‬َ‫ر‬ ِ‫ر‬ْ‫م‬َ‫أ‬ ْ‫ن‬ِ‫م‬ ُ‫ح‬‫ُّو‬‫ر‬‫ل‬‫ا‬ ِ‫ل‬ُ‫ق‬ ِ‫ح‬‫ُّو‬‫ر‬‫ل‬‫ا‬ ِ‫ن‬َ‫ع‬ َ‫ك‬َ‫ن‬‫و‬ُ‫ل‬َ‫أ‬ْ‫س‬َ‫ي‬َ‫و‬
‫ًا‬‫ل‬‫ِي‬‫ل‬َ‫ق‬ ‫َّا‬‫ل‬ِ‫إ‬ ِ‫م‬ْ‫ل‬ِ‫ع‬ْ‫ل‬‫ا‬ َ‫ن‬ِ‫م‬ ْ‫م‬ُ‫ت‬‫ِي‬‫ت‬‫ُو‬‫أ‬(58)
‫لعظيم‬‫ا‬ ‫لعلي‬‫ا‬ ‫اهلل‬ ‫صدق‬
(‫اإلسراء‬ ‫سورة‬)

Dedication
This humble work is dedicated to those who never left my mind a moment;
Granter of humanity science , culture and morals …
Messenger of Allah and Ahl al-Bayt,
To my parents who supported and kept supporting me when needed.
To my wife who helped a lot and continued in every step to
complete my work.

Acknowledgment
Praise be to Allah, the Almighty who blessed me with the wisdom to
perceive ideas for understanding this project.
I humbly pay my great gratitude and respects to the Holy Prophet “Peace
be upon Him and his family for their care throughout the duration of this
study.
It is my pleasant duty to express gratitude to my supervisors Prof .Dr.
Mohammed H. Khudor and Prof .Dr. Basil A. Abbas for their constructive
guidance and constant cooperation throughout my study.
I extend my thanks and appreciations to Assist.Prof.Dr.Ghazi Al-emarah
dean of College of Veterinary Medicine , Assist.Prof.Dr.Rasha Munther
Othman, head of department of microbiology and parasitology , and the
academic staff of Microbiology and parasitology department for awarding me
this valuable opportunity to complete my M.Sc. study.
I say my sincerest thanks to Prof. Abdullah Al Sadoon from Biology
department – College of Science for his valuable guidance, encouragement
help in the fungi identification throughout my research.
I express my gratitude to Prof. Dr. Atef A. Hassan , professor of
Mycology and Mycotoxins, Animal Health Research Institute, Dokki, Egypt,
for his valuable guidance for useful suggestions and moral support.
I am also thankful to Prof. Dr. Muslem Abdulrahman Mohamed, assist.
Prof. Dr. Kareem Hilal Thamer from Biology department – College of Science
and Dr. Ali Abdulrazzaq chemistry department, College of Science for their
efforts and assistance in chemicals preparations .
I would like to appreciate the help extended to me by Prof. Dr. Adnan
Albadran cell and biotechnology researches unit , for extending lab facilities
for my research work.
I feel great pleasure in expressing my sincerest thanks to Assist. Prof .Dr.
Munaf Jawdat from Biology department –College of Science and Dr.
Labeed Abdullah Najim Al-Saad, Agriculture College for their expert
guidance and cooperation in analysis of results of DNA sequencing .

Special thanks to all my friends , especially Hayder Abbas, Amged
Abdalrazaq , Hussein Jabbar Alrekaby and Jalal Afandi , M.S.c students and
Hayder Alhassanee M.S.c in engineering for their help and encouragement.
Finally, I appreciate any support, help and/or advice provided by any
generous person may I forget to list his name here and ask him/her to forgive
me.

I
Summary
The present study aimed to the occurrence of mycoflora in poultry feed,
determination of aflatoxigenic Aspergillus flavus and compatible homology
aflatoxigenic A.flavus strains with other strains in gene bank. A total of 180 samples
of concentrated poultry feed pellet were collected from different broilers, broiler
breeders and layers farms and local market of poultry in Basrah province . Feed
samples were collected during the period from Sep. 2014 to Apr. 2015. About 10 -
30 representative samples of 1 kg were collected from several locations. They were
cultured on Potato dextrose agar(PDA) and malt extract agar (MEA) and then
subcultured on Sabouraud dextrose agar (SDA) and coconut medium agar(CAM)
.Seven genera were recovered from 180 samples of poultry feed .The most genera
which recovered were Aspergillus (frequency(Fr) 62.77% - Relative density(RD)
52.03%), followed by Penicillium (Fr 47.77% - RD 17.01%) were the predominant
genera isolated from poultry feed, while Fusarium isolates were less frequency and
relative density(Fr.1.66%, RD 2.11%) .The most frequently isolated Aspergillus
spp.was Aspergillys flavus ( Fr 65.48%) and had the most RD (27.55%) , followed
by A.niger (Fr. 58.40%, RD14.23%),the less occurrence of Aspergillus was
A.paraciticus(Fr.1.76%, RD0.89%) .Fifty isolates of A. flavus were detect by UV
light (365nm) and ammonia vapor to detect aflatoxigenic A.flavus on CAM by
colored with blue –green on reverse of glass petri dish under UV light and produce a
pink to red color by exposure to ammonia vapor. The detection by fluorescent blue
revealed that 26 (52%) of isolates were aflatoxigenic (positive)by produce

II
fluorescent color under UV (356nm) light , and also 26 (52%) of isolates were
aflatoxigenic (positive) by ammonia vapor test. The molecular assessment was done
on 50 isolates of A.flavus by using primers pair for the aflatoxin regulatory gene
aflR in polymerase chain reaction (PCR). Five isolates of aflatoxigenic A. flavus
positive identified isolates by PCR were randomly selected to sequence and analyze
by basic local alignment search tool analysis (BLAST) to confirm the aflatoxigenic
strains. Five isolates were positive and confirmed approximately compatible(100%
and 99%) homology with other A.flavus strains on NCBI .

III
List of Contents
Numbers Subject Page
Summary I
List of contents III
List of tables VI
List of figures VII
List of abbreviations VIII
Chapter one: Introduction
1.1 Introduction 1
1.2 The aim of study 3
Chapter two: Review of literatures
2.1 Important mycotoxigenic fungi 4
2.1.1 Aspergillus species 4
2.1.2 Fusarium species 5
2.1.3 Penicillium species 6
2.1.4 Other toxigenic fungi 6
2.2 Mycotoxins 7
2.3 Mycotoxin occurrence 11
2.4 Factors influencing growth of fungi and production of
mycotoxin
12
2.4.1 Abiotic factors 14
2.4.1.1 Water activity 15
2.4.1.2 Temperature 16
2.4.1.3 Hydrogen ion potential (pH) 16
2.4.1.4 Oxygen supply 17
2.4.1.5 Carbon dioxide 18
2.4.2 Biotic factors 18
2.5 Regulation of mycotoxin biosynthetic genes cluster 19
2.6 Pre- and postharvest of mycotoxin contamination 20
2.7 Mycotoxin exposure , mechanisms of action and effect 21
2.7.1 Effects of mycotoxin on human 24
2.7.2 Effects of mycotoxin on animals 24
2.7.3 Effects of mycotoxin (phytotoxin) on plant 26
2.8 Methods to detect mycotoxigenic fungi 27
2.8.1 Detection by conventional microbiological methods 27
2.8.2 Analytical detection of mycotoxin production 28
2.8.2.1 Mycotoxins analysis by methods of chromatograph 28
2.8.2.1.1 TLC 28

IV
2.8.2.1.2 HPLC 28
2.8.2.1.3 GC 29
2.8.2.1.4 CE 29
2.8.2.2 Immunological methods for analysis of mycotoxins 29
2.8.2.2.1 ELISA 30
2.8.3 Cultural methods 30
2.8.3.1 Blue fluorescence 30
2.8.3.2 Ammonium hydroxide vapor-induced color change 31
2.8.4 Molecular detection of mycotoxin-producing molds 31
2.8.4.1 Polymerase Chain Reaction (PCR) 31
2.8.4.2 Real-time PCR 32
2.8.4.3 Future perspectives: new molecular methods 33
2.9 Main mycotoxins 33
2.9.1 Aflatoxins 33
2.9.1.1 Exposure and absorption into the organism 35
2.9.1.2 Metabolism of aflatoxin 36
2.9.1.2.1 Bioactivation 36
2.9.1.2.2 Conjugation 38
2.9.1.2.3 Deconjugation 38
2.9.1.3 The mechanism of toxicity 38
2.9.1.4 The role of aflR in aflatoxin pathway regulation 40
2.9.2 Fumonisins 40
2.9.3 Ochratoxins 41
2.10 The prevention and reduction of mycotoxins strategies 42
2.10.1 Practices of good agricultural quality 42
2.10.2 Biological control 44
2.10.3 Chemical control 45
Chapter three: Materials and methods
3.1 Materials 46
3.1.1 Instruments and equipment 46
3.1.2 Chemicals 47
3.1.3 Media 48
3.1.3.1 Coconut-Agar Medium (CAM) 48
3.1.3.2 Potato Dextrose Agar (PDA) 48
3.1.3.3 Malt Extract Agar (MEA) 48
3.1.3.4 Sabouraud Dextrose Agar (SDA) 49
3.1.4 Stains 49
3.1.4.1 Lactophenol cotton blue 49
3.1.4.2 Lacto-fuchsin 49
3.1.5 Kits 50
3.2 Methods 51
3.2.1 Collection of samples 51

V
3.2.2 Isolation and identification of fungi 51
3.2.3 Detection tools of aflatoxigenic A.flavus 52
3.2.3.1 Coconut based medium detection 52
3.2.3.2 Ammonia vapor detection 53
3.2.3.3 Molecular assay 53
3.2.3.3.1 Preparation of buffers, solutions and stains 54
3.2.3.3.1.1 TBE (1X) 54
3.2.3.3.1.2 Ethidium bromide 54
3.2.3.3.1.3 Agarose gel preparation 54
3.2.3.3.2 Preparing A.flavus mycelia for DNA extraction 55
3.2.3.3.3 DNA extraction 55
3.2.3.3.4 Polymerase chain reaction 57
3.2.3.3.5 PCR result analysis 59
3.2.3.3.6 Sequencing of PCR products for aflR gene 59
3.2.3.3.6.1 The basic local alignment search tool analysis (BLAST) 59
Chapter four: Results
4.1 Fungal isolation 60
4.2 Coconut based medium and ammonia vapor detection 69
4.3 Molecular detection 71
4.3.1 PCR 71
4.3.2 Sequencing analysis of PCR product 74
Chapter five: Discussion
5.1 Fungal isolation 82
5.2 Coconut based medium and ammonia vapor detection 84
5.3 Molecular detection 85
5.3.1 PCR 85
5.3.2 Sequencing and sequences analysis of PCR products for aflR
gene
86
Conclusions and recommendation 87
References 89
Appendix 116

VI
List of tables
Number Subject Page
1 Optimal conditions for fungal growth and mycotoxin production 14
2 Instruments and equipment with their remarks 46
3 Chemicals and biological materials 47
4 DNA extraction kit contents 50
5 PCR reaction kit and related materials 50
6 The sequences of the primers 54
7 Reaction components for PCR 58
8 PCR Program 58
9
Range and average count of cfu/g of recovered molds genera from
poultry feed samples
67
10
Frequency and relative density of recovered mold genera from
poultry feed samples
67
11
Range and average count of cfu/g of recovered Aspergillus
spp.from poultry feed samples
68
12
Frequency and relative density of recovered Aspergillus spp.
from poultry feed samples
68
13
Detection of aflatoxigenic and nonaflatoxigenic A.flavus isolates
from poultry feed by three methods
72
14
Aflatoxigenic and nonaflatoxigenic results obtained by CAM ,
ammonia vapor and PCR detection of A.flavus isolates recovered
from poultry feed samples
72
15
The compatibility of strains of A.flavus with other strains from
NCBI
76

VII
List of figures
Number Subject Page
1 Chemical structure of the different aflatoxins 35
2 Aflatoxin B1 pathways 37
3 Mechanisms of AFB1 toxicity 39
4 Fumonisins B1 structure 41
5 Ochratoxin A structure 42
6 The isolated molds genera from poultry feed on PDA medium 62
7 The isolated molds genera from poultry feed on PDA medium 63
8 The isolated Aspergillus spp. from poultry feed on PDA medium 64
11
The result of detection of aflatoxigenic A.flavus by CAM under
UV light (365nm)
70
12
The result of detection of aflatoxigenic A.flavus by ammonia
vapor
70
13
PCR products obtained through agarose gel electrophoresis from
DNA of A.flavus isolates showing amplicons for aflR primer
71
14 Sequence alignment of A. flavus isolate (Af1) 77

VIII
List of abbreviations
Abbreviation Full name
AF Aflatoxin
AFB1 Aflatoxin B1
AFB2 Aflatoxin B2
AFBS Aflatoxin B1 and B2
AFG1 AflatoxinG1
AFG2 Aflatoxin G2
AflR
Transcriptional (regulatory) gene in Aflatoxin B1 biosynthesis
genes cluster
aflC A structural gene in in aflatoxin B1 biosynthesis genes cluster
aflD A structural gene in in aflatoxin B1 biosynthesis genes cluster
aflM A structural gene in in aflatoxin B1 biosynthesis genes cluster
aflP Amplified fragment length polymorphism
AfP1 Aflatoxin P1
AAT Alternaria alternata Toxin
ALT Alternariol
APA Aflatoxin producing-ability medium
BEN Balkan endemic nephropathy

IX
CE Capillary electrophoresis
CIT Citrinin
D.W Distal water
Don Deoxynivalenol
DAS Diacetoxyscirpenol
DA Diode array
DNAse Deoxyribonuclease
ELISA Enzyme-linked immunosorbent assay
FBs Fumonisins B
FL Fuorescence
g gram
GC Gas chromatography
GLIO Gliotoxin
GST Glutathione S-Transferase
h hour
HACCP Hazard analysis critical control point
HPLC High-performance liquid chromatography

X
UHPLC High-performance liquid chromatography
IARC International agency for research on cancer
IgA Immunoglobulin A
ITS Internal transcribed spacer
kDa kilo dalton
LD50 Median lethal dose
lps Lipopolysaccharide
LAMP Loop-mediated isothermal amplification
MLN Mesenteric lymph node
mm millimeter
min minute
MPA Mycophenolic acid
nm nanometer
NRPSs Nonribosomal peptide synthetases
OTA Ochratoxin A
Pat Patulin
PA Penicillic acid

XI
PEN Penitrem
PKSs Polyketide synthases
PRT Penicillium roqueforti toxin
qPCR quantitative polymerase chain reaction
RNAse Ribonucleic acid (RNA) enzyme
RQ Roquefort
MS Mass spectrometry
ST Sterigmatocystin
MS/MS Tandem Mass Spectrometry
TZA Tenuazonic acid
LC/ESI-
QTOF-MS/MS
The liquid chromatography and electrospray ionization quadruple
time-of- flight mass spectrometry
TLC Thin-layer chromatography
TF Transcription factor
T-2 Trichothecene
UV Ultraviolet
VER Verruculogen

Chapter one Introduction
________________________________________________________________
1
1.1 Introduction
Poultry feed is food to poultry of farm such as ducks ,chickens , geese
,quails and domestic fowl . Poultry were mostly put on general farms before the
twentieth century, and feed of their, grain, eating insects, and plants near the
farm (Scherf, 2000; Romanov et al., 2009).Those of farms and hatcheries are
the source of the poultry feed .
The animal feeds purchased from abroad with large quantity and with the
increased confirmation on resources of animal, this quantity is predictable to
increase basically in coming years. Because the feed have a wide effect on the
birds ,it is necessary to have necessary quality control on them(Beg et al., 2005;
Shareef, 2010) . Poultry feed made for broiler: starter, finisher, and layer mash
(Beg et al., 2005).These feeds consist mainly of cereal, supplements of material
such as protein and as meal of soybean oil, vitamin (Steenfeldt et al., 2007;
Ravindran , 2013) .
Poultry feed industry is closely in relation with the primary agricultural
production and acts as an essential component of the food chain. Feed is
considered the major cost of poultry production that lies between 65 and 75%. ,
therefore , any effect on the feed leads to change on the performance of broilers
and layers (Ashraf et al., 2013).
The storage conditions are necessary to safe feed , so weather extremes
unsuitable storage practices and improper feeding conditions can cause feed –
fungal contamination that increase mycotoxins production (Dowd, 2004 and

________________________________________________________________
2
Hassan et al., 2012). Poultry feed is more susceptible to fugal growth during
processing, therefore identification of fungi with the ability to produce
mycotoxins is essential (Rosa et al. 2006).
Molds can grow and produce mycotoxins in preharvest and through
storage, convey, operations of processing or feeding . During these periods,
humidity and temperature play an important role in the fungi growth and
mycotoxins contamination (Krnjaja et al., 2008) . In wet feeds, increasing of
moisture levels help mold growth if oxygen is available (Lanyasunya et al.,
2005). Feeds with more than 12-15% moisture suitable to grow fungi . Because
aerobically growth of most molds, increasing of moisture concentrations can
eliminate air and prevent mold growth (Whitlow and Hagler, 2008).These
conditions are most appropriate for mold growth and for mycotoxin production
are not necessarily the same (Simpson et al., 2001). About 100.000 fungal
species are believed as natural contaminants of agricultural and products of
food.
There are general standard methods for determination of mycotoxins in food
including conventional methods and molecular methods. The conventional
methods such as culture and microscopic which are composed of culture-based
methods for detection and enumeration of fungi also determination and
identification of mycotoxins.The molecular methods including polymerase chain
reaction (PCR), pulsed-field gel electrophoresis(Yeni et al., 2014).

________________________________________________________________
3
1.2 The Aim of Study
1. Study the occurrence of mycoflora in poultry feed.
2.Determination of aflatoxigenic Aspergillus flavus which cause
contamination in poultry feed .
3. Compatible homology aflatoxigenic A.flavus strains with other strains in
gene bank.

Chapter two
Review of literatures

Chapter two Review of literatures
________________________________________________________________
4
2.1 Important mycotoxigenic fungi
Most filamentous fungi produce one or more mycotoxins (Demain and Fang
,2000) .Those of economic importance with respect to producing mycotoxins are
those belonging to the Fusarium, Aspergillus, Penicillium, Claviceps and Alternaria
, Although , one cannot forget the benefits derived from the use of fungi in the
food and pharmaceutical industry.
2.1.1 Aspergillus species
The Aspergillus spp. are filamentous and are among the most group of
microorganisms that are found in nature as in the soil, plant debris and indoor air
environments ((Myatt et al., 2008). The teleomorphic state has been described for
some of the Aspergillus spp. and others are without any known sexual spore
production. About 180 Aspergillus spp. exist (Klich, 2002) of which A. flavus and A.
parasiticus, are most widely studied because of their important role in AFs
production (Sánchez et al.,2005). Other spp. as A. sojae , A. oryzae and A.
awamori are economically important, they are used in industry as for enzymes
(amylase) and organic acids (citric acid) production or in the beverage and food
industries as flavourants and colorants (Klich, 2002). Several of these fungi act as
causative agents of opportunistic infections in human , animal and plant .They often
contaminate cereal grains, nuts and animal feeds . Within this genus, A. fumigatus is
the most commonly isolated spp., followed by A. niger and A. flavus (Eidi et al .,
2014) .

________________________________________________________________
5
This species of fungi are found mainly in tropical and subtropical regions and
their occurrence is more common than Penicillium (Samson et al., 2002). Among
this group , A .flavus and A. parasiticus are known to produce AFs (AFB1, AFB2,
AFG1 and AFG2), A. ochraceus, A. ostianus, A. sclerotiorum A. niger and A.
carbonarium have the ability to be producers of OTA and PA, while PAT was
produced mainly by A. clavatus and A. terreus., A. carneus and A. terreus are
known to produce CIT (Klich , 2002). In addition, several other mycotoxins such as
cytochalasin E, VER and GLIO produced by A. versicolor, A. fumigatus (Mwanza,
2011). A. flavus is widely distributed in nature and is largely found at cereal and
grains. Before harvest or during storage, A. flavus grows at agricultural crops (Saini
and Kaur, 2012). Its growth is affected by the environmental condition such as
temperature and relative humidity (Giorni et al., 2012).
2.1.2 Fusarium species
Fusarium is widely considered as an amorphic genus, and they are considered as
one of the most economically important genera of fungi common in tropical and
subtropical regions(Gräfenhan et al., 2011). Fusarium spp. are found in soil which
contaminate almost all plant spp.Some of them are pathogenic to human and
animals causing fusariosis in human , pulmonary edema in dairy cattle and necrotic
enteritis in poultry , other have to be plant pathogen and cause disease such as
crown and root rots (Marczuk et al ., 2012 ; Antonissen et al 2014) These species
include F. verticillioides , F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans
(Hsuan et al., 2011) .Most of these species are economically important as , F.

________________________________________________________________
6
verticillioides (F. moniliforme) which is known to produce FBs mycotoxins F.
graminearum is also an important Fusarium spp. known to produce zearalenone
(ZEA) and DON ,other Fusarium spp. of interest are F. proliferatum and F.
nygamai, also known to be producers of fumonisin mycotoxins such as
trichothecenes (Czembor et al., 2015).
2.1.3 Penicillium species
Penicillium spp. are widely found in soil, decaying vegetation and in the air and
food (Pitt and Hocking, 2009).Almost Penicillium spp. are looked to be
opportunistic saprophytes and many appear as habitat of primary natural on cereal
grains (Frisvad and Thrane, 2002).Several members such as P. digitatum, P.
expansum, P. italicum and P. roqueforti are pathogens on fruits capable of causing
food spoilage. Others may cause infections, particularly in immuno-compromised
hosts, as P. marneffei which is pathogenic particularly in patients with HIV-AIDS.
In addition, P. marneffei isolation from blood has been used as an HIV marker in
endemic areas (Pitt and Hocking, 2009). And also, some species act as poultry
pathogen and cause neurotoxic effect . Penicillium spp. are known to be producers
of OTA, CIT, PAT , PEN, RQ, PRT, PA and MPA in human , animals and
plants ( Bouhet and Oswald, 2005).
2.1.4 Other toxigenic fungi
Alternaria and Claviceps are the fungal genera of economic importance not only
recognized as plant pathogens (Dutton and Kinsey, 1996). The species A. alternata

________________________________________________________________
7
being the most common human pathogen . The common mycotoxins produced by
this genus are TZA, AAT and ALT ( Pose et al., 2010) .The fungus Claviceps
purpurea, is one of the most important and notorious fungi in human history (Alm,
2003). Claviceps purpurea is known to grow on the ears of rye and related cereal
and forage plants (Lev-Yadun et al., 2004) .
2.2 Mycotoxins
Mycotoxins are low molecular weight (Khayoon et al.,2014). Natural products
and toxic chemical secondary metabolites produced by filamentous fungi when they
grow under favorable conditions on foods and feeds (Aquino, 2011; Bolechová et
al.,2015; Cardoso et al.,2015). Chemical structure , they vary from simple C4
compounds, e.g., moniliformin, to complex substances such as the phomopsins
(O’Brien and Dietrich , 2005 ; Milićević et al., 2010 ) .
Mycotoxins resist decomposition or being broken down in digestion and
thermal stable , so they remain in the food chain even after heat treatment, such as
cooking and freezing (Milicevic,2009; Al-Kahtani ,2014; Czéh, 2014 ). There is no
reason of mycotoxins production known yet (Fox and Howlett , 2008 ; Suhaimi
et al., 2014 ), but they inhibit the physiological functioning of other organisms as
antibiotics which inhibit the growth of bacteria to provide a competitive advantage,
also may inhibit the growth of fungal species (Godish, 2001; Magan and Aldred
,2007). They do not have role in a normal metabolism containing growth and
development of the fungi (Mashinini, 2004;Keller et al.,2005 ; Al-Fakih, 2014) ,

________________________________________________________________
8
but they are produced after the fungus has completed its initial growth phase (Calvo
et al., 2002; Bhat et al., 2010 b).
Significantly , mycotoxins are non-volatile, therefore, they are non-airborne
except if they are attached with a particle and there are an aerosolization happening
,therefore , enough exposure through inhalation is uncommon (Fischer et al., 2000;
Jargot and Melin , 2013;Täubel and Hyvärinen, 2015) So, they remain in food or
feed products long after fungi have disappeared (Pscheidt and Ocamb, 2015).
Mycotoxins affect several agricultural products, including root crops ,cereals,
oilseeds, pulses, nuts, dried fruits, and coffee beans (Rohr et al ., 2015 ; McMullin
et al ., 2015; Stoev ,2015) . Contamination of agricultural products occurs because
of infection by toxigenic fungi under favorable environmental conditions in the field
at various stages in the food chain, e.g., pre-harvest, harvest, drying and storage
(Paterson and Lima,2010; Waliyar,2015). The presence of mycotoxins in feedstuffs
decrease the quality of feed in of both protein and value of energy (Pizzolitto et al.,
2013;Greco et al., 2014) ,therefore , they cause economic decline , an annual loss of
25percentage of the food production in world is rated to be lost because of spoilage
by mycotoxins. Additionally 5 - 10% of food losses can be attributed to fungal
spoilage (Pitt and Hocking, 2009; Wu, 2015).Annually, there are around 2.2 million
people died because of food contaminated with mycotoxins (WHO, 2013; Wu,
2015 ).
Fungi need moisture (relative >12%), oxygen as a minimum 1% - 2%, time and
temperature (changing depending on species; high temperature supports Aspergillus,

________________________________________________________________
9
low temperatures supports Fusarium) for growth (Magan et al.,2004; Pardo et al.
2005a,b ; Vieira et al ., 2015). Mycotoxins may get concentrated more in broken
grain than whole grain. (CAST, 2003; Chaytor et al. ,2011). Mycotoxins mainly
have synergistic effects, so the damage and spoilage caused by the combination is
more destructive (Ruiz et al ., 2011; Klarić et al., 2013 ; Li et al ., 2014). At low to
temperate levels, multiple mycotoxins can cause symptoms predominantly rather
than those associated with individual mycotoxins(Chaytor et al., 2011; Antonissen
et al ., 2014).The Immunosuppression is one of the serious outcomes of
contamination with mycotoxin, often is not noticed., making the bird or animal
susceptible to infection and problems of complex disease (CAST, 2003; Ramos et
al., 2010). Only about 100 of fungi are known to produce mycotoxin .There are
three main genera of fungi which produce mycotoxins: Aspergillus, Penicillium and
Fusarium (Dersjant-Li et al,. 2003; Gajęcka et al., 2011; Azaiez et al.,2014), the
global occurrence of them is considered to be a major risk factor, affecting human
and animal health. It is estimated that up to 25% of the world’s crop production is
contaminated to some extent by these toxigenic fungi (Larsen et al .,2004; Schmidt-
Heydt et al.,2011).Some fungal species are able to produce many mycotoxin , also
some mycotoxins are produced by more than one species (Pereyra et al., 2010;
Gutleb et al ., 2015).
Over 400 mycotoxins have been isolated and identified , but only a small
number of mycotoxins known to cause serious diseases in humans also animals
were studied (Kabak et al., 2006 ; Dzuman et al.,2015; Cao et al.,2015) . There are

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five major types of mycotoxins which infect human , animals health and
agricultural significance: (i) aflatoxins, (ii) fumonisins, (iii) ochratoxin A, (iv)
zearalenone and (v) trichothecenes (Marroquín-Cardona et al.,2014; Njumbe Ediage
et al., 2015; Degraeve et al., 2015). When mycotoxins ingested by animals or
humans, mycotoxins cause a toxic response known as mycotoxicosis (Richard and
Payne,2003; Pruimboom et al.,2014 ; Armenda´riz et al., 2014) , and cause acute
and chronic disease and effects on humans and domesticated animals as acute toxic,
carcinogenic, mutagenic, teratogenic and oestrogenic effects at the levels of
exposure (CAST, 2003; Gomaa et al., 2008 ;Cortinovis et al., 2013; Wcislo and
Szarlej-Wcislo 2015 ; Bennett and Moore ,2015) .
The presence of excessive mycotoxins can cause grain shipments to be
rejected by importing countries resulting in a loss in consumer confidence in the
importing country and severe economic losses for the exporting country. A main
possibility danger of mycotoxins is in the human diet, therefore, resides in the
disability in detection of them biologically( CAST,2003 ; Dohlman, 2003; Calvet et
al ., 2015). Mycotoxins can be classified into four kinds of toxicity: as hepatotoxins,
nephrotoxins, , immunotoxins and neurotoxins. Cell biologists place them in generic
toxins such as mutagens ,teratogens, carcinogens, and allergens (Omar, 2013;
Sorrenti et al., 2013). Kidney and liver function deterioration has been described as
the most common effect of acute mycotoxin poisoning, that were in excessive cases
may cause death (Voss et al .,2001; Zomborszky-Kovacs et al., 2002 ; Wild and
Montesano, 2009).

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11
Some of mycotoxins can interfere with synthesis of protein, and result in effects
fluctuating from sensitivity of skin or necrosis to extreme immunodeficiency
(CAST,2003 ; Li et al., 2011; Ferreras et al., 2013) . Others are neurotoxins, which
at low doses, can lead to continuous quiver in animals at high doses result in a brain
damage or death (Pitt , 2000; CAST,2003).
The primary effect of chronic of many mycotoxins is the induction of cancer,
particularly of the liver (O’Brien and Dietrich, 2005;Groopman et al., 2008; Ouko,
2014). Some mycotoxins inhibit DNA and RNA replication through impairment of
amino acid transport and m-RNA transportation cause antibody production in lower
level, and hence can cause mutagenic or teratogenic effects (Egner et al., 2001;Surai
et al., 2008; Liu et al., 2014; Hedayati et al.,2014).
2.3 Mycotoxin occurrence
Mycotoxin occurs in products of agricultural raw, processing foods, and
products of animal like meat, eggs and milk(CAST,2003; Streit et al., 2012).
Approximately 25% of crops in the world are affected by mycotoxins with variable
level annually (Lawlor and Lynch , 2005; Bhat et al., 2010b).
Mycotoxins contamination of samples feeds are six types: aflatoxin B1, ZEA,
DON, fumonisin, T-2 toxin and OTA ( Škrinjar et al .,2011 ; Rodrigues and Chin ,
2012). Feed of products obtain from production of ethanol can be contaminated up
to three times with mycotoxins than the main grain which product is derived from it,
because of removing of starch and mycotoxins would be concentrated in the
leftover(Zhang et al., 2009;Rodrigues and Chin,2012).Occurrence of mycotoxins

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12
and concentrations are changeable annually and related with changeable in
conditions of weather and stresses of plant (Coulumbe, 1993; Whitlow and
Hagler,2008).
Occurrence of mycotoxins is less frequent at greatly concentrations can be
caused instant and exciting damages in health and activities of animal, although they
happen considerably in a different feeds and in animals feeds (Danicke,
2002;Whitlow et al., 2010 ).
2.4 Factors influencing growth of fungi and production of mycotoxin
Growth of fungi is mainly dependent on several factors within the environment.
However, the moisture is very important for surviving them, but other
circumstances equally affect the development and mycotoxin synthesis (Atalla et al.
, 2003; Essono et al. ,2009) . The conditions production of mycotoxin are ordinarily
more finite than are the normal growth conditions of fungi (Magan et al., 2014).
Fungal colonization, development and subsequent mycotoxins production in foods
and feeds depend on different variables, which can be classed as abiotic and biotic
factors (Marin et al.,2012 ; Milani, 2013) .
The most important abiotic factor (environmental factors) are water activity
(aw) of the substrate and temperature that in combination provide ideal cases for
growth of fungi and production of mycotoxin (Guo et al., 2005). Other abiotic
factors are the gaseous composition of the surrounding atmosphere and pH of the
substrate. Respiration, insects and mites as well as competitions with other fungal
species and other microorganism are the most important biotic factors that

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13
influence fungal growth on food (Yu, 2012). Also, studies have shown that
interactions and combination between these factors influence the predominance of
fungi, particularly mycotoxigenic spp. (De la Campa et al., 2005).
Fungi foray only a secondary part of a commodity in which convenient
conditions for a growth exist (Murphy et al., 2006). CO2,O2 and genetic
characteristics also influence the production of mycotoxins and may obviously
differ from those for fungal growth (Atalla et al. , 2003). The optimal conditions of
mycotoxins production by some important fungi have been listed in table(1)
according to Murphy et al. (2006) , who showed that most mycotoxins are
produced under temperatures varying between 0-33C° and aw between 0.93-0.99. A
relationship that exists between these factors and fungal growth (Sautour, 2001a, b;
Astoreca et al., 2009).
The influence of abiotic factors on growth of fungi and mycotoxin production
can be an important consideration in expectation fungal contamination of foods both
in the field and during storage(Paterson and Lima, 2010 ; Lee et al., 2015). Food
and feed materials vary in their capability to assist growth of fungi because of their
variation in chemical and physical properties, as aw, O2 availability and surface
area (Tirado et al., 2010). Also other factors such chemical constituents (nutrient
composition) as fat, carbohydrates, protein and trace elements that support this
growth and production (Zaki et al., 2012).

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14
Table (1) Optimal conditions for fungal growth and mycotoxin production
Mycotoxigenic fungi
spp.
Mycotoxins
Temp
(°C)
Aw Reference
Aspergilluskflavus,
A.parasiticus
aflatoxins 33 0.99
Hill and others 1985
A. ochraceus. A. niger ochratoxin 31-37 0.98 Ramos and others 1998
A. carbonarius ochratoxin 15-20 0.85-0.90 Cairns and others 2003
Fusarium verticillioides fumonisin 10-30 0.93 Mitchell and others2003
F. proliferatum fumonisin 10-30 0.93 Marin and others 1999
F. proliferatum deoxynivalenol 11 0.90 Hope and Magan 2003
F. graminearum zearalenone 25-30 0.98 Sanchis 2004
Penicillium expansum patulin 0-25 0.95-0.99 Sanchis 2004
2.4.1 Abiotic factors
Abiotic factors are not living compositions environmental(chemical and physical)
factors. Abiotic factors are classed to four general types: physiographic factors
(topography and location); climatologic factors like light, temperature, air pressure ,
wind, , humidity and rainfall ; edaphic factors: soil composition such as sand, clay,
loam , mineral salts , pH of soil, and trace elements, water-holding capacity
(Mandeel, 2002 ; Cardwell and Henry, 2004) and gases factors such as vapour,
oxygen, carbon dioxide and nitrogen (Mwanza, 2011).

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15
2.4.1.1 Water activity (aw)
It is the pressure of vapor of food at same temperature and external
pressure(Chirife and Fontana, 2007) , and is unlinked water measurement in the
food obtainable for the growth of fungi (Zaki et al., 2012). In low aw , the fungi
ability to survive and adapt to environments varies from one species to
another(Leong et al., 2011). The required aw for fungal growth is between 0.61 and
0.91, and most storage fungi grow at aw below 0.75 (De Camargo et al., 2012;
Juneja et al ., 2012).
Water activity is affected by various small molecular and soluble compounds in
food. In case of spore formers, decrease of aw extends lag phase of growth and
extends the time to toxin production ,decreases growth rate, and reduces the
densities of maximum population (Juneja et al ., 2012).
The lowest aw for growth is 0.61 and below this value the spoilage of foods
cannot be fungal it may be insect damage or chemical (Adams and Moss, 2000).
Other factors such as temperature, pH, acid and nutrients can also interact with aw
and either inhibit or support fungal growth (CAST, 2003). Fusarium grows best at
higher aw of 0.98 whereas Penicillium and Aspergillus grow best at aw of 0.95,
(Choi et al.,2015). High moisture determines the extent of mycotoxin contamination
in stored food and feed and in the field and at storage (Hell et al., 2008) .
Aflatoxin production was highest at 0.98 and 0.95 aw at 25°C, ZEA at 0.98 aw
at 25°C and 0.95 aw at 16°C, the production of OTA was best at 25°C (Bhat et al.,

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16
2010 a). Thus, aw is very important for fungal growth and mycotoxins production.
2.4.1.2 Temperature
Temperature is an important factor for fungal growth and mycotoxin production
in food and feeds (Paterson and Lima,2010) . The role of temperature in the survival
of fungi by its influence on functioning of membrane-localized transporters and
enzymes activates the cell by effect on fatty acids incorporated into phospholipids in
the membrane (Maheshwari et al., 2000) .
The values of temperature are vary from one species of fungi to another , the
optimal temperature for production of most mycotoxins varies between 25-33°C
depending on the fungus and the type of mycotoxins they produce (Pitt and
Hocking, 2009). Aspergillus spp. require a narrower temperature range 15-40 C° for
growth and Penicillium spp. 25-30 C° , whereas the optimal temperature range of
37-47 C° for most Aspergillus and 28-30°C is ideal for Penicillium. Conversely,
Fusarium spp. can be considered as psychrophilic, because of its ability of growth
and reproduction in very low temperatures (Robinson, 2001; Francisco and Usberti,
2008).The suitable temperature of production of AFBs by A. flavus and A.
parasiticus occurred between 24-28 C° with optimum production at 25 C°, while F.
verticillioides have an best growth between 25 and 30°C (Marın et al., 2010). The
maximum production of OTA by A. ochraceus was around 30°C (Soso et al., 2012).
2.4.1.3 Hydrogen ion potential (pH)
The pH is a measure of the alkalinity or acidity of a substrate and expressed as
the negative logarithm of the H ion (H+) concentration . Food and feed materials

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17
vary with respect to their pH values and will host different fungal spp. and these
organisms have specific pH requirement for growth. There are a competition
between fungi and bacteria as food spoilers at high pH, but at lower pH, fungi can
out compete with most bacteria, and most of these fungi are less affected at pH
values, commonly 3 and 8 , other fungi can be grown at pH less than 2, (Pitt and
Hocking, 2009). However, the medium pH act as an necessary control over an
obtained morphogenic factors such as color, smell and density without affecting the
overall growth of some fungi(Zaki et al., 2012).
The H ion concentration is an important element in the mycotoxins production
, and it can influence the three dimensional structure of proteins, including the
enzymes , then affect on the cellular metabolism, the transport of nutrients and the
electrons transfer (Cojocaru, 2007),so the optimum pH for AF production by
Aspergillus spp. is between 3.5 and 8.0 (Oseni, 2011) while OTA production by A.
ochraceus is at minimum pH of 2.2 (Soso et al ., 2012) and for FB1 production by
F. verticillioides is at pH of 7.5 (Rao et al., 2010).
2.4.1.4 Oxygen supply
The most important and necessary element required for fungal growth is O2,
but also under anaerobic conditions , certain species can grow with the ethanol and
organic acids formation (Deacon , 2006). Almost fungi require 1-2% O2 in growing
(Forristal et al., 2000), while mycotoxins production influenced by the presence or
absence of O2 in the environment (Deacon , 2006). The growth of most Aspergillus
spp. is restricted at an O2 concentration of less than 1% ( Valero et al ., 2008),

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18
except F.moniliforme , it is able to grow at 60% CO2 and less than 0.5 % O2
(Bakutis et al ., 2006).
Penicillium roqueforti have the lowest growth requirement of oxygen than other
Penicillium species(Deacon , 2006).Production of FB1 under O2-limited conditions,
less growth occurred, while glucose consumption was increased with no FB1 being
produced. The entrance of oxygen will allow the growth of fungi , resulting losses in
the silage nutrient (McNamara et al., 2002).
2.4.1.5 Carbon dioxide
All fungi need carbon dioxide in small amounts to generate fatty acids,
oxaloacetate, etc. Anaerobic fungi grow in conditions often have a high CO2
requirement, whereas several aerobic fungi can be grown by high concentrations of
CO2(Deacon , 2006 ).Slightly increased CO2 concentrations ,in addition to elevated
temperature and water providing may induce some mycotoxigenic fungi growth,
particularly with stress of water (Magan et al.,2011).
2.4.2 Biotic factors
They are an important factors can influence fungal growth and mycotoxins
production , and are mainly living organisms effect on the growth, structure, and
composition of the fungi and mycotoxins (Magan and Aldered, 2007). Filamentous
fungi respond to numerous biotic signals that come from other organisms (fungi,
bacteria, animals and plants) in the natural environment . Other organisms can
produce a variety of physical and chemical signals, which influence the growth,
behavior, metabolism and gene expression of filamentous fungi. Physical and

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19
chemical signals from plant or animal hosts can assist the invasion and penetration
of hosts by fungi (Gow, 2004; Lucas, 2004).
Insect pests are among common biotic factor , causes problems in grains and
grow and multiply at water availableness much drier than those at which fungal
growth in grain (Magan et al., 2004). They can generate heat by metabolism of
organic material to generate water and can condense on grain surfaces due to
temperature differentials to induce fungal growth and grain spoilage (Magan et al.,
2004).
Post-harvest production of aflatoxin in maize increase by pre-harvest insect
infection as damage to the host plant, susceptible crop growth stages, poor soil
fertility, high crop density, and weed competition (Bruns, 2003) .Some storage
insects are provider of storage fungi by carrying the spores (Magan et al., 2004).
2.5 Regulation of mycotoxin biosynthetic genes cluster
Mycotoxin producer fungi have complex genomes, with species variations
predominantly in regulation of genes of the secondary metabolites synthesis, as
mycotoxins (Moretti et al ., 2013). The pathways of mycotoxin biosynthetic and the
producing fungi identification have relation with health interest that associated with
mycotoxin contamination (Wang and Tang, 2005).The genes responsible for
biosynthesis of mycotoxin are often clustered (Bhatnagar et al., 2006) that can
occupy several kilo-bases on the genome (Alkhayyat and Yu, 2014). At least one
pathway-specific (TF) in each mycotoxin gene cluster is often contained that
regulates genes within the cluster. AflR gene is the most studied which regulates AF

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20
biosynthetic genes in Aspergillus flavus and A. parasiticus and (ST) gene cluster in
A. nidulans (Ehrlich et al., 2005) , while FUM genes cluster regulate fumonisin
biosynthesis in Fusarium verticillioides (Brown et al., 2007) .
Many gene clusters contain one or several enzymes that belong to PKSs or
(NRPSs) family act as colossal multimodular enzymes which facilitate the structure
of the main scaffold of many secondary metabolites . These enzymes encoding by
key structural genes(Brodhun and Feussner, 2011). Other enzymes can provide
various modifications to the original structure (Karlovsky, 2011).
2.6 Pre- and postharvest of mycotoxin contamination
ontamination with mycotoxin is a collective process which starting in the
field , increasing during harvest, drying, and storage Mej a-Teniente et al ., 2011).
Fungi which colonize grain classed into two groups: field fungi and storage fungi.
Field fungi affect the seeds before the harvest ,while the crop is found in the field
and need high conditions of moisture about 20-21% to grow, but the storage fungi
are fungi which invade grains or seeds during storage and could grow at moisture
contents in equilibrium with relative humidity of 70 to 90% (Bakutis et al ., 2006).
By this dividing, Alternaria, Cladosporium, Fusarium, and Helminthosporium were
classified as field fungi; Aspergillus and Penicillium were dividing as storage fungi.
Even in climates of temperate when the growth season is often dry and hot,
Aspergillus and Penicillium species can invade seeds in the field (CAST,2003).
Aspergillus flavus is an important species which affect seeds in the field and storage
(Horn, 2005) .

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21
Fungus is predominately a storage fungus in temperate climates. Many
Fusarium species as well as some Penicillium species, effect grain in the field and
in storage (Atanda et al., 2011) . Many mycotoxigenic fungi grow in saprophytic
form , and these fungi may be contaminate grain by contact with soil or debris of
plant. Inoculum in field with affected seeds would be transmit to other kernels in
storage (CAST,2003).
Mycotoxin contamination widespread during cultivation, harvest, drying,
storage, transit and distribution. Preharvest infection by A. flavus is the major cause
of aflatoxin contamination in peanut. Mycotoxins have been contaminated a wide
range of commodities with both pre- and post-harvest ,this concerns both farmers
and consumers (Milicevic et al ., 2010) . The fungi species contaminant pre-harvest
crops are Fusarium, Alternaria and Aspergillus, while post-harvest infection fungi
species is most often caused by Penicillium roqueforti, P. paneum, Zygomycetes,
Aspergillus fumigatus, Byssochlamys nivea and a few other fungi(Storm et al .,
2014).
2.7 Mycotoxin exposure , mechanisms of action and effect
Growth of mycotoxin concerns occur increasingly because of their effect on
human , animal health and plant. There are four main routes of mycotoxins
exposures in human and animals: ingestion, inhalation, dermal and parental routes.
The main route of exposure to mycotoxins is ingestion, which come from
consuming of mycotoxin contaminated food or feed, while inhalation or bioaerosol
route is more severe route than others , by which airborne mycotoxins can be

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22
inhaled during the breathing process. In case of dermal route is exposure of the skin
to mycotoxins , via handling material contaminated with mycotoxins (Zain , 2011).
Parental exposure is transmission of mycotoxin from a mother to child through the
placenta or during breast feeding (Njobeh et al.,2010).
There are many factors affecting the toxicity quantity of human or animal
consuming foods or feeds contaminated with mycotoxin, they include species,
action mechanisms/modes, metabolism, and defense mechanisms(Hussein and
Brasel , 2001) .
Direct effect of mycotoxin range from acute to chronic disease . At acute ,
severe conditions as poisoning of liver or kidney function may lead to health as a
result of exposure to high levels of a mycotoxin (Pitt, 2000) . Chronic conditions
have a much greater impact , immunosupression and cancer are chronic effects ,
reduced growth and development, that have a greater happening continual exposure
to minimum level ingestion of mycotoxin (Bryden, 2007).
Other conditions(e.g., growth retardation, impaired immunity, immunosupression
and cancer are chronic effects, reduced milk or egg production) or more chronic
manifestations of disease (e.g., formation of tumor) because of protracted exposure
to small quantities of toxin. The exposure at low level is concern where food and
feeds are in a better quality. When the mycotoxin effected the contaminated foods
is aflatoxin , The diseases caused called aflatoxicoses (CAST, 2003) .

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23
Combined effect of more mycotoxins or with bacterial toxins is very hard to
predict than the effect of a single mycotoxin because it is influenced by several
factors, including chemistry and mechanism of action, toxicodinamics and
toxicokinetics as synergistic effect (Šegvić Klarić, 2012) . The combination of DAS
with AF suggesting a synergistic effect and have been confirmed as acutely and
fatally hepatotoxic and nephrotoxic (Zain , 2011) . Increasing of mortality can be
occur partly by the synergistic effects of mycotoxin combination with bacterial toxin
, (LPS) and T-2 at the late phase of murine salmonellosis. Also Salmonella
Typhimurium, DON with Salmonella Enteritidis decreased the resistance to oral
infection in mice by promoting translocation of Salmonella to MLN, spleen and
liver (Antonissen et al ., 2014).
Mycotoxicoses are diseases caused by mycotoxins , occur by ingestion routes
, also dermal and inhalation routes, range from tumor formation to rapid death, and
related with feed or food, considered as non-transferable ,non-contagious , non-
infectious, and non-traceable to microorganisms other than fungi mycotoxins (Zain ,
2011). Several commodities may be contaminated with mycotoxins in both pre-
and post-harvest ( CAST, 2003) .
More obscure disease happened after interferes of mycotoxin occur with
immune system , a high susceptible of performance of the patient take place to
infectious diseases.A fundamental event of mycotoxin can be increased by the
infectious disease.Epidemiological,clinical and histological results in mycotoxicoses
outbreaks coming from exposure to aflatoxins, ergot, ochratoxins ,trichothecenes, 3-

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24
nitropropionic acid, zearalenone and fumonisins(Bennett and Moore , 2015). A
wide range of actions of mycotoxins on animals and humans to include cytotoxic,
nephrotoxic and neurotoxic, carcinogenic, mutagenic, immunosuppressive and
oestrogenic effects have been characterized (Krska et al., 2007).
2.7.1 Effects of mycotoxin on human
Mycotoxins related with a number diseases of human.Their effects and
symptoms will equally vary significantly ,although , mycotoxins have highly
variable structural chemistry and different toxicological properties, (Njobeh et al.,
2010).Mycotoxins effects include toxigenic activities in sensitive species which
include carcinogenicity, immunosuppression, protein synthesis inhibition, dermal
irritation, and other metabolic perturbations depending on the type of toxin duration
and amount of exposure(Turner et al., 2003).
The synergistic effect of mycotoxin exposure with some important diseases
such as kwashiorkor, tuberculose malaria and HIV/AIDS have been recorded
(Turner et al., 2003; Gong et al., 2004).The important role of mycotoxicosis is in
suppression of immune in populations of human , this exposure was associated with
decreased levels of secretory IgA (Turner et al., 2003).
2.7.2 Effects of mycotoxin on animals
Contamination of mycotoxins in cereals and related products used in animal
feeds production may cause poisoning , particularly in farm animals (Krska et al.,
2007), causing loss of animals and equally reduces the economic output of the

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25
farm enterprise. Ruminants (cattle, sheep and goats) are less sensitive to mycotoxins
than non-ruminants (Hussein and Brasel, 2001).When the animals consume high to
moderate amounts of mycotoxins, this may induce acute mycotoxicosis leading to
different clinical manifestations of diseases including hepatitis, haemorrhage,
nephritis, oral necrosis, epithelial cells enteric and even death (Rohit Talwani et al
.,2011). In the case of mycotoxicosis in animals, symptoms may include poor milk
production, poor feed consumption, poor body weight gain and diarrhoea, anoestrus,
poor reproductive performance, abortion, feed refusal, vomiting ,high disease
incidence and general lethargy (CAST , 2003).
Poultry are the most susceptible to mycotoxins contamination than ruminants ,
and they are more resistant to FBs than are equines and swine, but to induce
measurable effects , doses should be high as 450 and 525 ppm of FBs for 21 days in
feed , this cause weight gains (Whitlow et al., 2010). Aflatoxicoses made great
economic losses in the poultry industry, affecting broilers,ducklings, layers, quail
and turkeys causing clinical signs include anorexia, decreasing in weight gain,
decreasing in egg production, hemorrhage, embryotoxicity, and increasing in
susceptibility to environmental and microbial stressors (CAST ,2003 and Cegielska-
Radziejewska et al.,2013).
At high level (1.5 ppm) of dietary aflatoxin in chicken , histopathologic effects
occur as fatty liver, necrosis and hyperplasia of bile duct (Dhanasekaran et al.,
2011). Aflatoxicoses also contribute in decreased activities of pancreatic amylase,
trypsin, lipase, RNAse, and DNAse when exposed to 1.25 and 2.5 mg AFB1/kg

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26
diet (Yunus et al., 2011). In contaminated corn with aflatoxin near LD50 levels
cause decreasing of egg production to 5% in laying hens, also decreasing the
percentage of total yolk weight from total egg weight (CAST,2003). The exposure
to lower levels of OTA in poultry may reduce consumption of feed in addition to
weight gain and immunosuppression which increase susceptibility to infection
(Murugesan et al., 2015).
2.7.3 Effects of mycotoxin (phytotoxin) on plant
Phytotoxins are fungal metabolites which are toxic to plants (Lou et al., 2013) .
They act as a pathogenic or virulence factors, cause a plant disease or they can play
a role in increasing various plant diseases. The phytotoxins made by fungal
pathogens as Alternaria, Aspergillus, Fusarium, and Penicillium are the most
common fungi of contamination of crop plants with mycotoxin, they infect many
different field crops including wheat, maize, rice, barley as well as peanuts, tree
nuts, coffee, grapes, and cotton (Amalfitano et al., 2002; Horbach et al.,2011). They
can lead to a high range of diseases of plant as stalk rots , crown and rots of root
(Stergiopoulos et al ., 2012).
Aflatoxins occurrence in agricultural raw materials depends on factors such as
season ,region and the conditions under which an appointed crop is grown,
harvested or stored. The harvests of crop plants are endangered because of plant
diseases lead to losses of at least 10% of the harvest in the world ( Endah ,2011).
Fungal Infection to plant pathogens occurs via several pathways such as seeds,
stems, roots, flower and fruit (CAST,2003). Most phytotoxins are organic acids,

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27
polyketides , cyclic polypeptides and cyclic terpenoids (Pusztahelyi et al., 2015).
Phytotoxins differ in the way they act but the main effects of their activities are
damage to the cell membrane as well as abnormalities and biochemical changes in
plant cells. Generally , effects of phytotoxins are wilting and growth suppression, as
well as spotting of aerial portions (Andolfi et al, 2011). Phytotoxins are divided
into host-specific and non-host-specific.At low concentrations , host-specific toxins
act as pathogenicity factors ,while non-host-specific phytotoxins act as virulence
factors (Taj et al .,2015) .
2.8 Methods to detect mycotoxigenic fungi
The detection and quantification of mycotoxins and mycotoxigenic fungi in
food and feed are preprocessed for the safe foods production. Rapid methods of
theses fungi presence within (HACCP) systems take suitable corrective actions to
avoid and prevent risks of mycotoxin accumulation in foods , thus prevent
economic damages such as resulting of removing of foods contaminated with
mycotoxins.These strategy can be dividing into which depending on description of
fungi by conventional strategy, analytical methods and molecular techniques
(Galaverna et al., 2009; Berthiller et al., 2013).
2.8.1 Detection by conventional microbiological methods
This method includes processes comprised of sampling, culture, isolation, and
characterization of production of mycotoxin which including extraction of toxin,

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28
procedures of cleanup and at end mycotoxins analysis qualitatively or quantitatively
(Yeni et al., 2014) .
2.8.2 Analytical detection of mycotoxin production
The method in which the procedure of sampling is the largest source of
mycotoxin variance test and the generality definitive step to obtain trustworthy
results (Köppen et al., 2010).There are several factors determine the of preparing a
representative sample difficulty such as a size of particle or the number of particles
in the mass or culture media complication or food models.(Whitaker et al.,
2009).These factors do it unattainable to determinate all mycotoxins. ( Shephard et
al., 2013).
2.8.2.1 Mycotoxins analysis by methods of chromatograph
After using cleanup or pretreatment methods, mycotoxins should be metabolites
free which may be interfere in their analysis (Cheli et al., 2012) .
2.8.2.1.1 TLC
It is a rapid and low-cost analytical technique and by visual inspection , it
offers capability to screen of yielding qualitative samples as a large numbers or semi
quantitative assessments(Cigic and Prosen, 2009;Turner et al., 2009).It is applied
for separation by screening , purity estimation and identification of mycotoxin.
2.8.2.1.2 HPLC
These methods are depend on coupled detector based on HPLC. The most
generally detection methods are DA ,UV, FL, MS, and MS/MS. The method of
HPLC-MS/MS is used for the simultaneous determination of different chemical

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29
families .The recent UHPLC technique has provided more features and adventages
in the mycotoxins determination in food by applying of columns stuffed with 2 μm
particles, to get tight peaks differs from which obtained by traditional HPLC
columns (Beltrán et al., 2013).New techniques of LC/ESI-QTOF-MS/MS give a
great sample throughput, great resolution, to obtained complete range data of
spectral mass as an alternative of only a single ion. This look as active instrument
for the detection of mycotoxins in foods, as aflatoxins (Sirhan et al., 2013).
2.8.2.1.3 GC
A method used to analysis only the thermally stable and volatile products.
Several mycotoxins do not volatile compound ,but they are to be derivative and
would be analyzed by GC (Cigic and Prosen, 2009). This method is joined to FID,
MS or FTIR detection techniques.
2.8.2.1.4 CE
It allows separation of a many mycotoxins, but it is not depended as HPLC. CE
has a serious problem and is performing a low limits detection , so the detection of
mycotoxins with this method have been developed by using fluorescence detection
(Cigic and Prosen, 2009(.
2.8.2.2 Immunological methods for analysis of mycotoxins
It is performed by the capability of a specific antibody to characterize the
structure-three dimensional of specific mycotoxin from among molecules. It is

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30
important before immunization , bind mycotoxins to a carrier protein because they
do not have immunogenicity and low-molecular substances , this achieved by
reaction results a specific monoclonal and polyclonal antibodies against the toxin,
therefore, just commercial kits can be developed for specific mycotoxins.These
method are rapid as the results are gotten within two hour to a few minutes , they
are used for a single requirements, which can raise the screening cost.
2.8.2.2.1 ELISA
Commercially kits for the detection of mycotoxins depend on a direct
competitive assay of primary antibody which consider as specific for the target
molecule or a link of an enzyme and the desired target.The formed compound react
with a chromogenic substrate to obtain commensurable results in one to two hour
(Turner et al., 2009).
2.8.3 Cultural methods
2.8.3.1 Blue fluorescence
This method is using for developing qualitative cultural methods for
aflatoxigenic Aspergillus species detection which grown on appropriate media. This
techniques use either solid media, such as CAM and PDA or liquid media, like
APA medium and a medium supplied with steep liquor (Abbas et al., 2004(a,b)
and Atanda et al., 2005), and achieved by cut a small plugs from Aspergillus
colonies on medium to culture on the other media.The aflatoxins producer
Aspergillus were detected under long-wave UV light (365nm)This rapid

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31
identification to determine aflatoxigenic isolates from non-aflatoxigenic by appear
blue to blue –green fluorescent to aflatoxigenic,and nonaflatoxigenic is non-produce
fluorescent (Rodrigues et al., 2007).
2.8.3.2 Ammonium hydroxide vapor-induced color change
A rapid and sensitive method for detection of aflatoxigenic and nontoxigenic
strains of Aspergillus (Yazdani et al., 2010). A single colony was grown in the
center of Petri dish. The reverse of colony of aflatoxigenic Aspergillus strain turned
to pink color when their medium were exposed to ammonia vapor by dropped of
ammonia hydroxide on it but nonaflatoxigenic is no color production( Saito and
Machida , 1999 ).
2.8.4 Molecular detection of mycotoxin-producing molds
The alternative technique used to detect genes by involved in the metabolites
biosynthesis by nucleic acid–based methods and give a characterization of mold
plus mycotoxins evaluation for mycotoxin-producing molds detection in
foods(CAST,2003). PCR-based techniques are among these methods.Recently,new
molecular techniques, such LAMP is used as example of this method (Notomi et
al., 2000).
2.8.4.1 Polymerase Chain Reaction(PCR)
It is a sensitive and specific technique using for early toxigenic molds detection
to control or reduce mass of mold (Dao et al., 2005 ( . As result of its specificity

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32
and sensitivity , PCR is an most suitable technique for identification of fungi.(Atkins
and Clark, 2004).The variety in ITS sequences applies for PCR-based tests
development for detection of many species of phytopathogenic fungi (Hussain et
al., 2014). This method with specific primers of fungi is a potent technique in
diagnostics and in ecological reports for fungi screening in environments, like soil,
water, plant samples.A biomolecular technique (PCR) applied by using a set of
primers of aflatoxigenic genes (aflR, aflD and aflM) to distinguish between
aflatoxigenic strains and non-aflatoxigenic strains of A. flavus and A. parasiticus
contaminating food and feed (Criseo et al., 2001). Recently this test has been
advanced to detect the quantity of Fusarium producing trichothecene, depending on
primers came from from Tri5 gene which can encode synthase gene of trichodiene
(Edwards et al., 2001).
2.8.4.2 Real-Time PCR
Technique used to measure the amplified PCR product at each cycle throughout
the PCR reaction. Real-time quantitative PCR is considered as the most sensitive
and reproducible form of PCR-based quantification and assists in the continuous
collection of fluorescent signal from one or more polymerase chain reactions over a
range of cycles(Bernard and Wittwer,2000).The increase in the amplifiers number
during PCR is amplification based on the proportional increase in fluorescence
intensity can be detected by Real-time PCR machines. By these tools ,any change in
amplified product is indicated in a change in the fluorescence intensity measured .
In the early cycles of PCR, there is no detectable signal and the amplification is

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33
below the level of detection of the real time instrument.This knows the baseline for
the amplification plot. A raise in fluorescence above the baseline give the
accumulated PCR product detection.A fixed fluorescence onset can be adjust above
the baseline.The parameter beginning cycle is defined as the number of fractional
cycle at which the fluorescence passes the fixed beginning (Valones et al., 2009) .
2.8.4.3 Future perspectives: new molecular methods
New techniques are defined as new molecular procedures for mycotoxin-
producing molds detection. An alternative technique for PCR LAMP of DNA is
using in testing of food safety and which is utilized RNA and DNA amplification
with isothermal situations .This technique is using DNA polymerase in addition of
a set of four specific primers which distinguish an aggregate of six sequence
specificity on the target DNA (Notomi et al., 2000).The characteristics of this
method in which the reaction time is shorter, without requirements for certain
instruments, great specificity and sensitivity, also relatively less susceptibility to
inhibitors which found in feed and food, then detection of pathogens, and shorter in
analysis and preparation time (Niessen et al., 2013).This method is special for
several mycotoxin-producer Aspergillus and Fusarium species food detection.
2.9 Main mycotoxins
2.9.1 Aflatoxins
Aflatoxins are difuranocumarin derivatives. They are classified to six main
toxins (figure 1) , based on their fluorescent features(blue or green) under UV of

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34
length wave 365 nm, and their chromatographic mobility(1 or 2) : B1, B2 (blue ),
G1, and G2 (green) , M1 and M2 a derivative of AFB1 monohydroxylated,present
in the milk of lactating animals by formation and excretion (Yao et al., 2015).AFs
are somewhat soluble in water about 10 to30 μg/ml, and they are in insolubility
form in solvents with non-polarity , while soluble in middling polar organic
solvents like chloroform and methanol ,but they have highly solubility in dimethyl
sulfoxide (Bertuzzi et al., 2012). Under the influence of ultraviolet light they are
unstable and with presence of O2, to severe of pH (< 3, > 10) and to oxidizing
agents (Khalil et al., 2013).
A group of aspergilli: A. flavus, A.parasiticus, and A.nomius strains Aflatoxins
are the main source of aflatoxin production(Moss,2002).Species as A.bombycis, ,
A.pseudotamari and A.ochraceoroseus can also producing aflatoxin, while they are
present in less considerably(Ito et al ., 2001). Aflatoxin cause a problem to several
commodities ,and AFB1 act as mutagenicity ,carcinogenicity and acute
toxicology.The IARC classified it as a human carcinogen .

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35
Figure (1): Chemical structure of the different aflatoxins(C17H12O6 of AFB1)
2.9.1.1 Exposure and absorption into organism
Aflatoxins are general incident in feeds, foods and milk products, so they act
as a dangerous threat to humans and animal(Zain,2011). Main contamination means
is oral route, also inhalation which occurs when people or animals have beeng
exposed to the dust of grains (Nuntharatanapong et al., 2001).During respiratory
exposure, AfB1 may occur in the blood more rapidly than after oral exposure. After
ingestion, Af B1 is well absorbed in the intestinal tract, and major site of absorption
is duodenum. Because of the particle with low molecular weight, the essential
mechanism of mycotoxin absorption is passive diffusion(Silvia, 2007).

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36
2.9.1.2 Metabolism of aflatoxin
Liver is the general metabolizing organ for aflatoxin, this also can be happen
directly in the blood or in many extra-hepatic organs. AfB1metabolism can be
divided into 3 phases( Yiannikouris and Jouany, 2002) :
1) Bioactivation.
2) Conjugation.
3) Deconjugation.
2.9.1.2.1 Bioactivation
In which AfB1 is oxidized into several hydroxylated metabolites. The pathways
of metabolites for AfB1 contain O-demethylation to AfP1(figure 2), reduction to
aflatoxicol and hydroxylation to AfB1-8,9-epoxide which is considered as acutely
toxic, mutagenic and carcinogenic, AfM1 (acutely toxic) and AfFQ1 or AfB2 ,both
somewhat non-toxic ( Yiannikouris and Jouany, 2002) .

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37
Figure (2) : Aflatoxin B1 pathways
Aflatoxin B1- 8,9 epoxide is greatly unstable, hence many reactions can happen,
according on the presence of the second molecule:
Biological nucleophils as nucleic acids – stable links to RNA and DNA are
resulted, inducing mutations of point and breaking of strands of DNA. These
reactions and the formation of AfB1-DNA adducts are highly correlated with the
AfB1carcinogenic effect in human and animal cancer. When water molecules
present, Aflatoxin B1– 8,9 epoxide will be hydrolyzed into AfB1– 8,9- dihydrodiol

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38
and become ready to be linked with proteins of serum, like albumin and
lysine(Friedman and Rasooly,2013).
2.9.1.2.2 Conjugation
It is responsible for phase I metabolites reaction in which the biotransformation
involving the enzymes (GST), β-glucuronidase, and/or sulfate transferase produce
conjugates of AfB1-glutathione, AfB1-glucuronide, and AFB1-sulfate, respectively
(Valko et al., 2006). The main identified of conjugate of AfB1-epoxide is the AfB1-
glutathione conjugate. This conjugation is the essential pathway of detoxification of
activated AfB1 in several mammals which is main in the AfB1 induced
carcinogenicity reduction and prevention.The forming conjugates are easily excreted
via the bile into the intestinal tract. The activity of cytosolic GST is inversely
correlated to the several animal species susceptibility to carcinogenicity of AfB1.
(Shetty and Jespersen, 2006).
2.9.1.2.3 Deconjugation
It can be happen in the intestinal tract as result of efficiency of bacteria. It acts
as a part of the large intestine flora metabolic role.
2.9.1.3 The mechanism of toxicity
The main target organ to AfB1 is liver, so the protein metabolism, lipids and
carbohydrates in liver will be affected . After AfB1conversion to AFB1-Epoxoide
by cytochrome P450, it will link with the guanine in DNA and RNA resulting

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39
depuration. The primary influence of this mechanism is DNA synthesis inhibition in
most active tissues, involving liver, intestine and bone marrow which leads to DNA
damage in the form of mutation (GC to AT mutation) and/or carcinogenic as liver
cancer after long-term exposure. The fast rate metabolism of aflatoxin B1 in some
animals like ducklings result in fast formation of AFB1-8,9-epoxoid which inhibits
RNA polymerase and protein synthesis subsequently. Other effects are represented
by affecting liver microsomal enzymes expressed as depletion of hepatic glycogen
stores (figure 3).The toxin also reduce activity of microsomal glucose-6-
phosphatase (Williams et al., 2004; Santella, 2007; Yunus et al., 2011).
Figure(3) : Mechanisms of AFB1 toxicity

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40
2.9.1.4 The role of aflR in aflatoxin pathway regulation
The activation of transcriptionl of most structural genes in the pathway cluster
of aflatoxin needed a 47 kDa sequence-specific zinc-finger DNA-binding protein, a
Gal 4-type 47-kDa polypeptide is encoded by aflR gene. The aflatoxin pathway
genes transcription will be activated after this protein links to the palindromic
sequence 5'-TCGN5CGA-3' (also called aflR-binding motif) in the promoter region
of the structural genes in A.flavus, A.parasiticus and A.nidulans. In case of more
than one motif in the promoter region, just one of them will be preferred as a
binding site such as in the case of aflC (pksA). (Yu et al., 2004; Yu and Ehrlich,
2011; Yu, 2012).
2.9.2 Fumonisins
Fumonisins are diester compounds with variaty tricarboxylic acids and
polyhydric alcohols and primary amine moiety (Oancea and Stoia , 2008).
Fumonisins B1 (FB1) and B2 (FB2) are examples of this group and have been found
in significant amounts.FB1 is formed by F.moniliforme and F.proliferatum. High
concentrations of fumonisins are related with hot and dry weather, and the periods
of high humidity(Fandohan et al., 2005). This species cause fusariotoxicoses to
human and animals, act as a carcinogenic effect on human and necroses and crusts
of the buccal mucosa in poultry , in plant may cause root disease (CAST, 2003).

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41
Figure (4): Fumonisins B1 Structure ( C34H59NO15)
2.9.3 Ochratoxins
Ochratoxins are produced of A. ochraceus and P.verrucosum in an enormous
diversity of feeds and foods.There are four types of ochratoxins : A, B, C, and D.
OTA is the main mycotoxin among them. (CAST,2003).Their chemical structure
contains an isocoumarin moiety bounded by a peptide bond to phenylalanine (figure
5) (Gallo et al., 2012) . They responsible for several disease, in humans, ochratoxins
are the essential agent causing the fatal disease of kidney, affecting villager
communities in the central Balkan regions , as Bulgaria or Croatia, caused (BEN) ,
and this is distinguished by a reducing in size of kidney .This toxin is a contaminant
of beans ,cereals and other products of plant, also cause nephrotoxicity to farm
animals.

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42
Figure(5): Ochratoxin A Structure (C20H18ClNO6)
2.10. The prevention and reduction of mycotoxins strategies
There are many strategies realized to reduce mycotoxins levels in foods which
must be taken to prevent or reduce the effect of mycotoxins .
2.10.1 Practices of good agricultural quality
Agricultural practices have been applied to decrease contaminated mycotoxins
effect on crops in the field.
(i) Early harvesting:
This decrease crops infection happen by fungi in the field before harvest and
harvested product contamination. The early harvesting results decreasing level of
aflatoxin and increasing in gross returns of 27% than in belated harvesting
(Rachaputi et al. 2002).

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43
(ii) Correct drying:
Reducing the moisture level of agricultural products by rapid drying .It is
critical to create less appropriate fungal growth and proliferation conditions. The
drying harvested corn to 15.5% moisture or less within 24-48 h may be reduce the
danger of growth of fungi and production of aflatoxin (Lanyasunya et al., 2005) .
(iii) Physical treatment:
This strategy elucidated that sorting, winnowing, washing, crushing joint with
hulling of grains of maize were influential in removing of mycotoxins.This have
been achieved by contaminated grain separation from the bulk that rely on the
heavy contamination of only a small part of the seeds (Park,2002) .
(iv) Sanitation:
The debris removing and destruction from previous harvest may be helped in
decreasing infection in the field. Cleaning of the stores before freight new produce
is indispensable to be correlated aflatoxin levels reduction (Hell et al., 2000).
(v) Proper storage:
Preventing of biological activity is necessary to preserve quality in storage,
through sufficient drying to lower than 10% moisture, insect activity removal which
raise moisture content through respiration, decrease temperatures, and inert
atmospheres ( Turner et al., 2005).

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44
(vi) Insect management:
Insect damage of maize is best foreteller of contamination by Fusarium
mycotoxins, so the level of its damage influences the extent of mycotoxins
contamination by carrying mycotoxins producing fungi spores from surfaces of
plant to the stalk interior or kernels or create infection wounds through their feeding
behaviors (Avantaggio et al. 2002 ; Munkvold, 2003).
(vii) Other methods:
Containing tillage , rotation of crop, date of planting and management of
irrigation and fertilization, have finite effects on infection and accumulation of
mycotoxins (Munkvold, 2003; Champeil et al., 2004).
2.10.2 Biological control
Progressive strategies would made in forming different bio control strategies
like atoxigenic bio-control fungi development that can out-compete their closely
related, toxigenic strains in field then they decreasing the mycotoxins levels in the
crops (Cleveland et al., 2003). Application of atoxigenic A. flavus and A. parasiticus
strains which decreased aflatoxin contamination of post-harvest by 95.9%. Use of
biological agents to suppress growth of fumonisin production by atoxigenic F.
verticillioides strains( Dorner and Cole , 2002). The endophytic bacteria can be
used as control of fumonisin producing fungi and also lactic acid bacteria as
Lactobacillus reuteri strain provided good control to fungi by lactic mixture
production (0.9% w/w), acetic (0.2% w/w), and succinic acids (0.2% w/w)

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45
(Plockova et al., 2001).Masoud and Kaltoft )2006) confirmed in vitro inhibition of
production of OTA by A. ochraceus by three yeasts (Pichia anomala, P.kluyveri and
Hanseniaspora uvarum).Other mechanism of use Trichoderma spp. to control
pathogenic fungi through competition for nutrients and space, fungistasis, antibiosis,
modification of rhizosphere, mycoparasitism, biofertilization and the plant-defense
mechanisms stimulation (Benitez et al., 2004).
2.10.3 Chemical control
A suitable pesticides using during the process of production may be minimized
the infection of fungi or infestation by insect of crops and resulting mycotoxin
contamination. fungicides application could reduce fumonisins contamination by
using chemical compound as propiconazole,prochloraz,epoxyconazole,
cyproconazole ,tebuconazole and azoxystrobin (Haidukowski et al., 2004).While
fungicides as amphotericin B and itraconazole have been appered to actively control
the aflatoxin-producing Aspergillus species (Ni and Streett, 2005). However, the
fungicides using is thwarting because of the economic reasons and growing related
with environment and food safety problems.

Chapter three
Materials and methods

Chapter three Materials and methods
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46
3 Materials and methods
3.1 Materials
3.1.1 Instruments and equipment
The instruments and equipment which were used in this study are listed
below (table 2 ) .
Table (2): Instruments and equipment with their remarks.
N0. Name of equipment Manufacturer / State
1 Autoclave Monarch MSI/ Germany
2 Centrifuge Hettich / Germany
3 Compound light microscope Olympus /Japan
4 Cooled centrifuge Hettich/ Germany
5 Cooled incubator Binder / USA
6 Digital camera HD Sony / China
7 Electric oven Memmert/ Germany
8 Electrophoresis apparatus MD-300N/ UK
9 Hood Cruma / Spain
10 Thermocycler apparatus Techne/ UK
11 Vortex Memmert/ Germany

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47
3.1.2 .Chemicals
The chemicals and biological materials which were used throughout the
study are listed in (table 3) below:
Table ( 3 ) : Chemicals and biological materials
No. Type of chemical or biological Manufacturers/ state
1 Absolute ethanol Fluka / Germany
2 Agarose gel Promega /USA
3
Chloramphenicol
Grand
Pharmaceutical/China
4 Coconut-agar medium Local market
5 Ethidium bromide Sigma-Aldrich/Germany
6 Free water nucleas Promega / USA
7
Lactofuchsin
AEML, Inc.
microbiological
laboratories/USA
8 Lactophenole blue stain Hardy Diagnostics/USA
9 Liquid nitrogen Local factory
10 MEA agar LabM/United Kindom
11 PDA medium Titan biotech / India
12 SDA medium Titan biotech / India
13 TBE(10 X) solution Bio basic / Canada
14 Tween 80 Sigma-Aldrich/Germany

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48
3.1.3 Media
3.1.3.1 Coconut-Agar Medium (CAM)
It was prepared by weighing of 100 g of ripped coconut , then it was
mixed with 300 ml of hot distilled water for 5 min for homogenizing. Through
four layered cheesecloth , the homogenate was filtered. The clear filtrate was
regulated to pH 7.0 with 2 N of NaOH. About 20 g/l of agar was added, and
chloramphenicol (500 mg / l) for prevention of bacterial growth. By
autoclaving at 121 °C for 15 min ,the mixture was sterilized. (Davis et al.,
1987).
3.1.3.2 Potato Dextrose Agar (PDA)
About 39 g. were dissolved in 1000 ml. distilled water. Adding of
chloramphenicol (500 mg / l), then stirring of suspension to dissolve
completely. Sterilizing was done by autoclaving at 15 psi (121°C) for 15
minutes. After this , cooling at room temperature to dispense(Beuchat and
Cousin, 2001) .
3.1.3.3 Malt Extract Agar (MEA)
Suspending of 50 g in 1000 ml. of distilled water, about 500 mg /l of
chloramphenicol were added. Bring to the boil to dissolve. Sterilized by
autoclaving at 115°C for 10 minutes, then remain to cool.

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49
3.1.3.4 Sabouraud Dextrose Agar (SDA)
Dissolving of 65 g. in 1000 ml. distilled water. Adding of chloramphenicol
(500 mg /l), then boil of suspension to dissolve completely. Sterilizing was
done by autoclaving at 15 psi (121°C) for 15 minutes . After this , cooling at
45-50 C◦ mixing well and dispense (Tokhadze et al., 1975). .
3.1.4 Stains
3.1.4.1 Lactophenol cotton blue
This stain is prepared over two days.
a. Dissolving of the cotton blue in the D.W. Leaving it to remove insoluble
dye overnight, this was done at first day.
b. Second day, in a glass beaker phenol crystals was added to the lactic acid. It
must be placing on magnetic stirrer to dissolved the phenol .
3. Then glycerol was added .
4. Filtering the cotton blue and D.W. solution into the solution of glycerol
/phenol/ lactic acid. Mixed , then stored at 25◦ C.
3.1.4.2 Lacto-fuchsin
Is prepared by adding of 0.1 g acid fuchsin with 100.0 ml. (85% ) of lactic
acid (McGinnis , 1980)

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50
3.1.5 Kits
The Kits were used in the diagnosis are listed below (table 4,5) :
Table (4) : DNA extraction kit contents (Bio basic / Canada) .
NO. Component
1 EZ-10 column
2 Collection tube
3 Buffer of universal digestion
4 Buffer PF
5 Buffer BD
6 PW solution (concentrate)
7 wash solution (concentrate)
8 buffer of TE
9 Proteinase K
Table (5): PCR reaction kit and related materials
N0. Component Manufacturer / state
1 DNA ladder 100 bp Bioneer / South Korea
2 DNA purification kit Bio basic / Canada
3 Green master mix Bioneer / South Korea
4 Oligonucleotide primers Bioneer / South Korea

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51
3.2 Methods
3.2.1 Collection of samples
A total of 180 samples of concentrated poultry feed pellet from different
breeders broiler farm and local markets of poultry feed were collected in
Basrah province . Feed samples were collected during one year from Sep. 2014
up to Apr. 2015. The feed stored for 2-3 days in sterile containers at room
temperature (22-25ºC). After that, they were prepared for fungal isolation and
identification( Shareef, 2010).
3.2.2 Isolation and identification of fungi
Twenty gram of the poultry feed samples were suspended with 180 ml of
saline solution (0.85% sodium chloride) in addition to 0.05% Tween 80
(polyoxyethylene sorbitan monoleate) on a horizontal shaker for 30 min. to
liberate the spores from fruiting bodies and to break the spore clumps (Mishra
et al.,2013) , then 0.1 ml of suspension was inoculated on PDA and MEA
media(Pitt and Hocking ,2009 and Greco et al., 2014) . The distinct colonies
were stained on a slide using lactophenol cotton blue and lacto-fuchsin , then
morphological characteristics of fungal isolates were described under
microscope ( Domsch and Gams. 1980 and Klich, 2002) The colony color
and conidia morphology were investigated. Each colony type was counted for
individual cfu/g counts and were recorded (Beuchat, and Cousin, 2001). The
frequency (Fr.) and relative density (RD) of isolation of genus and species

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52
were calculated ( Gonzalez et al., 1995; Pacin et al. , 2003 ; Saleemi et al. ,
2010) as follows :
samples number with a genus or specie
Fr. (%) = ___________________________________ X 100 ,
samples total number
isolates number of a genus or specie
RD(%) = ______________________________________ X 100 .
fungi isolated total number
3.2.3 Detection tools of aflatoxigenic A.flavus
These tests carried out on 50 isolates of A.favus to detect aflatoxigenic or
nonaflatoxigenic isolates by UV light , ammonia vapor and molecular detection
by PCR.
3.2.3.1 Coconut based medium detection
The detection of aflatoxigenic isolates was done by blue-green
fluorescence on CAM. A preliminary screen for aflatoxin producer A.flavus
was done on the basis of blue to blue – green fluorescence emission by light
of UV irritation at 365 nm when the isolate was grown on CAM ,this agar is
inductive of aflatoxin production (Dyer and McCammon , 1994). The isolates
can be identified by fluorescence in the reverse side of the culture because of
the reaction with coconut fats (Lin and Dianese, 1976 and Davis et al., 1987)

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53
in glass Petri dishes . Use 5 mm diameter sterile cork borer to make a hole in
the center of CAM medium in Petri dish . A mass of conidia of isolates were
inoculated of by cork borer to hole at the centric point of CAM in glass Petri
dish, then they were incubated at 28 °C for 7 days. The isolates of
aflatoxigenic A.flavus appeared blue to blue-green fluorescence under UV light
with long wavelength 365 nm, while the isolates of non aflatoxigenic A.flavus
remain colorless. Isolates of A.niger under the same conditions, was used as
nonaflatoxigenic control (Hara et al ., 1974 and Davis et al., 1987).
3.2.3.2 Ammonia vapor detection
The isolates of A.flavus were inoculated on CAM by cork borer (5mm)
diameter in the center of plate and incubated in the dark at 28 °C. for 7 days .
The dish was upended , then 1 or 2 drops of ammonium hydroxide solution
(concentrated) are put on the the lid inside of petri dish. The Petri dish inverted
over the lid containing the ammonium hydroxide.The colonies of aflatoxin-
producer A.flavus rapidly turn reddish pink after the bottom of the culture. No
color change occurs in colonies of non aflatoxins producer A.flavus (Saito and
Machida 1999) . A control as was mentioned in previous test was prepared.
3.2.3.3 Molecular assay
This assay is designed to include the examination of A.flavus isolates (table
6) by extraction of their DNA and using PCR technique , depending on primers
sequences of aflatoxin regulatory gene aflR (Manonmani et al., 2005).

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54
Table (6): The sequences of the primers
primer primer Sequence Characterized
Molecular
weight(bp)
aflR-1 Forward ´5 -AACCGCATCCACAATCTCAT-3´
A.flavus 798
aflR-2 Reverse ´5-AGTGCAGTTCGCTCAGAACA-3´
3.2.3.3.1 Preparation of buffers, solutions and stains
3.2.3.3.1.1 TBE (1X)
It was prepared by mixing 100 ml of stock TBE-10X ,the volume was
completed to 900 ml with D.W., and stored at 4°C until use in electrophoresis
( Sambrook et al., 2000) .
3.2.3.3.1.2 Ethidium bromide (0.5% (
Ethidium bromide stain was ready to be use .
3.2.3.3.1.3 Agarose gel preparation
The agarose gel was prepared according to the method of Sambrook et. al.,
(2000 ). The protocol of electrophoresis consists of two step:
A-Making the Gel
1- Twenty five ml. of TBE buffer(1X) was taken in a beaker .
2- About 0.175 g. of agarose was added to the buffer.
3- Agarose was melting in the microwave for 1 min. until the gel particles
dissolve .
4- Molten agarose was allowed to cool.

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55
5- The ethidium bromide was added to the buffer , then mixed before pouring
in casting apparatus.
B- Casting of the horizontal agarose gel
1- The previous mix was poured to casting tray at a depth of 4-8 mm and the
comb was positioned at one end of the tray.
2- The mix was allowed to hard at room temperature for 30 min .
3- the comb was carefully remove and the mix replaced in electrophoresis
chamber .The chamber was filled with TBE - electrophoresis buffer until
the buffer reached 3-5 mm over the surface of the gel.
3.2.3.3.2 Preparing A.flavus mycelia for DNA extraction
DNA was extracted from 0.5 g (wet weight) freshly growing cultures of
A.flavus mycelia harvested on PDA medium. Grinding the mycelium into a
fine powder by liquid nitrogen using a pre-cooled pestle,then transferred in an
Eppendorf tube (Arendrup et al., 2011) .
3.2.3.3.3 DNA extraction
The genomic DNA was extracted by using fungal genomic DNA extraction
mini-preps kit(Bio Basic / Canada ).
Procedures
1. Grinding cell pellets collected from 0 100-500 mg (wet weight)
mycelia/spores in liquid nitrogen using a pestle. grinded sample was
transferred to a clean 1.5 ml microtube.

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56
2. Universal digestion buffer (180 μl) and proteinase K (20 μl) were added to
the sample, and mix thoroughly by vortex. Incubate at 56°C for 30-60 min.
3. Universal Buffer PF (100 μl) was added, mix by inverting, and incubate at -
20°C for 5 min.
4. Centrifuge at 12,000 x g for 5 min at room temperature supernatant was
transferred the to a new 1.5 ml tube.
5. Universal Buffer BD (200 μl ) was added, mixed thoroughly by vortex.
6. ethanol 96-100% (200 μl) was added, mixed thoroughly by vortex.
7. The mixture from step 6 (including any precipitate) was transferred into EZ-
10 column was placed in a 2 ml collection tube. Centrifuged at 12000 rpm
for 1 min. Discarded the flow-through.
8. Universal PW Solution (500 μl) was added, centrifuged for 1 min. at 12000
rpm. Discarded the flow through. 18 ml of PW solution were diluted previously
with 12 ml of isopropanol.
9. Universal Wash Solution (500 μl) was added, centrifuged for 1 min. at
12000 rpm. Discarded the flow through.
10. empty column was placed the in the microcentrifuge and centrifuged for an
additional 2 min at 12000 rpm to dry the EZ-10 membrane. Discarded flow-
through and transferred the spin column to a clean 1.5 ml centrifuged tube.
11. Buffer TE (50-100 μl) was added directly onto the center part of EZ-10
membrane. Incubated at room temperature for 1 min, and then centrifuged for 1
min at 12,000 rpm to elute the DNA.

_______________________________________________________________
57
3.2.3.3.4 Polymerase chain reaction
The polymerase chain reaction (PCR)was used in amplification aflR
fragments of aflatoxigenic A.flavus genomic DNA. The forward and reverse
primers aflR sequence was mentioned prevously in table (6) , and were done
based on the sequence strand for A. flavus with size of 798 bp (Manonmani et
al., 2005). .
Protocol
-The following reagent was added for each tube of 20 µl (Bioneer /South
Korea) on ice (table 7( .

_______________________________________________________________
58
Table (7) : Reaction components of PCR (Bioneer /South Korea)
NO. Reagent Volume
1 Green master mix 5 µl
2 Upstream primer 1 µl
3 Downstream primer 1 µl
4 Template DNA 5 µl
5 Nuclease free water 8µl
- About 5 µl of template DNA were added plus 1µl of upstream primer and
1 µl of downstream primer to the tube containing (5 µl) green master . Mix
them thoroughly by vortex.
- To avoid contamination, all reagents were taken with separate clean tips.
- The volume of the mixture was completed to20µl with nuclease-free water.
- The tubes of PCR were put to preheated thermocycler to start the program
in table (8).
Table (8) : PCR Program
No. Step Temperature C° Time / Sec. No. of cycles
1 Initial denaturation 95 600 1
2
Denaturation
Annealing
Extension
94
50
72
18
45
75
30
3 Final extension 72 600 1

_______________________________________________________________
59
3.2.3.3.5 PCR result analysis
The products of PCR were analyzed by electrophoresis by a 1.2% mix of
agarose gel in TBE 1 X buffer stained with 0.25 µl ethidium bromide. The
positive and negative control were included .
3.2.3.3.6 Sequencing of PCR products for aflR gene
Five PCR products of aflatoxigenic A.flavus that identified by PCR were
randomly selected , sequenced at Macrogen company in South Korea .
3.2.3.3.6.1 The basic local alignment search tool analysis (BLAST)
The sequenced aflR products were analyzed homology with standard
sequences of aflR gene deposited to NCBI gene bank using BLAST analysis
software at (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).

Chapter four Results
__________________________________________________________________
60
4 Results
4.1 Fungal isolation
In the present study more than 10 fungi genera and more than 40 species were
recovered from 180 samples of poultry feed. Several species were identified in each
sample. Up to 20 different species were isolated in several samples were recovered.
The morphological characteristics of fungal isolates were described under microscope
.The colony color and conidia morphology were investigated. The most important
recovered genera of fungi were Aspergillus, Penicillium, Rhizopus, Cladosporium ,
Mucor , Alternaria and Fusarium (figure 6,7). There were 9 Aspergillus spp. recovered
: A.flavus , A.niger , A.fumigatus , A. terreus , A.flavipes , A.carbonarius , A.
ochraceus , A. candidus and A. parasiticus (figure 8,9,10 ) .
The total fungal counts cfu/g were ranged from 5X101
- 2.1X106
of feed sample,
with an average 1.5X 105
cfu/g sample ( tables 9) .The frequency(Fr.) and relative
density(RD) recorded a highest value which was belong to Aspergillus, while
Fusarium was identified as low value of Fr. and RD(table 10). The most frequent
mycotoxigenic fungi from 180 samples were those from the genus Aspergillus. This
genus recovered from113 samples ( Fr. 62.77%) also with most RD52.03% with a
range of 2.2X105
- 2.1X106
cfu/g and a mean value of 1.7X106
cfu/g , followed by
Penicillium which recovered from 90 samples (Fr.47.77%), in addition of RD17.01%
with a range of 7.1X103
- 8.0X104
cfu/g and a mean value 4.1X104
cfu/g , Rhizopus
was recovered from 86 samples with Fr. 50% and RD10.01% and had a range 4.2 X

__________________________________________________________________
61
103
- 2.1X104
cfu/g , with mean value 1.6X104
cfu/g . Fusarium, was reported as a low
ratio, it was recovered from 7 samples (Fr. 1.66%) with a range of 5X101
- 5.6X101
cfu/g and of a mean value of 7.8X101
cfu/g.
The total Aspergillus spp. counts cfu/g were ranged from (0.3X102
- 1.5X105
) of
feed samples, with an average 7.5X104
cfu/g sample (table11). The frequency and RD
recorded a highest value which was belong to A.flavus, while A. parasiticus was
identified as low value of Fr. and RD(table 12) .The most predominant Aspergillus
species recovered from 117 samples of Aspergillus was A. flavus , recovered from 74
(Fr. 65.48 %) with RD 27.55 % and with range 1.1X104
-1.5X105
and had mean value
of 8.6X104
cfu/g , followed by A.niger , ecovered from 66 samples (Fr. 58.40%) and
RD14.23 ,with a range 5.8X103
-7.4X103
and a mean value 9.5X103
cfu/g , A.fumigatus
, recovered from 19 samples (Fr.16.81%) with RD(7.60%) , recorded with range
1.7X103-3.0X103 cfu/g and mean value 3.2X103
cfu/g , A. parasiticus , were
recovered as low percentage and cfu/g , it was recovered from 2 samples with Fr.
1.76 % and with RD 0.89% , which recorded a range 0.2X102
- 0.4X102
cfu/g with
mean value 0.4X101
cfu/g .

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62
Figure(6): The isolated molds genera from poultry feed on PDA medium. a:
Aspergillus(*), b: Aspergillus (**), c: Penicillium (*), d: Penicillium(**),
e:Rhizopus(*), f: Rhizopus(**), g:Cladosporium(*), h:Cladosporium(**).
(*): In culture, (**): Microscopically,40X.
a
c
b
d
e f
g h
b
d
e f

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63
Figure(7): The isolated molds genera from poultry feed on PDA medium. i:Mucor(*),
j:Mucor(**),k:Alternarial(*), l:Alternaria(**), m:Fusarium(*), n:Fusarium(**).
(*):In culture ,( **):Microscopically , 40X .
i j
k l
m n

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64
Figure(8): The isolated Aspergillus spp. from poultry feed on PDA medium. a:
Aspergillus flavus(*), b: A.flavus(**), c: A.niger (*), d: A.niger (**), e:A.fumigatus(*), f:
A.fumigatus (**). (*): In culture, (**): Microscopically, 40X.
a b
c d
e f

Detection of Aflatoxigenic Aspergillus flavus in Poultry Feed

Detection of Aflatoxigenic Aspergillus flavus in Poultry Feed

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