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MYCOTOXINS IN POULTRY FEED AND FEED INGREDIENTS
IN SULAYMANIYAH, KURDISTAN REGION OF IRAQ
H. M. Rahim*, D.H. Othman*, R. K. Majeed*, N.M. Saeed**, D. O.
Ismaeel**
*Department of Basic Sciences, College of Veterinary Medicine, University of
Sulaimani.
**Department of Microbiology, College of Veterinary Medicine, University of
Sulaimani.
(Received 3 October 2020 , Accepted 26 October 2020)
Corresponding author: [email protected]
Keywords: Aflatoxin, Fumonisin, Mycotoxins,
ABSTRACT
Mycotoxins are fungal metabolic byproducts that can contaminate animal feed
and human food. To investigate the incidence of mycotoxins in poultry feed in
Sulaymaniyah Governorate, Iraq, 173 samples of feed (n =150) and feed ingredients
(n = 23) were collected from 49 poultry farms and feed mills. The samples were
analyzed to quantitatively determine the existence of five mycotoxins, namely
aflatoxin, fumonisin, ochratoxin, T-2, and zearalenone, using specific kits. All the
tested samples contained ochratoxin at concentrations ranging between 1.0 and 6.0
μg/kg and averaging 3.4 ± 0.1 μg/kg. About 94.8% of the feed and feed ingredient
samples contained a minimum of four mycotoxins in different concentrations. There
is a need to understand the cumulative toxic effects better when several mycotoxins
occur in animal feed, which may result in issuing new regulations about the maximum
allowed concentrations of mycotoxins in feedstuff.
INTRODUCTION
Mycotoxins are secondary metabolites synthesized by specific fungi such as
species of Aspergillus, Fusarium, and Penicillium (1). Over 200 species of fungi can
produce mycotoxins, and about 300 chemically different fungal metabolites have been
identified as mycotoxins. These metabolites can occur in a wide range of raw
materials originating from animal and plant sources, which are used in the preparation
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of animal feed among the
agriculturally-important, mycotoxins, the ochratoxins and aflatoxins are the most
catastrophic to the poultry industry (2). Other mycotoxins comprise zearalenone,
fumonisins, nivalenol, and T-2, which can negatively impact the health and
productivity of poultry farms (3).
Several factors influence the rate of contamination of poultry feed by
mycotoxins. These factors include weather conditions, species of fungi contaminating
the harvests, method of gathering the crops, seasonal variations, as well as the
methods for the storage of provender and ingredients (4). Fungal species may
contaminate poultry feed and ingredients, and remain dormant until suitable growth
requirements, such as high humidity and aerobic condition, become available. Under
desirable conditions, the contaminating fungi start to grow and produce different
mycotoxins as secondary byproducts (5).
Mycotoxins may cause detrimental effects on the health and growth
performance of poultry, which may involve inhibition of organ functions, immune
suppression, reduced egg production, and stunted growth (4, 6). These effects might
eventually lead to severe economic losses. Besides, fungal metabolites may also
threaten the health of humans that consume contaminated poultry meat. The presence
of multiple mycotoxins in human food and livestock provender can exacerbate the
toxicity problem through the development of a synergistic effect (7).
Poultry feed is prepared from a variety of ingredients that originate from
animals and plants. Cereals are added to poultry feed as a source of energy, while
proteins may originate from plant sources, such as soybean and peanut, or animal
sources, such as fish and bone meals. These ingredients may be contaminated with
mycotoxins such as aflatoxin B1, zearalenone, ochratoxin A, and fumonisins (8).
Agencies, for example, the United States Food and Drug Administration and the
European Commission, have established regulatory guidelines that determine the
maximal tolerated levels of several mycotoxins in food and animal provender.
Variations in the limiting concentrations occur according to the type of raw material,
species, age of the animal, and intended use (6).
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The incidence of mycotoxins in poultry feed has been reported throughout the
world (4, 9, 10). The poultry industry in the Governorate of Sulaymaniyah, Kurdistan
Region of Iraq, is an essential sector of agriculture that provides food for more than
1.5 million people and supports the region’s economy. However, there is little data
about the incidence of mycotoxins in animal feed and ingredients. Therefore, this
work intended to inquire about several mycotoxins, namely aflatoxin, ochratoxin A,
fumonisins, zearalenone, and T-2 in feed and feed ingredients used locally for poultry
in Sulaymaniyah. This study provides preliminary data about the magnitude of poultry
feed contamination for consideration of other ingredients that are less susceptible to
be contaminated by mycotoxins.
MATERIALS AND METHODS
Study area and sampling: Sulaymaniyah is one of 18 governorates of Iraq that
is located in the northeast of the country. The governorate consists of 12 districts,
namely Chamchamal, Darbandikhan, Dukan, Penjwen, Pishdar, Ranya, Slemani,
Mawat, Qaradagh, Said Sadiq, Sharazoor, and Sharbazher (Figure 1). The first seven
districts contain 99 poultry farms, which account for the majority of the farms in
Sulaymaniyah.
Samples of feed and feed ingredients were collected from several districts in
Sulaymaniyah. Farms were visited from January to December 2019 to gather
specimens of feedstuff. Some of the poultry farms were visited more than once for
sampling. Batches of feed that were recently imported were considered. Larger farms,
which had their feed mills in location, were preferably selected, and if several farms
were dependent on the same feed mill in a specific locality, samples were collected
only from the feed mill to ensure the coverage of a larger area.
Two to four kilograms of feedstuff and ingredients were taken from each farm
by smaller portions from several 50-kg capacity bags. Collected specimens from each
farm were put in tightly closed plastic bags and stored at -18°C until they were
analyzed for mycotoxin contents.
Quantification of mycotoxins in feed and ingredients: Samples of feedstuff
and ingredients that were taken from each poultry farm were mixed well, and a
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measured amount was ground using a laboratory grinder. Ten grams of the ground
specimen was taken and put in a suitable container, and 70% methanol was added at a
ratio of 3:1. The mixture was shaken for two minutes and filtered using a Whatmann #
4 filter paper. One milliliter of the filtered solution was put in a 1.5 mL-capacity
centrifuge tube, and the tube was centrifuged for one minute. Quantitative
determination of each of the five fungal metabolites in the poultry feed was then
conducted using different ROSA® WET® Quantitative Test kits from Charm
Sciences, Inc. (MA; USA). The names and order codes of the kits used for the
quantification of the mycotoxins are shown in Table 1. The instructions of the
manufacturer were followed to determine the amount of each mycotoxin. Each test
was repeated three times to confirm the consistency of the results.
Analysis of data: The concentrations of the different fungal metabolites in the
feed, wheat, and corn samples were compared statistically to determine the most
common cause of feedstuff contamination by mycotoxins. The Iraqi climate is
characterized by a hot and dry summer and a cold and wet winter. The weather in
spring and fall months is fair and characterized by a higher rate of humidity, which
favors the fungal growth and development in these seasons. Hence, the concentrations
of different mycotoxins were measured and compared according to the season to
determine the influence of seasonal changes of weather on the rate of feed
contamination by fungal metabolites. Statistical comparison was conducted using oneway
analysis of variance (ANOVA), followed by post hoc (Duncan). A probability
level lower than 0.05 was considered statistically significant.
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Table 1. Names and order codes of the kits used in the quantification of mycotoxins .
Mycotoxin Name of Kit Order Code
Aflatoxin FAST5 Aflatoxin Quantitative Test LF-AFQ-FAST5-100K
Fumonisins FAST5 Fumonisin Quantitative Test LF-FUMQ-FAST5-100K
Ochratoxin Ochratoxin Quantitative Grain Test LF-OCHRAQ-G-100K
T-2 T-2/HT-2 Quantitative Test LF-T2-HT2-100K
Zearalenone ROSA Zearalenone test for Grains and
Feeds
LF-ZEARQ-100K
RESULTS
Study area and sampling: Forty-five poultry farms and feed mills from seven
districts in Sulaymaniyah, namely Chamchamal, Darbandikhan, Dukan, Penjwen,
Pishdar, Ranya, and Slemani, were included in this study (Figure 1). These districts
contain the majority of poultry farms and feed mills in the governorate. Initially, it
was intended to take a consistent number of samples from each district, but only four
samples were taken from the district of Penjwen since the area contains only a few
poultry farms. However, the results indicated that the levels of mycotoxins in poultry
feed in Penjwen were close to those of other districts (Table 2). The total number of
collected samples from January to December 2019 was 173, which included 150 feed,
14 wheat, and nine corn specimens. Of the 173 samples, 26 were gathered in winter,
49 in spring, 77 in summer, and 21 in fall months.
Level of mycotoxins in the collected samples: All the tested specimens
contained two mycotoxins at least, and 99 samples comprised all five of the fungal
metabolites. Also, 65 samples were positive for 4/5, and 8 of the specimens comprised
3/5 of the mycotoxins. Ochratoxin A was present in all samples at a concentration of
3.4 μg kg-1. Further, T-2 was detected in 170 of the collected specimens at an average
concentration of 15.7 μg kg-1. Zearalenone and aflatoxin were detected in 162 and
157 samples, respectively (Table 2). The average concentration of zearalenone in the
positive samples was 37.8 μg kg-1, and the concentration of aflatoxin was 4.5 μg kgBas.
J.Vet.R es.Vol.19, No.2, 2020.
1. Moreover, 119 (68.8%) of the collected specimens contained fumonisins at an
average concentration of 1160.0 μg kg
poultry feed in all districts included in our study. The incidences ranged from 58.3%
to 100.0% (Table 3). However, the levels of the individual mycotoxins in the poultry
farms were not statistically different in the different districts. Further, the
concentrations of mycotoxins did not differ statistically (p > 0.05) in different seasons
(results not shown).
The concentration of mycotoxins in feed, wheat, and corn were also compared
statistically, and the results are illustrated in Table 4. Results revealed that the
mycotoxin contents of the different specimens did not differ statistically (p > 0.05).
However, the cumulative concentration of all mycotoxins in feed, corn, and wheat
samples averaged at 61.1, 69.2, and 75.4 μg kg
metabolites were more abundant in wheat than in poultry feed or corn samples tested
in our investigation.
Figure 1. Iraq map, showing the seven districts of Sulaymaniyah, in the northeast of
the country, that were included in the study
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kg-1.Fungal metabolites were detectable in
kg-1, respectively. Hence, fungal
study.
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Table 2. Mean concentrations of mycotoxins in animal feed in Sulaymaniyah, Iraq
Mycotoxin Occurrence† (%)
Concentration (μg kg-1)
Range Mean
Aflatoxin 90.7 1.0–16.0 4.5 ± 0.3
Fumonisins 68.8 500.0–5000.0 1159.0 ± 84.0
Ochratoxin A 100.0 1.0–6.0 3.4 ± 0.1
T-2 98.3 1.0–80.0 15.7 ± 1.2
Zearalenone 93.6 1.0–180.0 37.8 ± 2.5
Values indicate the percentage of occurrence of the fungal metabolite in 173 samples.
Table 3. Occurrence and concentration of mycotoxins in specimens from seven
districts of Sulaymaniyah Governorate.
District
Num
ber of
sampl
es
Occurrence (%) Mean concentration (μg kg-1) ± SE
Afl.
Oc
h.
T-2
Fu
m.
Zea
.
Afl. Och. T-2 Fum. Zea.
Chamcha
mal
40
90.
0
100
.0
97.
5
70.
0
100
.0
4.6 ±
0.5
3.5 ±
0.1
21.2 ±
3.0
1038.0 ±
165.0
39.0 ±
5.0
Darbandi
khan
15
80.
0
100
.0
100
.0
86.
7
86.
7
5.0 ±
0.9
3.6 ±
0.2
10.4 ±
2.8
1654.0 ±
369.0
52.0 ±
9.9
Dukan 58
93.
1
100
.0
96.
6
58.
6
96.
6
4.6 ±
0.5
3.2 ±
0.1
17.1 ±
2.1
1187.0 ±
150.0
37.5 ±
4.4
Penjwen 4
75.
0
100
.0
100
.0
75.
0
100
.0
1.3 ±
0.3
3.5 ±
0.3
9.5 ±
3.7
1083.0 ±
693.0
23.5 ±
9.0
Pishdar 12
100
.0
100
.0
100
.0
58.
3
100
.0
6.2 ±
1.0
3.5 ±
0.2
12.8 ±
2.4
950.0 ±
152.0
35.0 ±
6.4
Ranya 14
85.
7
100
.0
100
.0
85.
7
92.
9
3.5 ±
0.6
3.1 ±
0.1
9.7 ±
2.0
1050.0 ±
187.0
37.9 ±
7.7
Slemani 30
93.
3
100
.0
100
.0
73.
3
80.
0
4.1 ±
0.5
3.7 ±
0.1
13.4 ±
2.9
1116.0 ±
202.0
32.3 ±
6.2
The total number of samples is 173, including 150 feed, 14 wheat, and nine corn samples. Afl. =
aflatoxin, Och. =ochratoxin A, Fum. = fumonisins, Zea. =zearalenone
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Table 4. Occurrence and concentration of mycotoxins in feed, wheat, and yellow corn
in Sulaymaniyah Governorate.
Mycotoxin Percentage of occurrence* Mean concentration (μg kg-1) ± SE
Feed Wheat Corn Feed Wheat Corn
Aflatoxin 90.6 92.9 88.9 4.6 ± 0.3 3.5 ± 0.6 4.8 ± 1.1
Fumonisins
72.0
100.0 33.3 1190.0 ± 90.0 860.0 ±
194.0
850.0 ±
407.0
Ochratoxin A 100.0 100.0 100.0 3.4 ± 0.1 3.4 ± 0.3 3.0 ± 0.3
T-2 99.3 92.9 88.9 15.3 ± 1.3 20.2 ± 4.5 16.4 ± 2.3
Zearalenone 92.7 100.0 100.0 36.6 ± 2.6 41.2 ± 10.6 50.3 ± 10.7
*Percentage of occurrence was calculated based on the total number of each sample type (150 feed, 14
wheat, and nine corn samples). No difference was observed in the concentration of the mycotoxin in
the different samples at a probability level of 0.05 (in the same row), using a one-way analysis of
variance, followed by Duncan’s post hoc.
DISCUSSION
Fungal metabolites are a significant threat to the health and productivity of
poultry. Hence, the availability of data about the extent of contamination of poultry
feed is a crucial step towards finding ways to reduce the burden of mycotoxins on the
health of birds. This study investigated the incidence of mycotoxins in poultry feed in
Sulaymaniyah, Iraq, using special kits manufactured by Charm Sciences Inc. These
tests are approved by UDSA-FGIS (United Stated Department of Agriculture-Federal
Grain Inspection Service) to quantitatively determine the level of each mycotoxin in
feed and feed ingredients.
Aflatoxins are metabolic byproducts of Aspergillus flavus and A. parasiticus
and are known to be hepatocarcinogenic in humans (11, 12). The risk of
contamination of feedstuff by aflatoxins is not only a problem of developing
countries. It is estimated that the potential economic losses in the corn industry in the
USA due to aflatoxin contamination might reach up to $1.68 billion (13). In Europe,
the rate of contamination of maize by aflatoxin has increased in recent years, owing to
climate change and global warming (14).
In our study, aflatoxin was found in 90.7% of the samples. The concentration
ranged between 1.0 and 16.0 μg kg-1 and averaged 4.5 ± 0.3 μg kg-1. This
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concentration is much lower than the recommended maximal allowed levels in
feedstuff by the United States’ FDA and the EU, which are 300.0 μg kg-1 and 50.0 μg
kg-1, respectively (15, 16). However, the probability of feedstuff contamination by
aflatoxin is high when conditions are suitable for fungal growth since the vast
majority of the samples were positive for this mycotoxin.
Fumonisins are byproducts of Fusarium fungi, including F. nygamai, F.
proliferatum, and F. verticillioides. These fungal metabolites cause severe diseases in
humans and animals, such as the liver and esophageal cancers (17). The maximum
allowed concentration of fumonisin in poultry feed is 20,000.0 μg kg-1, based on the
recommendation of the European Union Commission (18). In our study, only one
sample of feed contained fumonisins at a rate of 5,000.0 μg kg-1, while the rest of the
119 positive samples contained less than 2,500.0 μg kg-1 of the mycotoxin and
averaged 1159.0 ± 84.0 μg kg-1.
Ochratoxin A is a critically harmful mycotoxin produced by A. ochraceus.
Research has revealed that ochratoxin is carcinogenic, genotoxic, hepatotoxic,
nephrotoxic, neurotoxic, and teratogenic (19). Ochratoxin A is the most harmful of
the twenty types of ochratoxins to poultry, causing oxidative stress and many
hematological and immunological alterations (2, 20).
The upper limit on this mycotoxin in unprocessed cereals in the EU is 5.0 μg
kg-1, under the Codex General Standard for Contaminants and Toxins in Food and
Feed (21). Ochratoxin A was detectable in all the assayed specimens in this study, and
the concentration ranged between 1.0 and 6.0 μg kg-1 with an average of 3.4 ± 0.1 μg
kg-1. Nine of the tested samples (5.2%) contained ochratoxin A at a density of ≥ 5.0
μg kg-1. Comparison between the whole feedstuff, wheat, and corn samples showed
no significant differences in ochratoxin A content. In a recent study in the neighboring
country of Turkey, from the north of Iraq, ochratoxin A was present in laying hen
feed with an average concentration of 27.3 μg kg-1 (22), which means that Fusarium
fungi are widespread in the area and might contaminate all cereal products with their
metabolic byproducts. Hence, control measures should be taken to reduce the
contamination of poultry feed and other cereals by ochratoxin A.
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About 98.3% of the samples contained T-2 in different concentrations, between 1.0
and 80.0 μg kg-1. T-2 toxins are a family of more than twenty chemically related
compounds produced by several genera of fungi, including Fusarium and
Myrothecium. T-2 might cause inappetence, oral and enteral lesions, and impaired
immune responses in poultry (23). Destruction of the hematopoietic system, reduced
egg production, thinning eggshells, ruffled feathers, and reduced weight gain might
also occur as a consequence of T-2 toxicity (24). The overall outcome of T-2 toxicity
on several organ systems in poultry is reduced productivity, leading to economic
losses in broiler farms. In the European Union, the maximum permitted content of T-2
in feedstuff ranges between 100.0 and 200.0 μg kg-1 (25), while China has restricted
the presence of this mycotoxin to 80.0 μg kg-1 (23). None of the samples in the
current survey exceeded the maximal allowed T-2 content. However, the fact that
98.3% of the tested specimens were positive for this mycotoxin is an indication that
precaution measures are requisite to keep feedstuff free from fungal contamination.
Many species of Fusarium produce zearalenone, such as F. graminearum and
F. culmorum. In our survey, zearalenone was detectable in the tested samples at an
average concentration of 37.6 ± 2.5 μg kg-1. The European Commission recommends
that the maximal limit of zearalenone in cereals and cereal products used as feedstuff
is 2,000 μg kg-1 25). The positive samples in this study comprised zearalenone at
concentrations ranging from 1.0 to 180.0 μg kg-1, which means that none of the
samples contained exceeding concentrations of zearalenone. However, zearalenone
was present in 93.8% of the provenders, which implies that contamination of cereals
is likely to occur if suitable conditions for fungal growth become available.
The vast majority (94.8%) of feedstuff samples in this study contained at least
four of the five tested mycotoxins, including ochratoxin, which is considered one of
the most harmful fungal metabolic byproducts. Only 5.2% of the samples contained
higher concentrations of ochratoxin than the recommended limits, while none of the
other mycotoxins reached the internationally recognized maximal limits in the tested
feed samples. However, the cumulative toxic effects of these mycotoxins have not
been established yet. Therefore, future research might address studying the synergistic
toxic impact of two or more fungal metabolites on the growth and production of
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poultry. Understanding the toxic effects of simultaneously administered two or more
mycotoxins might result in the emergence of new regulations about the maximal
limits of fungal metabolites in livestock feed.
Author contribution: All authors contributed equally in the study design, collection
of data, and interpretation of results. HO Dyary prepared the manuscript.
Conflict of interest: The authors declare no conflict of interest.
مسح تقییم السموم الفطریة فی علف الدواجن ومکوناتھا فی السلیمانیة، إقلیم کردستان العراق
ھرسین محمود رحیم*، دیاری ھیوا عثمان*، ریبین کانبی مجید*، نھلة محمد سعید**
دانا عمر اسماعیل**
١فرع العلوم الأساسیة، کلیة الطب البیطری، جامعة السلیمانیة، ٢فرع الأحیاء المجھریة، کلیة الطب البیطری،
جامعة السلیمانیة، السلیمانیة ، إقلیم کردستان ، العراق
الخلاصة
السموم الفطریة ھی نواتج استقلابیة فطریة یمکن أن تلوث النظام الغذائی للحیوان والغذاء البشری، وللتحقق من
وجود السموم الفطریة فی علف الدواجن فی محافظة السلیمانیة، العراق، تم جمع ١٧٣ عینة مقسمة على العلف
الغذائی (عدد = ١٥٠ ) ومکونات العلف (عدد = ٢٣ ) من ٤٩ مزرعة دواجن ومطاحن علف. تم تحلیل العینات
،T- لتحدید وجود خمسة أنواع من السموم الفطریة وھی الأفلاتوکسین، الفومونیزین، الاوکراتوکسین، 2
والزیرالینون، وذلک باستخدام کتات خاصة. کل العینات احتوت علی الاوکراتوکسین بترکیز 1.0 الی ٦.٠
٠.١ میکروغرام/کغ، احتوت حوالی ٩٤.٨ ٪ من عینات العلف ومکونات ± میکروغرام/کغ وبمتوسط ٣.٤
بترکیزات zearalenone و ،T-2 ،aflatoxin ،ochratoxin الأعلاف على ما لا یقل عن أربعة سموم فطریة
مختلفة وتخلص الدراسة إلى أن توجد السموم الفطریة فی علف الحیوانات ومکوناتھا فی السلیمانیة، العراق،
وھناک حاجة لفھم التأثیرات السمیة التراکمیة بشکل أفضل عند ظھور العدید من السموم الفطریة فی العلف
الحیوانی، والتی قد تؤدی فی النتیجة فی إصدار لوائح جدیدة حول الحد الأقصى المسموح بھ من ترکیزات
السموم الفطریة فی الأعلاف.
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