Egypt. J. Exp. Biol. (Bot.

), 15(2): 145 154 (2016) Th e Eg yptian Society of Exp erimen tal Biolog y
DOI: 10.5455/eg yjebb.20160710115119
RESEARCH ART ICLE

D a li a A . M . A bd o u
R a sh a M . O t hm an
N a s r . E . El - Bo r d en y
N e vi n A . Ib r a h im
M o h am e d A. Ab o u zei d

M ON IT OR ING IM POR T ED GR AIN - B AS ED IN G R ED IEN T S U SED IN F EED

PR OC ESSIN G F OR T OXI G EN I C M OU LD S AN D N AT U R AL L Y OC CU R R ING
MY C OT OXIN S

ABSTRACT:
Samples of imported feeds and feed ingredients
were collected every three months during the study
time from feed factories in two governorates, Egypt.
Samples were cultured and the recovered isolates
of Fusarium and Aspergillus were mycologically
identified using the universal keys. Species of
Fusarium were the prevalent with relative density of
52.9%. The majority of samples analyzed (67.7%)
had moderate fungal counts (around 8.9 10 3 CFU
g -1) whereas 26.2% contained counts over the feed
hygienic quality limits of 110 4 CFU g 1. Based on
frequency in tested feed samples as well as relative
density, F. proliferatum was the predominant
Fusarium species (23.8%) whereas 36.9% yielded
isolates of A. flavus. Applying high performance
liquid chromatography technique, Applying
chromatographic techniques, T-2 toxin (0.0493
mmol/L) was detected in the culture filtrate of a
single isolate of F. sporotrichioides whereas two
different isolates of F. verticillioides and an isolate I NTRODUCTI ON :
of F. proliferatum produced fumonisin B1 in
amounts of 0.094 mmol/L, 0.1530 mmol/L, and
0.212 mmol/L. Two A. flavus isolates were capable
of producing aflatoxin B1 (0.0580 mmol/L and
0.0446 mmol/L) and aflatoxin B2 (0.0202 mmol/L
and 0.0236 mmol/L). Aflatoxins G1 (0.0744 mmol/L)
and G2 (0.0433 mmol/L) were produced in the
culture filtrate of a single isolate of A. parasiticus. stocks and poultry industries are imported from
Only three feed samples were naturally Latin America, Eastern Europe, Canada and
contaminated with traces of mycotoxins, a sample
of Argentinean corn which contained T-2 toxin in
addition to two locally produced samples of broiler
finisher feed which contained traces of fumonisin
B1 and dairy cattle CFM which contained aflatoxin
B2 and a trace amount of aflatoxin B1.
KEY WORDS:
feed stuf fs, t oxigenic moulds, grain's major important genera of mycotoxin -producing
contami nation l evel, F us arium and
Aspergillus m yc otoxi ns.
CORRESP ONDEN CE:
M o h am e d A. Ab o u zei d
Department of Micr obiolog y, F ac ult y of
Science, Ain S hams Univ ersit y, 11566
Abbassia, C airo, Egypt, 11566 Abbassia,
Cairo, Eg ypt.
E-mail : m_abouzaid@sci.asu.edu.eg
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D a li a A . M . A bd o u *
R a sh a M . O t hm an *
N a s r . E . El - Bo r d en y * *
N e vi n A . Ib r a h im *
* Dep artment of Microbiolog y, F acult y of
Science, Ain S hams Univ ersit y, 11566
Abbassia, C airo, Egypt, 11566 Abbassia,
Cairo, Eg ypt
** Departm ent of Anim al Prod ucti on, F ac ult y
of Agricult ure, Ai n Shams U niv ersit y, 11241
Hadaek S houbra, Cair o, Egypt .
ARTI CLE CODE: 15.02.6
In Egypt, local poultry production was on
average 874 thousand tons (Hassan, 2013)
whereas total beef production is anticipated to
reach 300 thousand metric tons (Egypt:
Livestock and Products Annual, 2014). Most
feed ingredients used in the processing of live
USA (El-Sayed, 2012). Such imported
ingredients are prone to fungal contamination
either when transported or stored in Egyptian
factories.
Fungal contamination in animal feeds of
cereal grains, oil-seed meals and forages
represents the major animal health risk
throughout the world (DMello, 2004). Three
fungi, Aspergillus, Penicillium and Fusarium
appear at different stages of grain production.
Fusarium species invade grains during growth
while species of Aspergillus and Penicillium
generally develop during grain storage (Miller,
1995). Mold growth can affect grain's nutritional
quality in several ways and may adversely affect
health of animals, some species are able to
produce highly toxic compounds called
mycotoxins (Marguardt, 1996). However, the
presence of fungal growth doesn't necessarily
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146
mean that mycotoxins are present. As
mentioned by Lawlor and Lynch (2005),
mycotoxins are estimated to affect as much as
25% of the worlds crops each year. Mycotoxin
production is unavoidable, unpredictable and
contamination by such toxic compounds leads to
greater losses in production, even when mold is
not readily apparent (Adams et al., 1993). They
have negative impacts on animal performance,
growth and reproductive rates, immunological
defense, as well as being carcinogenic,
mutagenic and teratogenic (Ratcliff, 2002).
Many of such natural toxic compounds become
concentrated in meat, egg and even milk of
animal and consequently may pose a threat to
human health, this explains the major concern
of food and feed industries in preventing them
from entering the food chain (Akande et al.,
2006). The most commonly encountered
mycotoxins in feedstuffs are aflatoxins,
fumonisins, zearalenones, ochratoxin, and T2-
toxin (Surai et al., 2008), this wide range of
mycotoxins contaminating animal/poultry feed in
addition to the variation in their chemi cal
compositions make protection against their
hazards a difficult task.
Also, there are many problems
complicating safety in feed manufacturing
process: 1) the mixing of various batches of
different raw materials together thus producing
a totally new matrix with a new risk profile; 2)
mycotoxins can contaminate practically all feed
ingredients, for example, Fusarium species
have been found in wheat, maize, barley, oats
and rye whereas aflatoxins contaminate
oilseeds and other feed ingredients; 3) most of
mycotoxins are stable compounds that do not
degrade during storage, milling or high-
temperature feed manufacturing processes
(Surai et al., 2008). The present study was
designed in the interest of safety and quality
assurance, with the aim of providing useful
information on the mycological and toxicological
risks associated with consumption of some
imported agricultural raw materials locally used
in animal feed processing. Objectives were: (i)
developing a sampling plan to include all
imported feed ingredients used in Egyptian feed
factories (ii) isolating, enumerating, identifying
toxigenic Fusarium and Aspergillus species in
collected feed samples (iii) analyzing the most
significant mycotoxins contaminating imported
and processed feed.
M ATERI AL AND M ETHODS:
Sam pl e col l ecti on pl an:
A t ot al of one hundred and thirt y
samples i ncluding proc ess ed f eed s amples
(84) and feed ingredients (46) were c ollec ted
from diff erent f eed fac tori es i n El -Fayoum and
El-Qal ubea gov ernorates , Egypt . Samples of
feed ingredient s wer e divided into 2 groups
acc ording to t heir origin: t he first group
consisted of 22 samples ( 47. 8%) of imported
feed ingredients (Ukrai ne, Croatia, Argentine,
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Egypt. J. Exp. Biol. (Bot.), 15(2): 145 154 (2016)
Brazil, US A). T he s ec ond group c omprised of
24 samples (52. 2%) of imported s oybean and
corn whic h ar e loc all y us ed in processi ng of
soybean meal and c orn glut en. Moist ure
content w as det ermined immediat ely af ter
collecti on; s amples wer e divided i nto tw o
parts: one w as c ultur ed immediatel y for
myc ological examination and t he ot her w as
stored at - 20 o C f or m yc ot oxin anal yses .
M ycol ogi cal anal yses:
Quantitative enumeration of fungal
propagules was performed on solid media using
the surface spread method. 10 g of each sample
were homogenized in 90 mL 0.1% peptone
water solution for 30 min. Serial dilutio ns (10- 2 -
10- 4) were prepared and 0.1 mL aliquots were
inoculated in triplicates onto the following
media: dichloran rose bengal chloramphenicol
agar (DRBC) (Pitt and Hocking, 1997) for total
culturable mycobiota and dichloran
chloramphenicol peptone agar (DCPA)
(Andrews and Pitt, 1986) for Fusarium species.
DRBC and DCPA plates were incubated at 25 C
for 710 days and for 7 days, respectively. Only
plates containing 10100 CFU were used for
counting and the results were expressed as
CFU per gram of sample; feed hygienic quality
limit is 1 10 4 CFU g -1 (GMP, 2008). Selected
colonies were sub-cultured on plates with fresh
agar media for purification then transferred on
slants of potato dextrose agar (PDA) and
Czapek yeast extract agar (CYA). Fusarium
species were identified on the basis of the
macroscopic and microscopic characteristics
using the universal taxonomic keys of Leslie and
Summerell (2006), whereas Aspergillus species
using Klich (2002).
Mycotoxin analysi s:
Reagents and standards:
All reagents w ere of anal ytical grade.
Solv ents used f or c hrom atograp hy w ere of
HPLC grade (ELG Trad e P harmac eutic als and
Chemic als C o, Egypt). Myc otoxi n s tandards:
aflatoxi n B1, B 2, G1, G 2, Fum onisi n B 1, T -2
toxin, and m onilif ormin w ere p urchased from
Sigma Aldrich, St Louis e, U SA . S epar ation
was carried out b y using HP LC (S himadz u,
K yoto, J apan) consisti ng of an LC -10 AD V P
pump and a S urveyor FL spec trophot ometer
detec tor. S upelc o, U SA . T he immune - affi nit y
colum ns f or was h up of the isol at ed toxi ns
were purc hased from Vicam L. P., US A and
Ver atox, N eog en, US A.
Toxigenicity of the recovered Fusarium isolates:
For solid culture, corn and rice grains were
prepared according to the method described by
Idris et al. (2003). 100 g of corn/rice were placed
in 500 mL Erlenmeyer flasks with 45 mL of
distilled H 2 O. Flasks were stored overnight at
room temperature, steamed for 20 min, and then
sterilized for 20 min at 121C before inoculating
each with 5 squares (1 cm 2) of agar containing
actively growing mycelia and incubated at 2 5C
for 4 weeks. Duplicates were made for each
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Abdou et al., Monitoring imported grain-based ingredients used in feed processing for toxigenic moulds and naturally occurring mycotoxins
isolate. Cultures of each isolate were dried in a
forced-air draft oven at 55C for 48 hours,
crushed with a mortar and pestle, packed in
plastic bags, labeled and stored at 4C until use.
Extraction was carri ed out as follows: 25 g
sample were dried and ground, extracted with
100 mL methanol/water (55:45, v/v, 1% NaCl)
and filtered through a Whatman grade no. 1 filter
paper. The filtrate (50 mL) was defatted twice
with n-hexane and extracted three times with
methylene chloride. Extracts were combined,
dried (Na 2 SO4), evaporated under reduced
pressure and dissolved in 1.0 mL methanol for
further analysis.
For liquid culture, 500 mL Erlenmeyer
flasks containing 200 mL of potato dextrose broth
medium were used. Each flask was inoculated
with 3 squares (1cm 2) of actively growing mycelia
selected from the margin of cultures. Flasks were
incubated in static conditions at 25C for 4 weeks
in the dark, then filtered and kept lyophilized until
extraction. Lyophilized culture filtrate
corresponding to 100 mL was dissolved in 100
mL ultra-pure water, and extracted (4 100 mL)
with ethyl acetate under acidic conditions (pH 2)
using HCOOH 1M. Organic extracts were
combined, dried and then evaporated under
reduced pressure ( Idris et al., 2003).
Toxigenicity of the recovered Aspergillus isolates:
Erlenmeyer flasks each containing 50 mL
of the YES broth medium described by Scott et
al. (1970) and Ezzat and Sarhan (1991) were
inoculated with 3 squares (1 cm 2) cut from the
margin of actively growing colonies of the tested
isolates grown on PDA medium. Three flasks
were prepared for each isolate and all flasks
were incubated at 25 oC under static conditions
for 10 days. Aflatoxins were extracted by adding
equal amount of chloroform t o the obtained
culture filtrate. The lower chloroform layer was
collected and organic extracts were dried at 40 oC
using a rotary evaporator and the dry weights of
each extract were recorded. The samples and
standards were spotted on commercially pre-
coated silicagel 60 TLC glass plate, 20 x 20 cm,
0.25 mm thickness, (E. Merck, Germany) . The
plate was first eluted with anhydrous ethyl ether,
Table 1. HPLC instrumentation and running conditions used in analyses of organic extraxts* from solid and liquid culture
filtrates of some isolated Fusarium species.
MYCOTOXIN FUMONISIN
HPLC COLUMN reversed-phase C18 (4.6X250 mm) Catalog No. 1612124
Buffer (Sodium phosphate, pH 3.5): Methanol (MeOH):
Acetonitrile (ACN)
(85: 0:15 0.0 min)
(85: 0:15 5.0 min)
SOLVENT SYSTEM
(55: 30:15 5.1-20 min)
(40:6 0: 0 20.1-23 min)
(40:6 0: 0 23.1-40 min)
(20: 0:80 40.1-60min)
FLOW RATE 1 mL/ minute
WAVE LENGTH 330 nm 465 nm
RETENTION TIME 7.85-8.60 min
TOTAL TIME RUN 30 min
*Three injections each of 20 L for each sample
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147
dried up in a fume hood for 5 min., and
developed with chloroform: acetone (9 :1, v /v)
(Gimeno and Martins, 1983) at s ame direction.
The TLC plate was visually examined under
ultraviolet light at 366 nm (Chromato-Vue C-
70G, Ultra-violet Products, USA). The R f values
for develop ed spots in comparison to authentic
samples of Aatoxin G2, G1, B2, and B1 (Sigma
Aldrich, St Louise, USA). The sample showed
similar R f values with those of the standards: B1:
0.7; B2: 0.64; G1: 0.61; G2: 0.52.
Extraction and analyses of naturally -
occurring mycotoxins from raw feed samples:
Extraction and cl ean-up of feed sam pl es for
HPLC analyses:
For Fusarium mycotoxins, Extraction and
purification were performed as described by
Hietaniemi and Kumpulainen (1991). After
extracting and filtering the sample, the filtrate
was defatted twice with n -hexane and
evaporated to dryness. The residue was washed
through a Florisil column with methanol,
mycotoxins were eluted with chloroform -
methanol (90: 10, vol/vol) and evaporated to
give a residue which was dissolv ed in 1 ml
methanol-water (60: 40, vol/vol) and filtered
through Sep-Pak C18 cartridges. Elution from
the cartridge with 15 ml methanol-water (60: 40,
vol/vol) followed with evaporation to dryness
yielded a residue which was reserved in
methanol for chromatographic analysis.
For Aspergillus mycotoxins, based on the
method described by Stroka et al. (2000). 50 mL
of n-hexane was added to the extract before
filtration, 25 mL of the filtrate was diluted with
150 mL of deionized water and was cleaned up
on a Afla test IAC column preconditioned with 10
mL of phosphate buffered saline (3 mL/min) and
washed with 25 mL water. Aflatoxin was eluted
from the column using HPLC grade methanol
followed by addition of HPLC grade water.
HPLC Analyses of Fusarium mycotoxins:
Chromatographic columns and running
conditions used in HPLC analysis of organic
extracts of various Fusarium solid and liquid
cultures are recorded in table 1.
T2 TOXIN
reversed-phase Hypersil C18-BDS
column (2504.60) mm, 5 m particle size
Methanol: Water (60:40, v/v)
1.5 mL/ minute
381 nm
26.13-26.30 min
55 min
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148 Egypt. J. Exp. Biol. (Bot.), 15(2): 145 154 (2016)

HP LC Anal yses of afl atoxi ns:

RESULTS:
Stock standard solutions of AFB 1 , AFB 2 ,
AFG 1 , and AFG 2 (~10 g/mL) were prepared by Mycological analyses:
dissolving the solid standard in benzene: One hundred twenty-seven (97.7%) out of 130
acetonitrile (98:2, v/v) and the exact tested samples were mould-contaminated. Total
concentration were measured by fungal counts observed on DRBC medium ranged
spectrophotometer (Shimadzu UV -1601PC, from < 102 to 7.4104 CFU g-1 with an average count
Shimadzu Scientific Instruments, Japan) . of 8.9 103 CFU g-1. The Majority of samples (67.7%)
Chromatographic run was proceeded through investigated had fungal counts of moderate values
Supelcosil GOLD Lc -18 (15 cm 4.7 mm) above the detection limit (1 102 CFU g-1). 26.2% of
column and solvent s ystem wat er: acetonitrile: samples contained fungal counts exceeding the feed
methanol (63: 22: 15 v/v/v ) at a flow rate of hygienic quality limits of 1 104 CFU g1. The
900 L/min. Det ection was at ex: 365 nm em: relationship between the different types of feed
455 nm. Order of elution (R t ) is: aatoxin G2: samples examined during the study and their level of
3.64 min; aatoxin G1: = 3.27 min; aatoxin fungal contamination was highly statistically
B2: = 4.18 min; aatoxin B1= 4.69 min. significant (p < 001). All corn samples showed fungal
Stati sti cal an al yses: contamination levels over the maximum
recommended level. Yellow corn contained the
IBM SPSS statistical software package (V. highest counts (ranging from 1.2 104 to 7.4 104
20.0, IBM Corp., USA, 2011) was used for data CFU g1) with an average 3.58 10 3 CFU g1. Corn
analyses. Data were expressed as both number gluten had the lowest mould counts ranging from 0 to
and percentage for categorized variables. Cross- 6.8 103 CFU g1 with an average 3.18 10 3 CFU
tabulation with Chi-square was used to study the g1. Mycological examination of the 130 samples
association between variables or comparison tested indicated the presence of 10 different species
between independent groups as regards the of the genus Fusarium and two species of the genus
categorized data. The probability of error ( P Aspergillus. Total fungal counts in the investigated
value) at 0.05 or less was considered significant, feed samples are illustrated in figure 1.
while at 0.01 and 0.001 highly significant .

35000
2.9 104
30000
25000
Average count CFUg-1

20000
1.5 104 1.5 104
15000
9.1103
10000 7.6 103
Maximum
recommended
3.9 103 5.1103 level (104 )
4 1 03 4.1103
5000 3.1 10 3
3.2103

Type of feed material

Fig. 1. Fungal counts* (CFUg -1) in the investigated feed samples.

*: Results are given as mean value standard deviation; Detection limit: 1 102 CFU g1; Maximum recommended
level: 1 104 CFU g1.

Frequ ency of o ccurr ence of th e recover ed ingredients . F usarium and As per gillus speci es
Fusari um and Asp ergi llu s speci es: were the predominant, a highl y s tatis ticall y
significant diff erenc e b etween the i ncidence of
Is olates of F us arium and As pergillus
both speci es and t ypes of feed as w ell as the
species w ere rec ov ered from 70 s amples
origin of import ed feed ingredients and
(53. 8%) out of 130 t ot al f eed s amples (46
incidenc e of differ ent F us arium and
samples out of 84 proc ess ed f eed s amples Aspergillus speci es r ec over ed from t hes e
and 24 samples out of 46 f eed ingredients).
ingredients (p < 0. 01) and (p < 0.05),
Tot al c ount of Fusarium and As per gillus
respectiv el y. N umber of is olat es, isol ation
isolat es r ec over ed from t hes e feed s amples frequenc y and relative densit y of t he identified
were 90 from proc ess ed f eeds and 48 from
Fusari um and As per gillus is olates are
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Abdou et al., Monitoring imported grain-based ingredients used in feed processing for toxigenic moulds and naturally occurring mycotoxins 149

calcul at ed in imported f eed i ngredients in in loc ally proc ess ed f eed c omposed of
relation t o t he countr y of origin (Table 2) and imported ingredients (T able 3).
Table 2. Isolation frequency and relative density of Fusarium and Aspergillus species in feed ingredient samples according
to the country of origin
Locally extracted
Argentineb Brazilb Ukraineb USAb Croatiab
ingredientsa
Fusarium species
No. of Frc RDd No. of Frc RDd No. of Frc RDd No. of Frc RDd No. of Frc RDd No. of Frc RDd
isolates (%) (%) isolates (%) (%) isolates (%) (%) isolates (%) (%) isolates (%) (%) isolates (%) (%)

F. andiyazi - - - - - - - - - - - - - - - - - -
F. fujikuroi - - - - - - - - - - - - - - - - - -
F. globosum 1 4.3 11.1 - - - - - - - - - - - - - - -
F. napiforme - - - 1 33.3 25 - - - 1 7.7 11.1 - - - 1 100 50
F. nygamai - - - - - - - - - 1 7.7 11.1 - - - - - -
F. oxysporum 1 4.3 11.1 - - - 1 7.7 11.1 - - - - - -
F. proliferatum 2 8.7 22.2 1 33.3 25 1 100 100 4 30.8 44.4 1 20 25 - - -
F. solani 1 4.3 11.1 - - - - - - - - - - - - 1 100 50
F.sporotrichioides - - - 1 33.3 25 - - - - - - - - - - - -
F. verticillioides 4 17.4 44.4 1 33.3 25 - - - 2 15.4 22.2 3 60 75 - - -

Total isolates 9 4 1 9 4 2
Total samples 23 3 1 13 5 1
A. flavus 6 26.1 100 1 33.3 33.3 - - - 3 23.1 60 2 40 100 1 100 50
A. parasiticus - - - 2 66.7 66.7 1 100 100 2 15.4 40 - - - 1 100 50
Total isolates 6 3 1 5 2 2
Total samples 23 3 1 13 5 1
b c
: Seeds from USA and processed into meal in Egypt; : country of origin; : Frequency = (ns/N) 100, ns: the number of
samples where a s genus/species occurred, N: the total number of collected samples; d: Relative density = (ni/Ni)
100, ni: the number of isolates of a genus/species, Ni: the total number of fungal isolates
Table 3. Isolation frequency of Fusarium and Aspergillus species recovered from different processed feed materials

Poultry layer Broiler finisher Broiler starter Rabbit growing Rabbit does Dairy cattle Beef cattle
feed feed feed mixture mixture CFM CFM
Fusarium
species
No. of Fra No. of Fra No. of Fra No. of Fra No. of Fra No. of Fra No. of Fra
isolates (%) isolates (%) isolates (%) isolates (%) isolates (%) isolates (%) isolates (%)

F. andiyazi - - - - - - - - - - - - 1 8.3
F. fujikuroi - - - - - - - - - - 1 8.3 - -
F. globosum 2 1.7 - - 1 8.3 - - - - - - - -
F. napiforme 4 33.3 1 8.3 - - - - - - 1 8.3 - -
F. nygamai - - - - - - - - - - - - 1 8.3
F. oxysporum - - - - - - - - - - 1 8.3 - -
F. proliferatum 3 25.0 6 50.0 4 33.3 2 1.7 2 1.7 1 8.3 4 33.3
F. solani - - - - - - - - - - 1 8.3 - -
F. sporotrichioides - - - - - - - - - - - - -
F. verticillioides 1 8.3 2 1.7 3 25 1 8.3 1 8.3 - - - -
Total isolates 10 9 8 3 3 5 6
Total samples 12 12 12 12 12 12 12
A. flavus 7 58.3 6 50.0 7 58.3 3 25.0 2 17.6 6 50.0 5 41.7
A. parasiticus 3 25.0 2 16.7 1 8.3 - - 3 25.0 1 8.3 1 8.3
Total isolates 10 8 8 3 5 7 6
Total samples 12 12 12 12 12 12 12
a
: Frequency = (ns/N) 100, ns: the number of samples where a genus/species occurred, N: the total number of collected
samples

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150 Egypt. J. Exp. Biol. (Bot.), 15(2): 145 154 (2016)

M ycotoxi n producti on by Fu ariu m and

Aspergillu s i sol at es:
In comparison to authentic samples of
different mycotoxins, chromatographic analyses
of aliquots of organic extracts from solid cultures
and liquid culture filtrates, eleven Fusarium
isolates b elonging to five species namely F.
proliferatum, F. verticillioides, and F.
sporotrichioides F. oxysporum, and F. nygamai
were found to be producers of 8 different types of
mycotoxins. Isolates of F. proliferatum were
producers of fumonisins, moniliformin whereas F.
verticillioides and F. sporotrichioides were
producers of fumonisin and T-2 toxin,
respectively. Only three isolates of A. flavus out
of the 12 tested were able to produce aflatoxins
B1 and B2 and one single isolate out of the 4
tested of A. parasiticus was found to be a
producer of aflatoxins G1 and G2.
Anal yses of m ycotoxi n's di stri buti on i n raw
feed sam pl es:
A c onc entration of 0. 0493 mmol/ L w as
recorded f or T-2 toxi n (Fig. 2) by an is olat e of
F. s por otrichi oides r ecov ered from a sample of
corn imported from Argentina whereas a higher
amount (0.212 mmol/L) was recorded for FB1
detected in the culture of F. proliferatum isolate
(Fig. 3) recovered from a sample of locally
processed broiler finisher feed. Compared with
authentics of aflatoxins (Fig. 4), only two isolates
contaminating two different samples of locally
produced broiler starter feed and dairy cattle
CFM produced aflatoxin B1 (0.0580 mmol/L and
0.0446 mmol/L) and aflatoxin B2 (0.0202 mmol/L
and 0.0236 mmol/L), a single isolate from soya
bean meal imported from America and prepared
in Egypt was aflatoxin B2 (0.0088 mmol/L)
producer and of A. parasiticus from a locally
processed sample of layer poultry feed produced
aflatoxin G1 (0.0744 mmol/L) and G2 (0.0433
mmol/L). Distribution of detected mycotoxins and
toxigenic isolates of Fusarium and Aspergillus
among the investigated feed samples is fully
illustrated in tables 4 and 5.

Fig. 2. HPLC pattern of the organic extract of F. sporotrichioides showing a characteristic peak of T-2 toxin at Rt of 26.26
min.

Fig. 3. HPLC pattern of the organic extract of F. verticillioides showing a characteristic peak of fumonisin B1 at Rt of 8.64
min.

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Abdou et al., Monitoring imported grain-based ingredients used in feed processing for toxigenic moulds and naturally occurring mycotoxins 151

Fig. 4. HPLC pattern of the reference samples of aflatoxins showing peaks of aflatoxins G2, G1, B2 and B1 at Rt of 3.27
min., 3.64 min. 4.18 min, and 4.69 min., respectively.
Table 4. Distribution of mycotoxins and toxigenic Fusarium isolates according to type and origin of feed samples
Type of Mycotoxin (s) in raw
Detection feed samples
Isolate mycotoxin Source Origin of
Species limit
code (s) sample samples Detection
(mmol/L) Occurrence
produced limit
Fumonisin Broiler Locally
F. proliferatum PRO.S16 0.212 + ve 0.107
B1 Finisher Feed processed
F. sporotrichioides SPR.S34 T-2 toxin 0.0493 Corn Argentine + ve 0.0255
Fumonisin Broiler Locally
VER.S28 0.0941 ND ND
F. verticillioides B1 Finisher Feed processed
Fumonisin Soya Bean USA (Locally
VER.S31 0.1530 ND ND
B1 Meal extracted)
Table 5. Distribution of aflatoxins and toxigenic isolates of A. flavus and A. parasiticus according to type and origin of feed
samples
Mycotoxin (s) in raw feed samples
Isolate Type of mycotoxin Detection limit Origin of
Species Source sample Detection limit
code (s) produced (mmol/L) samples Occurrence
(mmol/L)

Afla B1 (traces) 0.0446 ND ND

Broiler Locally
Flav.S02
starter feed processed
Afla B2 (traces) 0.0236 ND ND

A. flavus Afla B1 0.0580 + ve (traces) 0.0406

Dairy cattle Locally
Flav.S26
CFM processed
Afla B2 0.0202 + ve 0.0115

Soya bean America (locally

Flav.S50 Afla B2 0.0088 ND ND
meal extracted)

Afla G1 0.0744 Layer poultry Locally ND ND

A. parasiticus Par.S57
feed processed
Afla G2 0.0433 ND ND
remov ed from t he f eed (Bankol e and Kpod o,
DI SCUSSI ON: 2005). Ingredients us ed during proc essing
Ubiquitous nat ure of mold cont aminati ng may hav e various m yc ot oxins present i n a
feedstuf fs w orldwide r es ults m ainl y from the mixed feed. Suc h f ungal t oxins c an b e st ored
raw materials used i n their produc tion and in meat, milk and egg and fi nall y tr ans ferred
make t heir total eliminati on from f eed to hum an bei ngs (Brag ulat et al., 1995). In
impossible (Accensi et al., 2004). Pr ocessed this s tud y, m old and differ ent t yp es of
feed contai ns cor n, s oybean and oil s eed myc ot oxins cont aminating import ed grains
meals as m ajor ingredients w hich are locall y us ed in proc ess ed animal f ee ds in
exc ellent s ubstrates f or num erous fungi. W hen Egypt w ere inv estigated. Likewis e, m yc otoxi n
long -term f avor able c onditions f or fungal production b y s ome of the identifi ed fungal
growth are provided, m yc ot oxins may be species w as examined.
produced whic h c annot be complet el y
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152
According to G ood Manufacturi ng
Practic es ( G MP, 2008), t otal f ungal count
should not exceed 1 10 4 CFU g 1 . During
this s tud y, 26. 2% of c ollec ted feed s amples
(34 samples) did not m eet micr obiological
adequac y st andards and exc eeded t he f eed
hygienic qualit y limits. Tot al f ungal loads in
the anal yzed finis hed feed s amples w ere
around 8. 9 10 3 CFU g - 1 w hich is higher t han
those obt ained fr om Slov akian poultr y f eed
mixt ures examined b y Labuda and Tani nov
(2006) but w ere similar wit h findings on fungal
counts in f eed s amples from Argentina
(Mag noli et al., 1998) and Serbia ( Krnjaj a et
al., 2011).
Althoug h t he m ajorit y of t he anal yz ed
samples ( 67.7%) s how ed mod erat e fungal
counts , high fr equenci es of F us arium (50. 8%)
and Aspergillus (47.7%) wer e notic eable.
Many res earchers have prov ed t hat the
majorit y of feeds ar e cont aminat ed with
species from Aspergillus and/ or Fus arium
gener a ( Os ho et al ., 2007; Krnjaj a et al.,
2011; P ereyr a et al., 2011; R eddy and S alleh,
2011). Out of t he 130 inv es tigated s amples,
Fusari um is olates w ere rec ov ered from 66
feed s amples ( 50.8%). This c oincides wit h the
finding of Krnj aja et al. (2011) t hat Fus arium
species w ere t he m ost c ommon f ungi isolated
from poultr y f eed samples wit h frequencies of
56.09% in 2007 and 63. 40% i n 2008 at S erbia,
and with an earlier r eport by Osho et al.
(2007) w ho f ound t hat t he F usari um speci es
were t he s ec ond frequent isol at es (42%) in
poultr y f eed samples anal yzed in Nigeria.
In t he pres ent st ud y, F. prolifer at um was
found t o be the m ost predomi nant Fus arium
species (43%) f ollow ed by F. v erticillioides
(25%) of t otal Fus arium is olates and w ere
recov ered fr om 31 s amples ( 23.8%) and 18
samples (13. 8%), r espectiv el y out of t he total
feed samples . S uc h results s upport previous
findings or surv eys on t he mos t enc ount ered
Fusari um species eit her in Egypt (Abo El -
Yaz eed et al., 2011) or in ot her countries
including Argenti na ( D alcero et al., 1998),
Slov ekia (Labuda and Tani nov , 2006) and
Malaysi a (R eddy and S alleh, 2011). F.
prolifer at um and F. verticillioi des are
renowned for t heir role as important maiz e
ears pat hog ens t hat are comm onl y diff used in
maize-produci ng c ountries w orldwide
(Ghiasi an et al. , 2004; Glenn, 2007; Iglesi as
et al., 2010; Chil aka et al., 2012). In the
present st ud y, F. napiform e, F. oxy sporum, F.
solani, F. nygamai and F. s por otrichi oides
were also isol at ed from f eed samples , t his is
in acc ordance wit h ot her st udies c oncer ned
REFERENCES:
Abo El-Yazeed H, Hassan A, Moghaieb RE, Hamed Accensi F, Abarca ML, Cabanes FJ. 2004.
M, Refai M. 2011. Molecular Detection of
Fumonisin-producing Fusarium Species in
Animal Feeds Using Polymerase Chain
Reaction (PCR). J. Appl. Sci. Res., 7(4): 420-
427.
ISSN: 1687-7497 On Line ISSN: 2090 - 0503
Egypt. J. Exp. Biol. (Bot.), 15(2): 145 154 (2016)
with c ont aminated ani mal feed ( Labud a and
Tani nov 2006; Attit alla et al., 2010; Azizi et
al., 2012). Myc ological examinati on of 130
feed samples recov ered 48 As per gillus flav us
isolat es (36. 9%) and 17 As per gillus
par asiticus isol at es ( 13. 1%). T hes e r esult s are
almost identical t o thos e rec orded previousl y
in Eg ypt by A zab et al. (2005) w ho is olat ed A.
flavus and A. parasitic us fr om 36% and 13%
out of 500 of anim al f eed samples and
ingredients . They i nvestigat ed the level of
aflatoxi n B 1 c ontami nating anim al feeds and
recorded t he fr eq uenc y of t oxigenic A. flav us
and A. par asiticus in t he examined f eed
samples. W orldwide, sev eral s tudies on f ungi
contami nating f eedst uf fs prov ed onl y a high
frequenc y of A. flav us (Labuda and
Tani nov, 2006; Azar akhsh et al ., 2011;
Reddy and S alleh, 2011). A . par asitic us w as
onl y enc ount ered in feed mi xt ure s amples
studied b y D alc ero et al. (1998) and Mag noli
et al. (1998) in Argenti na but was missing in
the rest of st udies on Slov aki an, Ir ani an , and
Malaysi an samples .
Monit oring the pres enc e of m yc ot oxins
in various f eed samples is not onl y important
for cons umer prot ecti on, but also for
producers of the r aw produc ts prior t o
transp ort. Met hods bas ed on high
performanc e liquid chrom at ography (H PLC)
hav e been report ed (Ali et al., 2005; Manett a
et al., 2005). H PLC t echniques are simple,
eas y, rapid, w ell proven and widel y acc epted
even if havi ng some disadv antages (Bad ea et
al., 2004). In t he pres ent st ud y, HP LC
provided a rapid v aluable tool i n d et ecting
myc ot oxins including T - 2 toxi n, f umonisin, as
well as afl at oxins B 1, B2, G1, and G 2,
relativ el y measuri ng their content in fungal
cultur es and i n raw f eed. F our out of 73
Fusari um isolates ( 5. 5%) were found t o be
myc ot oxigenic. Thes e isolates w er e recov ered
from f our s amples (3. 1%) and bel onged t o
three speci es: F. pr oliferatum , F.
verticillioides and F. S por otrichi oides . Toxi ns
produced b y differ ent sp ecies of Fusarium are
of mos t conc ern f or animal healt h (Oliveira et
al., 2006). S urveys c onc erni ng Fus arium
myc ot oxins , even if s till limited, all cl earl y
show that feed should be acc urat el y
inves tigat ed fr om t he m ycotoxic ological poi nt
of view (Eriks en and P ett erss o, 2004; Lab uda
et al., 2005).
ACKNOWLEDGM ENTS:
The authors thank Dr Ahmad Kamal,
Consultant at ASSH for precious suggestions on
statistical analyses and statistical elaboration.
Occurrence of Aspergillus species in mixed
feeds and component raw materials and their
ability to produce ochratoxin A. Food
Microbiol., 21(5): 623-627.
http://my.ejmanger.com/ejeb/
Abdou et al., Monitoring imported grain-based ingredients used in feed processing for toxigenic moulds and naturally occurring mycotoxins
Adams RS, Kephart KB, Ishler VA, Hutchinson LJ,
Roth GW. 1993. Mold and mycotoxin problems
in livestock feeding. Pennstate. College of
Agricultural Sciences. Cooperative Extension.
www.das.psu.edu/teamdairy/, pp. 1-16.
Akande KE, Abubakar MM, Adegbola TA, Bogoro SE.
2006. Nutritional and health implications of
mycotoxins in animal feeds: A Review.
Pakistan J. Nutr., 5(5): 398-403.
Ali N, Hashim NH, Saad B, Safan K, Nakajima M,
Yoshizawa T. 2005. Evaluation of a method to
determine the natural occurrence of aflatoxins
in commercial trad itional herbal medicines
from Malaysia and Indonesia. Food Chem.
Toxicol., 43(12): 1763-1772.
Andrews S, pitt JR. 1986. Selective medium for
isolation of Fusarium species and
dematiaceous hyphomycetes from cereals.
Appl. Environ. Microbiol., 51(6): 1235-1238.
Attitalla IH, AL-An i LKT, Nasib MA, Bal al IAA,
Zakaria M, El-Maraghy SSM, Karim SMR.
2010. Screening of fungi associated with
commercial grains and animal feeds in Al -
Bayda Governorate, Libya. Word Appl. Sci. J.,
9(7): 746-756.
Azab RM, Tawakkol WM, Hamad AM, Abou -Elmagd
MK, El-Agrab HM, Refai M. 2005. Detection an
estimation of aflatoxin B1 in feeds and its
bioderaation by bacteria and fungi. Egypt. J.
Nat. Toxins, 2: 39-56.
Azarakhsh Y, Sabokbar A, Bayat M. 2011. Incidence
of the Most Common Toxigenic Aspergillus
Species in Broiler Feeds in Kermanshah
Province, West of Iran. Global Vet., 6(1): 73-77.
Azizi IG, Shateri S, Mousavizadeh M. 2012. Isolation
and identification of the mycotoxigen ic and
non-mycotoxigenic fungi from foodstuff and
feedstuff in Mazandaran Province, Northern
Iran. Afr. J. Microbiol. Res., 6(18): 3993 -3999.
Badea M, Micheli L, Messia MC, Candigliota T,
Marconi E, Mottram T, Velasco-Garcia M,
Moscon e E, Palleschi G. 2004. Aflatoxin M1
determination in raw milk using a flow-injection
immunoassay system. Anal. Chim. Acta,
520(1-2): 141-148.
Bankole SA, Kpodo KA. 2005. Mycotoxin
contamination in food systems in West and
Central Africa. In: Reducing the impact of
mycotoxin in trop ical agriculture with emphasis
on health and trade in Africa. Proceedings of
the Myco-Globe Conference, September 2005,
Accra, Ghana.
Bragulat MR, Abarca ML, Castella O, Cabaes J.
1995. A mycological survey on mixed poultry
feeds and mixed rabbit feeds. J. Sci. Food
Agric., 67(2): 215-220.
Chilaka CA, Kock S, Phoku JZ, Mwanza M, Egbuta
MA, Dutton MF. 2012. Fungal and mycotoxin
contamination of South African commercial
maize. J. Food Agric. En viron., 10(2): 296-
303.
Dalcero A, Magnoli C, Luna M, Ancasi G, Reynoso
MM, Ch iacch iera S, Miazzo R, Palacio G.
1998. Mycoflora and naturally occurring
mycotoxins in poultry feeds in Argentina.
Mycopathologia, 141(1): 37-43.
D'Mello JPF. 2004. Microbiology of animal feeds. In:
Assessing quality and safety of animal feeds.
FAO Animal Production and Health Paper
ISSN: 1687-7497 On Line ISSN: 2090 - 0503
153
(FAO), No. 160/FAO, Rome (Italy). Animal
Production and Health Division, pp: 89-106.
Egypt: Livestock and Products. 2014. USDA GAIN:
Egypt livestock and products annual report,
pp. 11.
El-Sayed REM. 2012. Analyses of poultry production
systems in rural sector. M.Sc. Agricultural
Sciences: poultry production. Department of
Animal Production, Faculty of Agriculture,
Cairo University, pp. 105.
Eriksen GS, Pettersson H. 2004. Toxicolog ical
evaluation of trichothecenes in animal feed.
Anim. Feed Sci. Tech., 114(1): 205 239.
Ezzat SM, Sarhan MM. 1991. Effect of different
concentrations of sodium chloride on the
growth and biochemical activities of
Aspergillus flavus link. Egypt. J. Microbiol., 26:
133-146.
Ghiasian SA, Kord-Bacheh P, Rezayat
SM, Maghsood AH, Taherkhani H. 2004.
Mycoflora of Iranian maize harvested in the
main production areas in 2000.
Mycopathologia, 158(1): 113121.
Gimeno A, Martins ML. 1983. Rapid thin layer
chromatographic determination of patulin,
citrinin, and aflatoxin in apples and pears, and
their juices and jams. J. Assoc. Off. Anal.
Chem., 66(1): 85-91.
Glenn AE. 2007. Mycotoxigenic Fusarium species in
animal feed. Anim. Feed Sci. Tech., 137(3 -4):
213240.
GMP. 2008. GMP + certification scheme animal feed.
Sector 2006, Appendix 1: p roduct standards:
regulations on product standards in the animal
feed Sector. Good Manufacturing Practices 14,
pp: 139.
Hassan MK. 2013. Promising future for the Egyptian
poultry industry. VIV EUROPE. May 20-22,
2014, Utrecht, Th e Netherlands.
Hietaniemi V, Kumpulainen J. 1991. Contents of
Fusarium toxins in Finnish and imported grains
and feeds. Food Addit. Contam., 8(2): 171-
182.
Idris AE, Abouzeid MA, Boari A, Vurro M, Evidente A. 2003.
Identification of phytotoxic metabolites of a new
Fusarium sp. inhibiting germination of Striga
hermonthica seeds. Phytopathol. Mediterr., 42:
6570.
Iglesias J, Presello DA, Botta G, Lori GA, Fauguel
CM. 2010. Aggressiven ess of Fusarium
section Liseol a isol ates causing maize ear rot
in Arg entina. J. Plant Pathol., 92(1): 205-211.
Klich MA. 2002. Identification of common Aspergillus
species. Utrecht: Centraalbureau voor
Schimmel cultures, pp. 116.
Krnjaja V, Stoj anovi L, Trenkovski S, Bijeli Z,
Tomaevi D. 2011. The frequency of
pathogenic fungi genera in poultry feed.
Maced. J. Anim. Sci., 1(1): 187190.
Labuda R, Tancinov D. 2006. Fungi recovered from
Slovakian poultry feed mixtures and their
toxinogen ity. Ann. Agric. En viron. Med., 13(2):
193200.
Labuda R, Parich A, Berthiller F, Tancinov D. 2005.
Incidence of trichothecenes and zearalenone
in poultry feed mixtures from Slovakia. Int. J.
Food Microbiol., 105(1): 19-25.
Lawl or PG, Lynch PB. 2005. Mycotoxin management.
Afr. Farming Food Process., 46: 12-13.
http://my.ejmanger.com/ejeb/
154 Egypt. J. Exp. Biol. (Bot.), 15(2): 145 154 (2016)

Leslie JF, Summerell BA. 2006. The Fusarium
Laboratory Manual. Blackwell Publishing, pp.
388.
Magnoli C, Dalcero AM, Chiacchiera SM, Miazzo
R, Saenz MA. 1998. Enumeration and
identification of Aspergillus group and
Penicillium species in poultry feeds from
Argentina. Mycopathol ogia, 142(1): 2732.
Manetta AC, Di Giuseppe L, Giammarco M, Fusaro
I, Simonella A, Gramenzi A, Formigoni A.
2005. High-performance liquid
chromatography with post-column
derivatization and fluorescence detection for
sensitive determination of aflatoxin m1 in milk
and cheese. J. Chromatogr. A, 1083(1-2): 219-
222.
Marguardt RR. 1996. Effects of molds and their
toxins on livestock performance: A western
Canadian perspective. Anim. Feed Sci. Tech.,
58(1-2): 77-89.
Miller JD. 1995. Fungi and mycotoxins in grain:
Implications for stored product research. J.
Stored Prod. Res., 31(1): 116.
Oliveira GR, Rib eiro JM, Fraga ME, Cavaglieri
LR, Direito GM, Keller KM, Dalcero AM, Rosa
CA. 2006. Mycobiota in poultry feeds and
natural occurrence of aflatoxins, fumonisins
and zearalenone in the Rio de Janeiro State,
Brazil. Mycopatholog ia, 162(5): 355-362.
Osho IB, Awoniyi TAM, Adebayo AI. 2007.
Mycological in vestigation of compounded
poultry feeds used in poultry farms in
southwest Nigeria. Afr. J. Biotechnol., 6(15):
18331836.
Pereyra CM, Cavaglieri LR, Chiacchiera SM, Dalcero
AM. 2011. Mycobiota and mycotoxins
contamination in raw materials and finished
feed intended for fattening pigs production in
eastern Argentina. Vet. Res. Commun., 35(6):
367379.
Pitt JI, Hocking AD. 1997. Fungi and food spoilage,
3r d Ed, springer US, pp. 593.
Ratcliff J. 2002. The role of mycotoxins in Food and
Feed Safety. Presented at AFMA (An imal Feed
Manufacturers Association) on 16 t h August,
2002. (http://www.facs.org.uk).
Reddy KRN, Salleh B. 2011. Co -occurrence of mold
and mycotoxin in corn grains used for animal
feeds in Malaysia. J. An im. Vet. Ad v., 10(5):
688-673.
Scott PM, Lawrence JW, VanWalbeek W. 1970.
Detection of mycotoxins by thin layer
chromatography: Application to screening of
fungal extract. Appl. Microbiol., 20(5): 839-
842.
Stroka J, Anklam E, Joerissen U, Gilbert J. 2000.
Immunoaffinity column clean -up with liquid
chromatography using post-column
bromination for the determination of aflatoxins
in peanut butter, pistachio paste, fig paste and
paprika powder. J. Assoc. Official Anal.
Chem., 83(2): 320340.
Surai PF, Mezes M, Fisinin V, Fotina T. 2008.
Mycotoxins and animal health: from oxidative
stress to gene expression. Krmiva, 50: 35 43.

* ** * *
*
*
**
F._sporotrichioides
) / 0.0493 ( T-2
F._verticillioides
F. proliferatum
/ 0.0153( .
.) / 0.212 / 0.094
A._flavus .

) / 0.0446 / 0.0580( B1
0.0236 / 0.0202 ( B2 .
G1 .) /
0.0433 ( G2 ) / 0.0744( ) 67.7%( .%52.9
) / 103 8.9 (
3 .A._parasiticus ) 26%( )
. 104 1(
) : (.)
, T-2 F._proliferatum
%23.8
B1 A.
. %36.9 flavus
. B1 B2 .HPLC

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