2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 133-144, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000018-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.18

Mycotoxins Contamination Levels in Broiler Feeds and Aflatoxin

Residues in Broiler Tissues

Anwaar M. El-Nabarawy^1*, Elshaimaa Ismael², Khaled A. Shaaban¹, Sawsan S. El Basuni^3,Mohamed M. Batikh⁴

and Mohamed Shakal¹

¹Poultry Disease Department, Faculty of Veterinary Medicine, Cairo University, Egypt.

²Department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo University, Egypt.

³Avian and Rabbit Diseases Department- Faculty of Veterinary Medicine, Benha University, Egypt.

⁴Poultry Disease Department, Animal Health Research Institute, Kafr El-sheikh Provisional Lab, Egypt.

*Corresponding author’s E-mail: anwaar.elnabarawy@gmail.com; ORCID: 0000-0003-1618-6842

Received: 24 Feb. 2020

Accepted: 02 Apr. 2020

ABSTRACT

The need for regulations to limit the concentration of mycotoxins in feed and food requires the availability of data on

levels of contamination in different feedstuffs and estimation of the mycotoxin residues in animal meat. Therefore,

this study was conducted to determine contamination levels with different mycotoxins in broiler feed and aflatoxin

residues in broilers’ muscle and liver. A total of 194 feed samples, including 148 compound feeds and 46 feed

ingredients, were collected from feed manufacturing companies and broiler farms. Feed samples were analyzed for

detecting aflatoxins, ochratoxins, zearalenone, and fumonisins using an official analytical method. Moreover,

aflatoxin residues were estimated in 64 broiler’s muscle and liver tissues. Obtained results revealed that 100% of

compound broiler feed sampled from manufacturing companies were contaminated with aflatoxin and ochratoxin.

Also, 96.4% and 92.8% of compound broiler feed sampled from broiler farms were contaminated with aflatoxin and

ochratoxin, respectively. Furthermore, 30.6% and 91% of the feed samples were above the permissible levels of

aflatoxin and ochratoxin. Aflatoxin residues were detected in all meat and liver samples with levels above the

permissible limits. Large scale surveys for determination of different mycotoxins in poultry feed and mycotoxins

residues in poultry products are of national and international importance.

Key words: Aflatoxin, Broiler feed, Fumonisin, Mycotoxin residue, Ochratoxins, Zearalenone.

INTRODUCTION

al., 2016). Naturally contaminated broiler diet by

aflatoxin, ochratoxin, and zearalenone at permissible

levels resulted in a significant reduction in feed conversion

rate, body weight and antibody titers to infectious bursal

disease virus (El Nabarawy et al., 2020 ). Permissible

limits of mycotoxins in poultry feed and feed ingredients

are 20 ppb for aflatoxins (FDA, 2000; van Egmond and

Jonker, 2004a), 25 ng/g for ochratoxins (EC, 2006 ), 10

ppm for zearalenone (FDA, 2010) and 100 ppm for

fumonisin (FDA, 2001 ). Besides, zearalenone is receiving

serious attention for control, since it is considered a

mycotoxin indicator in addition to its synergistic action

with other mycotoxins, but its regulation needs further

attention (Park and Troxell, 2002).

The contaminated animal feed is the major cause of

exposure to mycotoxins in animals and therefore

ultimately in humans (Bryden, 2012 ). In the last few

decades, the increase in the incidence of various types of

cancers between various categories of people may be

contributed to dietary factors, aflatoxins and agrochemical

Mycotoxins are secondary metabolites produced by

mycotoxigenic fungi infecting feed ingredients under field

and storage conditions and they remain long after the

death of the mold (Aravind et al., 2003). Moreover, the co-

occurrence of mycotoxins in poultry feed is more

prevalent than a single mycotoxin (Atalla et al., 2003;

Kana et al., 2013; Kovalsky et al., 2016). Concomitant

contamination by several mycotoxins may augment their

toxic effects (Huff and Doerr, 1981; Chandrasekaran

1996; Pappas et al., 2014 ). Aflatoxin, ochratoxin A,

zearalenone, T-2 toxin, vomitoxin, and fumonisin are the

most significant mycotoxins affecting poultry species

through naturally contaminated feeds and have serious

toxic effects and probable synergistic properties (Njobeh

et al., 2012 ). The combined effect of ochratoxin and

aflatoxin at a dose of 23 and 16 ppb; respectively, resulted

in depressed T and B lymphocytes activity, suppressed

immunoglobulin and antibody production (El Nabarawy et

To cite this paper: El-Nabarawy AM, Ismael E, Shaaban KhA, El Basuni SS, Batikh MM and Shakal M (2020). Mycotoxin Contamination Levels in Broiler Feeds and Aflatoxin

Residues in Broiler Tissues. J. World Poult. Res., 10 (2S): 133-144. DOI: https://dx.doi.org/10.36380/jwpr.2020.18

133

El-Nabarawy et al., 2020

contaminated foods (Maiyoh and Tuei, 2019 ). In the same

the FAO for detection and determination of aflatoxins,

ochratoxin A, zearalenone, and fumonisin mycotoxins.

Requirements and consumable materials

respect, van Egmont and Jonker (2004b) reported that

dietary contamination of aflatoxins represents a major risk

to public health and aflatoxins are known to have a strong

hepatotoxic and carcinogenic effect. In general, the

consumption of contaminated food induces neurotoxic,

A) Mycotoxins columns: Aflatest, ochratest,

zearalatest, and fumonitest, each type of toxin has its

specific column which is consumed for one sample.

B) Chemicals and reagents: Methanol, HPLC grade

(4X4L), Distilled deionized water, and Nonionized sodium

chloride (salt, NaCl), Afla test developer, Phosphate

buffered saline (PBS) Lot: 17021PBS, Ochratest eluting

solution Lot: 17061E, 0.1 tween PBS Lot: 17011G2,

Zearalatest developer Lot: 102594-4, and fumonisin A and

B developer, were utilized in the analysis.

C) Mycotoxins calibration standards: One vial;

each of 3 levels, for aflatest, ochratest, zearalatest, and

fumonitest calibration.

D) Fluorometer series 4: Fluorometer series 4

provides an accurate and sensitive measurement for

aflatoxin, ochratoxin, zearalenone, and fumonisins

mycotoxins.

immunosuppressive,

teratogenic,

mutagenic

and

carcinogenic effects in humans (Fernandez et al., 2000 ).

Mohd-Redzwan et al. (2013) reported on cumulative

evidence from humans revealed a strong linkage occurs

between aflatoxin and hepatic chronic carcinoma (HCC).

Also, acute aflatoxicosis induced abdominal pain,

vomiting, edema, and death. Moreover, aflatoxicosis

outbreak was recorded four times in Kenya from 2004 to

2014, with mean 600 individuals were affected, and 211

deaths were estimated from this outbreak (Awuor et al.,

2017 ). Hence, the European community and many other

countries have determined 2 ng/g aflatoxin B1 (AFB1) and

4 ng/g total aflatoxin as maximum tolerance levels in

human food products (Van Egmond and Jonker, 2004b;

Wild and Gong, 2009 ). The accumulation of AFB1

residues in broiler meat and liver leads to the toxin

carryover through the food chain. AFB1 residues may

persist unchanged in the liver even when exposure levels

are relatively low (Magnoli et al., 2002 ). The occurrence

and incidence of aflatoxins, ochratoxins, and zearalenone

in chicken meat are alarming and urged the need for

continuous monitoring for these toxins in chicken meat

and eggs (Iqbal et al., 2014 ).

Extraction of mycotoxins

Mycotoxins were extracted from representative

samples of broilers feed where 50g of each ground sample

was mixed with 5g sodium chloride analar and 100 ml

methanol: water (80: 20 by volume) solution. The mixture

was blended at high speed for 1 minute and then the

extract was filtered through fluted filter paper. For

aflatoxin; 10ml filtered extract was mixed with 40ml

distilled water, then filtered through a glass microfiber

filter (VICAM, 1999). For ochratoxin; 10ml filtered

extract was mixed with 40ml phosphate-buffered saline

(PBS), then filtered through a 1.5 µm glass microfiber

filter (VICAM, 2008 ). For zearalenone, 1ml filtered

extract was mixed with 49ml distilled water and then

filtered through microfiber filter (VICAM, 2013 ). For

fumonisin, 10ml filtered extract was mixed with 40ml of

0.1% Tween-20/2.5%PEG/PBS wash buffer, then filtered

through a 1.5 µm microfiber filter (VICAM, 2015).

For aflatoxins, 10ml of the filtered diluted extract

was passed through the affinity column at a rate of about 1

drop/second (10ml = 1.0g sample equivalent). Then, the

column was washed with 10ml distilled water at a rate of

1-2 drops/second. The affinity column was then eluted

with 1.0ml HPLC grade methanol at a rate of 1

drop/second, and the elute was collected in a glass cuvette,

to where 1ml of freshly made test developer solution was

added (VICAM, 1999). For ochratoxin, 10ml of the

filtered diluted extract was passed through the affinity

column, then the column was washed with 10ml 0.1%

There are very limited data on the epidemiological

status of mycotoxins in broilers feed and meat in Egypt.

Therefore, the objective of this work was to study the

situation of the contamination levels of aflatoxin,

ochratoxin, zearalenone, and fumonisins in broilers feed

and the level of aflatoxin residues in broiler tissues.

MATERIALS AND METHODS

Sampling, extraction and determination of

mycotoxins in broilers feed and feed ingredients

Sampling of broiler feed

A surveillance study was carried out on different

mycotoxins contamination levels in broilers feed in 2014

and 2018. A total of 194 broiler feed samples including

148 compound broilers feeds and 46 feed ingredients were

collected from feed manufacturing companies (n=37) and

broilers farms (n= 111) for mycotoxins detection and

determination. The samples were collected by

representative method according to the recommendation of

134

J. World Poult. Res., 10(2S): 133-144, 2020

Tween 20/PBS followed by 5ml purified water, and 1.5ml

different governorates (1, 2 and 3), to determine aflatoxin

residues.

Extraction of aflatoxin residues

The aflatoxin residues were extracted from Liver and

muscles where 20g of each ground sample was added to

100ml of the extraction solvent (70% methanol), in which

the ratio of sample to extraction solvent is 1:5 (w/v). After

blending for 2min, 5-10ml of the extract was filtered

through a Whatman filter paper (Kensler et al., 2003;

Williams et al., 2004; Klich, 2007 ).

OchraTest^TMElution Solution was used to elute the

column (VICAM, 2008 ). For zearalenone, 1ml of the

filtered diluted extract was passed through the affinity

column, then the column was washed with 10ml distilled

water, and eluted with 1ml HPLC grade methanol, on

which a 1ml ZearalaTest

Developer was added

(VICAM, 2013 ). For fumonisin, 5ml of the filtered diluted

extract was passed through the affinity column, then the

column was super-washed with 5ml of 0.1% Tween

20/2.5% PEG/PBS followed by 5ml of PBS, and 1ml

HPLC grade methanol was used to elute the column, and a

1ml mixture of Developers A and B was added (VICAM,

2015 ).

ELISA screening of total aflatoxin residues in the

muscles and liver of broiler chickens

The concentration of total aflatoxin residues in the

tissue of muscles and liver of broiler chickens was

determined by a solid-phase competitive inhibition

enzyme-linked immune-assay (ELISA), using HELICA®

Low Matrix Total Aflatoxin Assay Kits (HELICA

Biosystems, Inc. Santa Ana, CA). The extracted filtrate

and the aflatoxin- horse-radish peroxidase (HRP) enzyme

conjugate were mixed and added to the antibody-coated

microwell. After a step of 5 washes, an enzyme-substrate

was added, and the blue color was developed. This was

followed by the addition of a stop solution. Absorbances

were read at 450 nm by a computerized microplate reader

and the optical densities (OD) of the samples were

compared to the ODs of the kit standards and a result was

determined by interpolation from the standard curve and

the total concentration expressed in ng/g (Kensler et al.,

2003; Williams et al., 2004; Klich, 2007 ).

Standardization of Fluorometer series 4

Mycotoxin calibration standards (1 vial each of 3

levels). For aflaTest, calibration settings are adjusted to -1,

27, and 13±2, with detection range 0 – 100 ppb, and limit

of detection 1ppb (VICAM, 1999). For Ochratest,

calibration settings are adjusted to -1.3, 30, and 14±2, with

detection range 0 – 100 ppb, and limit of detection 2ppb

(VICAM, 2008 ). For ZearalaTest, calibration settings are

adjusted to 16, -2, and 8±2, with detection range 2 – 100

ppm, and limit of detection 2ppm (VICAM, 2013 ). For

FumoniTest, calibration settings are adjusted to -0.50, 12,

and 5.8±0.3, with detection range 0 – 10 ppm, and limit of

detection 0.25ppm for corn (VICAM, 2015).

Determination of mycotoxins in broilers feed

Fluorometer series 4 provides accurate and sensitive

measurement of mycotoxins. AflaTest® WB SR,

Statistical analysis

OchraTest^TM, ZearalaTest ^TM, and FumoniTest

have

Descriptive analysis of mycotoxin levels was

performed using PASW Statistics software, version 18.0

(SPSS Inc., Chicago, IL, USA).

been used for quantitative measurement of aflatoxins,

ochratoxin A, zearalenone, and fumonisins in broilers feed

and feed ingredients. These test kits are based on

immunoaffinity chromatography. The fluorescence of the

mycotoxin in the elution solution can then be measured in

a fluorometer series 4. Quality assurance and validation of

Series-4 Fluorometer procedures were validated by the

AOAC Research Institute under the Performance Tested

Program to detect and determine mycotoxins, and were

licensed under certification mark no. 940801.

RESULTS AND DISCUSSION

Feedstuffs contamination by mycotoxins represents a great

threat to broilers industry and public health. As shown in

Table 1 and Figures 1 and 5, rates of mycotoxins in

compound broiler feed in 2014 and 2018 revealed that all

37 analyzed samples were positive to aflatoxin and

ochratoxin and their levels ranged from 1 to 55 ppb (mean

= 14.33 ppb in 2014 and 20.36 ppb in 2018) and 1.8 to 71

ppb (mean = 27.85 ppb in 2014 and 3.12 ppb in 2018),

respectively. In addition, zearalenone and fumonisins were

detected in 21 (56.8%) and 6 (16.2%) of the examined

samples, respectively, with levels range of 0.48 to 10 ppm

(mean = 1.06 ppm in 2014; 3.80 ppm in 2018) and 1.2 to

12 ppm (mean = 7.17 ppm), respectively.

Sampling, extraction and ELISA screening of

total aflatoxin residues in the muscles and liver of

broiler chickens

Sampling of broilers liver and meat

Upon obtaining the approval of the Institutional

Animal Care and Use Committee (IACUC) on the Animal

Use Protocol (AUP) (VetCU10102019093); 64 broilers’

muscles and liver were collected from markets located in

135

El-Nabarawy et al., 2020

Table 1. Levels of mycotoxins contamination in broiler feed sampled from manufacturing companies in 2014 and 2018

No. of

sample

Aflatoxin Ochratoxin Zearalenone Fumonisin

No. of

sample

Aflatoxin Ochratoxin Zearalenone Fumonisin

Year

ppb

ppm

ppb

ppm

8.9

3.3

7.5

8.9

3.3

1.2

0.79

1.2

0.79

8.3

1.2

8.3

2.9

2.3

1.8

1.9

2.8

2.3

2.7

2.2

2.5

3.9

5.1

7.2

8.6

8.2

0.62

0.52

0.48

0.59

4.2

2014

2018

1.2

- Not analyzed.

Table 2. Levels of mycotoxins contamination in broiler feed sampled from broiler farms in 2014.

Sample

No.

Aflatoxin

ppb

Ochratoxin

ppb

Zearalenone

ppm

Fumonisin

ppm

Sample

No.

Aflatoxin

ppb

Ochratoxin

ppb

Zearalenone

ppm

Fumonisin

ppm

8.6

1.3

8.9

9.7

1.6

8.5

5.7

7.5

3.4

3.5

1.3

7.5

8.9

3.3

1.1

1.4

0.98

2.4

2.3

6.1

3.6

5.2

3.8

4.1

5.6

1.1

0.96

1.3

1.4

1.2

0.79

136

J. World Poult. Res., 10(2S): 133-144, 2020

Table 3. Levels of mycotoxins contamination in broiler feed sampled from broiler farms in 2018.

Sample

No.

Aflatoxin

ppb

Ochratoxin

ppb

Zearalenone

ppm

Fumonisin

ppm

Sample

No.

Aflatoxin

ppb

Ochratoxin

ppb

Zearalenone

ppm

Fumonisin

ppm

8.6

1.2

1.4

8.5

5.6

1.9

3.6

4.9

2.3

3.8

4.1

1.1

0.96

8.1

6.2

7.8

8.4

7.9

1.5

100

140

560

1.7

3.8

2.1

1.6

1.4

3.7

2.4

8.9

1.1

0.98

2.4

6.1

5.2

Table 4. Levels of mycotoxins contamination in feed ingredients

Type of

feed

ingredient

Type of feed Sample Aflatoxin Ochratoxin Zearalenone Fumonisin

Sample Aflatoxin Ochratoxin Zearalenone Fumonisin

ingredient

No.

ppb

ppm

No.

ppb

ppm

8.5

3.3

8.5

3.3

8.5

5.2

1.3

5.5

Yellow corn

5.2

1.3

Soya bean

3.3

5.5

3.5

Wheat germ

1.3

5.2

1.3

5.2

Nutritive

concentrates

Lysine

Feed additive

- Not analyzed.

137

El-Nabarawy et al., 2020

Table 5. Mean level of total aflatoxin residues in liver and muscle samples collected from commercial broiler chickens from

different provinces of Egypt

Total aflatoxin residues (ng/g) in tissue samples (n)

Breast (15)

Thigh (28)

Mean ± SE

Liver (21)

Mean ± SE

Province

Mean ± SE

Range

20.30±2.80

17.00±0.90

20.30±1.10

19.2±1.6

13.15-30.00

12.90±4.90

37.18±4.22

19.20±1.40

23.09±3.51

0.20-27.10

22.30±4.40

33.66±3.42

17.70±2.70

24.55±3.51

11.30-34.90

16.05-17.85

17.20-23.60

13.15-30.0

13.15-69.40

13.35-24.80

0.20-69.40

6.80-4.20

7.10-28.70

7.10-54.20

Total

SE: Standard error. n: number

Aflatoxin (ppb)

Ochratoxin (ppb)

0.1 to 10 11 to 20

Zearalenone (ppm)

31 to 50 > 51

Fumonisin (ppm)

21 to 30

Figure 1. Mycotoxins contamination levels in compound broilers feed sampled from feed manufacturing companies, Egypt

Aflatoxin (ppb)

0.1 to 10

Ochratoxin (ppb)

Zearalenone (ppm)

Fumonisin (ppm)

11 to 20 21 to 30

31 to 50 51 to 99 100 to 600

Figure 2. Mycotoxins contamination levels in compound broilers feed sampled from broiler farms, Egypt

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J. World Poult. Res., 10(2S): 133-144, 2020

Aflatoxin (ppb)

Ochratoxin (ppb)

1 to 10 11 to 20

Zearalenone (ppm)

31 to 50

Fumonisin (ppm)

21 to 30

Figure 3. Mycotoxins contamination levels in feed ingredients

Within

permisible

range

Ochratoxin in compound feed

Aflatoxin in compound feed

Above

permissible

limit

30.6%

Within

permisible

range

Above

permissible

limit

69.4%

91%

Ochratoxin in Feed ingredients

Aflatoxin in Feed ingredients

Above

permissible

limit

Within

permisible

range

19.6%

22.7%

Within

permisible

range

Above

permissible

limit

80.4%

78.3%

Figure 4. Percentage of aflatoxin and ochratoxin contamination exceeding the permissible limits (20 and 5 ppb; respectively

according to FDA, 2000 and EC, 2006) in compound broilers feed and feed ingredients samples, Egypt.

139

El-Nabarawy et al., 2020

38.49

29.19

27.85

22.40

20.36

15.45

14.33

9.65

7.17

6.19

5.38

3.80

3.12

2.41

2014

1.06

2014

2018

Factories

Farms

Aflatoxin (ppb)

Ochratoxin (ppb)

Zearalenone (ppm)

Fumonisin (ppm)

Figure 5. Mean levels of mycotoxins contamination in compound broilers feed in 2014 and 2018, Egypt

As illustrated in Tables 2 and 3 and Figures 2 and 5, rates

of mycotoxins in compound broiler feed in 2014 and 2018

revealed that 96.4% and 92.8% of totally analyzed

samples were positive to aflatoxin and ochratoxin with

levels ranged from 1 to 560 ppb (mean = 15.45 ppb in

2014; 38.49 ppb in 2018) and 1.7 to 90 ppb (mean = 29.19

ppb in 2014; 22.40 ppb in 2018), respectively. Also,

zearalenone and fumonisins were detected at a rate of

45.5% and 23.4%, respectively, with levels range of 0.79

to 70 ppm (mean= 2.41 ppm in 2014; 9.65 ppm in 2018)

and 1.2 to 19 ppm (mean= 6.19 ppm in 2014; 5.38 ppm in

2018), respectively. Table 4 and Figures 3 and 5 revealed

that aflatoxin and ochratoxin in different types of feed

ingredients were detected with rates 54.5% and 95.5%,

respectively and their levels ranged from 7 to 44 and 1.3 to

48 ppb, respectively. In addition, the prevalence of

zearalenone and fumonisin was 20.5% and 4.5% in the

analyzed samples with a range of 5.5 to 19 and 3.5 to 15

ppm, respectively. The obtained results revealed that the

contamination levels of aflatoxins and ochratoxins were

above the permissible values (20 and 5 ppb, respectively)

in compound broilers feed at 30.6% and 91% and in feed

ingredients at 19.6% and 78.3%, respectively (Tables 1-4,

and figure 4).

levels of aflatoxins in poultry feed and their ingredients

are parallel to those previously reported in Egypt and other

countries. In a previous study, from 87-broiler feed

samples collected from a poultry feed production unit in

Kuwait, aflatoxin was detected in broiler starter at 0.48

ppb level (range 0 to 3.26 ppb), and in broiler finisher at

0.39 ppb level (range 0 to 1.05 ppb) (Beg et al., 2006 ).

Moreover, in Kuwait, aflatoxins were detected in 63.9% of

poultry feed; with range 6 to 201 ppb for AFB1, and 8 to

335 ppb for aflatoxin B2 (Natour et al., 1983 ). In Egypt,

80% of the sampled maize contained aflatoxins at 480 ppb

level (Mahmoud, 1993 ). In Turkey, 71% of layer feed

samples showed an aflatoxin level of less than 5 ppb, and

only 2 samples exceeded acceptable levels (20-ppb)

(Nizamlýolu and Oguz, 2003). In Bangladesh, poultry feed

showed aflatoxin levels ranged from 7 to 160 ppb

(Dawlatana et al., 2002 ), while 216- feed ingredients from

a poultry feed factory in India, showed contamination with

aflatoxin in 60% of the mixed feed samples, with range 10

to 1500 ppb (Thirumala-Devi et al., 2002 ). In Nigeria,

analysis of 102 samples of poultry feed and feed

ingredients from poultry farms showed AFB1 in 83% of

feed samples (range, 0.5–760 ppb; mean, 74 ppb)

(Akinmusire et al., 2019 ). In South Africa, aflatoxins

reported the lowest prevalence (30% of samples) with

levels ranged between 0.2 to 71.8 ppb (mean: 9.0 ppb)

(Njobeh et al., 2012). In Cameroon, Abia et al. (2013)

analyzed 20 feed samples pools collected from different

The FDA restricts levels of aflatoxin in food

and animal feeds to 20 ppb and the EU limits levels

of aflatoxin to 15 ppb (Yang et al., 2020 ). The reported

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J. World Poult. Res., 10(2S): 133-144, 2020

poultry farms and reported the rate of contamination with

detected Zearalenone in 86% of the poultry feed samples

(median 50ꢀppb).

The EU has set a maximum limit of fumonisin in raw

aflatoxins of 75-95%. In Argentina, Greco et al. (2014)

detected aflatoxins in 90% of poultry feed samples

(median 2.685ꢀppb).

corn 4000 ppb (Yang et al., 2020). The high incidence of

detectable fumonisin is similar to that found by previous

studies conducted in several countries. In Kuwait,

fumonisin ranged from 1.4 to 3.2 ppm in broiler feed

samples collected from a feed factory (Beg et al., 2006). In

Nigeria, fumonisin B1 was detected in most of the samples

(97%) (Range, 37–3760 ppb; mean, 1014 ppb)

(Akinmusire et al., 2019 ), as well as Ezekiel et al. (2012)

detected fumonisins in 83% of 58 commercial poultry feed

samples in Nigeria, with concentrations range, 31– 2733

ppb; and mean, 964 ppb. In South Africa, Njobeh et al.

(2012) detected fumonisins in 87% of 92 compound feeds

samples with concentrations range, 104–2999 ppb; and

mean 903 ppb. In Cameroon, Abia et al. (2013) analyzed

20 feed samples pools collected from poultry farms and

reported fumonisins in 100% of samples, with

concentrations range, 16– 1930 ppb; and mean, 468 ppb.

In Taiwan, Tseng and Liu (2001) detected fumonisin in

few samples of imported maize at level exceeded 0.3 ppm.

In Iran, Shephard, et al. (2002) detected fumonisin B1 in

maize at average levels of 3.18 ppm (range 0.68 to 7) and

0.22 ppm (range <0.01 to 0.88) in two areas. Likewise, In

the United Kingdom, maize feedstuffs were reported to

frequently contain fumonisin B1 and B2 at levels up to 5

ppm (Scudamore et al., 1997 ). In Argentina, Greco et al.

(2014) detected Fumonisins in all the samples in a range

of 222–6,000ꢀppb (median 1,750ꢀppb).

The combined toxic effects of aflatoxin, ochratoxin,

zearalenone, and fumonisins in feed and food might pose a

veterinary and public health risk. Overall, results indicated

that 86.60% of compound feed and feed ingredient

samples contained two or more mycotoxins. Combined

contamination with aflatoxin and ochratoxin was detected

in 76.8% of the samples. In Argentina, Greco et al. (2014)

found that 90% of poultry feed samples were

contaminated with ochratoxin and aflatoxins. Beg et al.

(2006 ) detected the coexistence of ochratoxin A,

fumonisin, and zearalenone in poultry feed from Kuwait,

although in lower concentrations than the permissible

limits defined for the poultry feed. In Bangladesh,

Dawlatana et al. (2002) confirmed the possibility of

multiple mycotoxins contamination in poultry feed and

detected five mycotoxins in one sample of maize. While in

Nigeria, Akinmusire et al. (2019) reported contamination

with at least four mycotoxins in 102 samples of feed and

feed ingredients collected from poultry farms, as they

detected fumonisin B1 in most of the samples (97%) and

Since ochratoxin A was discovered in 1965, it has

been ubiquitous as a natural contaminant of moldy food

and feed. The multiple toxic effects of ochratoxin A are a

real threat to human beings and animal health. Humans

exposed to ochratoxin A can develop a range of chronic

disorders and plays the main role in the pathogenesis of

some renal diseases including Balkan endemic

nephropathy, kidney tumors occurring in certain endemic

regions of the Balkan Peninsula, and chronic interstitial

nephropathy occurring in Northern African countries and

likely in other parts of the world (Malir et al., 2016 ).

Worthwhile, the EU has set a maximum limit of 5 ppb for

cereal products (Yang et al., 2020 ). In our study, the vast

contamination of the poultry feed and feed ingredients

with ochratoxin-A agrees with several previous reports. In

Kuwait, broiler feed showed ochratoxin levels ranged

from 4.6 to 9.6 ppb (Beg et al., 2006 ). In Argentina,

ochratoxin was found in 38% of the poultry feed samples

with levels ranged from 25 to 30 ppb (mean 27 ppb)

(Dalcero et al., 2002 ). In South Africa, ochratoxin reported

the lowest prevalence (4% of samples) with levels ranged

between 6.4 and 17.1 ppb (mean: 9.9 ppb) (Njobeh et al.,

2012). In Cameroon, Abia et al. (2013) analyzed 20 feed

samples pools collected from different poultry farms and

reported the ochratoxins rate of 80-90%. In Argentina,

Greco et al. (2014) detected ochratoxin in 90% of the

poultry feed samples (median 5ꢀppb) and aflatoxins

(median 2.685ꢀppb).

The EU has set a concentration limit for zearalenone

in raw maize to 100 ppb and in cereal products to 20 ppb

(Yang et al., 2020 ). The incidence of detectable

Zearalenone is similar to that found by previous studies

conducted in several regions. In Cameroon, Abia et al.

(2013) analyzed 20 feed samples pools collected from

different poultry farms and reported feeds contamination

with zearalenone in 100% of samples, with mean

concentrations 155 (range 0.7-600) ppb. In Kuwait and

Egypt, zearalenone ranged from 46.4 to 67.6 ppb in broiler

feed samples collected from a poultry feed production unit

(Beg et al., 2006), and 40 ppb in 80% of the maize

samples integrated in poultry feeds (Mahmoud, 1993 );

respectively. In Swedish, zearalenone was detected in 2 of

68 mixed feed samples, with one showed a very high level

(1200 ppb) and the other was 100 ppb (Pettersson and

Kiessling, 1992 ). In Argentina, Greco et al. (2014)

141

El-Nabarawy et al., 2020

AFB1 in 83% of feed samples. Also, Scudamore et al.

ingredients and the combined contamination with aflatoxin

and ochratoxin was found in 76.8% of positive samples.

Aflatoxin residues that exceeded the recommended

permissible limit for human consumption were detected in

100% of broiler meat and liver samples.

(1997) detected multi-mycotoxin contamination with both

aflatoxin and fumonisin in some samples of maize.

Magnoli et al. (2002) reported that fumonisins had the

highest incidence (97%) followed by AFB1 (46%) and

zearalenone (18%). Moreover, Aravind et al. (2003)

DECLARATIONS

analyzed

contaminated with mycotoxins in India, and reported

contamination with aflatoxins, ochratoxin, and

zearalenone toxins, and suggested the possible synergistic

toxic effect from the combination of multiple mycotoxins

offered in the contaminated feed.

commercial broilers diet naturally

Competing interests

The authors have no competing interests.

Acknowledgment

This study was carried out in Mycotoxin Research

Lab and partially supported by the project titled

"Mycotoxicosis, the natural potent immunosuppressive

carcinogen of veterinary and public health concern

scientific research sector of Cairo University.

The presence of mycotoxins in animal products is the

most critical aspect and a serious factor affecting meat

quality that has a special public health concern. As

presented in Table 5, aflatoxins residues were detected in

all examined tissue samples that collected from the

commercial broiler chickens in three provinces, Egypt

with detectable levels above the recommended permissible

limit for human consumption (4 ng/g) according to FDA

regulations. These results came in agreement with

Herzallah (2009) who documented the levels of AFB1,

AFB2, aflatoxin G1 and G2 ranged from 1.10 to 8.32

mg/kg and 0.15 to 6.36 mg/kg in imported and fresh meat

samples. Moreover, Iqbal et al. (2014) and Markov et al.

(2013) reported that AFB1 was detected in 10% and 35%

of the collected chicken samples from Croatia and

Pakistan, with maximum levels 3.0 mg/kg and 8.01 mg/kg,

respectively.

Collectively, mean total aflatoxin residues in the

liver of all examined samples were higher than their levels

in the breast and thigh muscle tissues (Table 5). Similarly,

Magnoli et al. (2011) and Herzallah et al. (2014) reported

aflatoxin and its metabolites residues in the different

tissues of broiler chicks fed aflatoxin-contaminated diets

with different concentrations and various treatments.

These recorded results related to the ability of poultry to

metabolize and eliminate aflatoxin from their tissues.

Authors’ contribution

Anwaar Mettwally El-Nabarawy designed the

experiment, provided the facilities and the material

needed,

performed

mycotoxins

detection

and

determination, wrote and revised the manuscript.

Elshaimaa Ismael contributed to mycotoxins detection and

determination, designed the figures, wrote and revised the

manuscript. Sawsan El Basuni collected broiler tissue

samples, performed the extraction of aflatoxin residues

and wrote the manuscript. Khaled Shaaban contributed to

mycotoxin detection and determination. Mohamed

Mohamed Ismail Batikh collected feed and broiler tissue

samples

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