Food Additives and Contaminants: Part B Vol. 5, No. 1, March 2012, 11–15

Natural occurrence of aflatoxins (B1 and M1) in feed, plasma and raw milk of lactating dairy cows in Beja, Tunisia, using ELISA Samir Abbe`sab*y, Jalila Ben Salah-Abbe`say, Yousra Bouraouia, Sarra Oueslatia and Ridha Oueslatia a Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences Bizerte, University of Carthage, Tunisia; bAnimal Biotechnology Department, Higher Institute of Biotechnology of Beja, University of Jendouba, Beja, Tunisia

(Received 13 January 2011; final version received 10 November 2011) Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B1 (AFB1). Milk samples were analysed for aflatoxin M1 (AFM1). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB1 and AFM1, respectively, while the limit of detection (LOD) was 0.01 mg/kg or mg/l for both mycotoxins. Results revealed the presence of AFB1 in 84.4% of the feed samples (mean 18.7  1.4 mg/kg), and 39.2% of the plasma-examined samples (median 7.1  1.0 mg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5 mg/kg in animal feed, respectively. AFM1 was detected in 60.7% of the cow raw milk samples examined (median 13.6  1.4 mg/l). Contaminated levels were higher than the EU limit of 0.05 mg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination. Keywords: raw milk; feedstuff; plasma; aflatoxin B1; aflatoxin M1; occurrence; ELISA; risk

Introduction In Tunisia, aflatoxins (AFs) have been found to contaminate different agricultural commodities such as pistachio nuts, grain and feedstuffs (Ghali et al. 2008, 2009; Bensassi et al. 2010). They are causative agents in human hepatic and extrahepatic carcinogenesis (Kamakar 2005; Abbe`s et al. 2008). Aflatoxin M1 (AFM1) is derived from AFB1 following ingestion of feed contaminated with AFB1 and transferred to milk and consequently milk products destined for human consumption (Trucksess et al. 1983). The kind of animal feed and the harvesting time and temperature could be effective parameters in regard to contamination (Kuiper 1999). The limits for AFB1 fixed by the Tunisian National Standard (TNS 1983) and the European Commission in animal feeds and complete feeding-stuffs for dairy animals are 20 and 5 mg/kg, respectively (European Commission 2003). In milk, the European Commission set the AFM1 maximum limit at 0.050 mg/kg (European Commission 2006), while in Codex the maximum AFM1 concentration is set at 0.5 mg/kg

*Corresponding author. Email: [email protected] yThese two authors have contributed equally to the manuscript. ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis

(Codex Alimentarius 1995). It is resistant to thermal inactivation, pasteurisation and autoclaving (Deshpande 2002; Soha et al. 2006). Production of yoghurt, cheese, cream, milk powder or butter does not lead to loss of AFM1, although it is redistributed differentially into the products resulting from these processes (FAO/WHO 2002). Many researchers have reported a linear relationship between the amount of AFM1 in milk and AFB1 in feed consumed by animals (Bakirci 2001), as it will be metabolised and excreted as AFM1 in the urine and also in milk (Gurbay et al. 2006). Apart from this, exposure to AFs can be through ingestion of contaminated mothermilk containing AFM1. Sources affecting levels of AFM1 contamination in milk are of animal feed, ecologic and economic factors on the farm and also farm management. The region of Beja is the main agricultural area in Tunisia and produces more than 33% of the national need of milk. Meanwhile, there is little information about the natural occurrence of AFB1 in feedstuff and AFM1 in milk produced in this region.


S. Abbe`s et al.

Consequently, the aim of the present study was to analyse, in randomly collected samples, the presence of AFB1 in feed and plasma of dairy lactating cows, together with AFM1 in raw milk, by ELISA method.

Then, the chloroform phase was separated and evaporated with a Rotavapor. The extract was dissolved in 1 ml methanol and 35 ml water. Then a similar procedure as mentioned for feed samples was followed. In some cases, the milk, plasma or feed samples were diluted 1 : 10.

Materials and methods Chemicals and kits AFB1 and AFM1 were purchased as pure crystal from Sigma Chemical Co. (St. Louis, MO, USA). The RIDASCREEN AFB1 and AFM1 test kits were purchased from R-Biopharm AG (Darmstadt, Germany) and the immunoaffinity columns AFs Easy-extract were obtained from Rhoˆne diagnostics technologies (Glasgow, UK). All other chemicals used were of analytical grade.

Samples A total of 226 samples (112 raw milk samples, 56 blood samples and 58 feed samples) were collected from cows at farms and local markets in Beja province, Tunisia. Samples were collected from November 2008 until April 2010. All samples were kept in refrigerator before analysis.

Preparation of samples Feed samples AFB1 was extracted using immunoaffinity technique according to Bircan (2005) and Arranz et al. (2006). Briefly, 10 g of dried feed was dissolved in a methanol:water solution (80:20 v/v), shaken at 150 rpm for 45 min and filtered. Then, 5 ml were eluted with 45 ml of bidistilled water through an immunoaffinity column previously washed with 20 ml of phosphate-buffered saline solution (pH 7.4). The column was washed with 5 ml water and slowly eluted with 2.5 ml methanol. The extract was dried under nitrogen, redissolved in 1 ml methanol:water (70:30) and filtered. An aliquot of 100 ml per well was used for the ELISA.

Milk samples AFM1 was extracted from milk of lactating cows according to Mortimer et al. (1987) and Kussak et al. (1995). Briefly, in order to defat the milk, 10 ml was centrifuged at 7000 rpm for 10 min at 4 C and the upper oily phase was collected. Then a similar procedure as mentioned for feed samples was followed.

Plasma samples AFB1 was extracted from plasma samples by putting it in a separatory funnel with 35 ml of chloroform.

Analytical procedure Quantitative analysis was carried out using a commercial ELISA kit (RIDASCREEN, Darmstadt, Germany), which also contained most of the reagents used and the aflatoxins standard solutions used for the construction of the calibration curve, at levels of 0, 0.005, 0.01, 0.02, 0.04 and 0.08 mg/l. The 100 ml standard solutions and prepared sample portions were added into the wells, mixed by shaking gently and incubated for 30 min at room temperature in the dark. At the end of the incubation time the liquid in the wells was poured out, and the microwell holder was tapped upside down on an absorbent paper to remove the remainder of the liquid. The wells were washed twice with 250 ml washing buffer. Enzyme conjugate (100 ml; peroxidase conjugated AFB1 or AFM1) was added to each well and mixed gently by shaking the plate manually and incubated for 15 min at room temperature in the dark. At the end of the incubation time, the liquid in the wells was poured out. The wells were washed three times with 250 ml washing buffer. Substrate/chromogen (100 ml) were then added to each well and incubated for another 15 min at room temperature in the dark. Following the addition of the stop solution (100 ml) to each well, the absorbance was measured photometrically at 450 nm against an air blank. The evaluation of AFB1 and AFM1 was carried out using the mean of the absorbance values obtained for the standards and the samples, divided by the absorbance value of the first (zero) standard and multiplied by 100. The zero standard is thus set to 100% and the absorbance values are expressed as percentages. The absorption is inversely proportional to the AFB1 or AFM1 concentration. The calibration curve was linear in the 0.01–0.08 mg/l range. To determine recovery, standard AFB1 or AFM1 solutions at different concentrations (2, 5 and 10 mg/kg or mg/l) were added to non-contaminated feed, plasma or raw milk samples in triplicate. After extraction of the spiked material, the residue was dissolved in 200 ml of toluene/acetonitrile (98/2, v/v) solution and analysed by ELISA at 450 nm.

Statistical analyses A randomised block experiment was used to evaluate the differences between AFB1 or AFM1 occurrence levels of the samples. Furthermore, Duncan multiple

Food Additives and Contaminants: Part B comparison test was applied to obtain significance level between sampling periods. A multi-sample ANOVA was used to determine statistical significance between differences of means. P value 50.05 was accepted as significant.

Results and discussion From Table 1 can be seen that recovery values of AFB1 obtained from spiked feed and plasma and of AFM1 obtained from spiked raw milk were in the range of 80%–95%, which is in accordance with the value of 95%  14% as given by the manufacturer. The highest recovery rate was obtained at 10 mg/kg. The recovery

Table 1. Evaluation of the extraction method by the determination of AFB1 and AFM1 recovery in spiked blank samples. AFB1 (mg/kg or mg/l) Number


Recovered (mean  SD)

% Recovery



2 5 10

1.60  0.04 4.33  0.06 8.89  0.08

80 87 89



2 5 10

1.66  0.05 4.73  0.03 9.56  0.03

83 95 96


AFM1 (mg/l) Number of analyses Raw milk


Recovered (mean  SD)

% Recovery

2 5 10

1.62  0.04 4.55  0.16 9.23  0.06

81 91 92


percentage from feed samples was slightly lower when compared to plasma or milk samples, especially at the highest spike level. It seems that recovery values are dependent on spiking level, matrix and acidification during extraction. Var et al. (2007) demonstrated the influence of the matrix on the recovery of AFB1 in helva. In addition, the extraction influences the recovery, especially the acidification effects, on the isolation, and analysis of mycotoxins is controversial. Acidification of the matrix before extraction with organic solvent has been described as induction (Sangare-Tigori et al. 2006; Nguyen et al. 2007) as well as suppression (Entwisle et al. 2000; Villa and Markaki 2009) for isolation and analysis. Acidification of the matrix can induce a further increase of the mycotoxin concentration, thus being a key parameter in the isolation and analysis of mycotoxins (Ghali et al. 2008). In our study, however, acidification decreased the extraction of both aflatoxins from liquid and solid matrix in a level-dependent manner (data not presented). The results of the analyses are presented in Table 2. They were not corrected for recovery. AFB1 was not detected in 9 of 58 feed samples and not in 25 of 56 plasma samples. The percentages of positive samples for AFB1 are 84.4% for feed and 55.3% for plasma samples. From 51 positive cow feed samples, 31 (84.6%) are exceeding the maximum level regarding Tunisian national standard (TNS 1983) and EU limit (European Commission 2003), which are 20 and 5 mg/kg in animal feeds and complete feeding stuffs, respectively. In addition, of 84.4% cow feed samples containing AFB1, 20% are in the range of 5–10 mg/kg, 22.4% of 11–30 mg/kg, 12.4% of 30–50 mg/kg and 22.4% above 50 mg/kg. In the literature, many papers indicated that crops such as barley, wheat, maize, sorghum, pistachios, molasses and their industrial

Table 2. Occurrence of AFB1 and AFM1 level in samples collected from the region of Beja, Tunisia. AFB1 (mg/kg or mg/l) Frequency distribution Samples


Positive samples





Mean  SD*

Feed Plasma

58 56

49 22

27 46

10 8

17 2

4 –

18.66  1.4 7.05  1.2

AFM1 (mg/l) Frequency distribution

Raw milk


Positive samples











Note: *of positive samples.


420 17

Mean  SD* 13.62  1.4


S. Abbe`s et al.

by-products are frequently contaminated by AFs (IARC 2002; Ghali et al. 2008, 2009) produced primarily by Aspergillus flavus and Aspergillus parasiticus, either in the field or during transportation or storage (Scheidegger and Payne 2003; Bensassi et al. 2010). With increasing levels of AFs in the diet, reduction in feed intake and growth rate become severe in herds, as also if AF levels are high enough, liver damage can occur (Williams et al. 2004). Moreover, AFB1 is considered the most toxic and carcinogenic aflatoxin (Roebuck and Maxuitenko 1994), once ingested by mammals is absorbed in the gastrointestinal tract and appears rapidly in blood (Gallo et al. 2008) and in milk (Masoero et al. 2007, 2009) as AFM1, the principal AFB1 hydroxylated metabolite. The results presented in Table 2 are in accordance with the cited literature. AFB1 was detected in plasma samples from blood of dairy lactating cows at a mean level of 7.05 mg/l. Moreover, AFM1 was detected in raw cow milk, in 60.7% of all samples. The overall mean level of AFM1 in the samples was 13.6  1.4 mg/l. This finding indicated that the concentration of AFM1 in the randomly collected samples was higher than the maximum level of AFM1 in liquid milk regarding Tunisian national standard and Codex standard. Five (4.4%) samples were higher than maximum limit of 0.05 mg/l of the European Union (European Commission 2006). The AFB1 carry-over rate into milk as AFM1 was determined to range from 1% to 3% in lactating dairy cows and principally to be affected by milk yield (Van Eijkeren et al. 2006; Masoero et al. 2007), with a reported maximum value of about 6% (Veldman 1992). This study indicated that cow feedstuff could be affected by mycotoxin contamination. Also, there are transfers of contamination to raw milk from lactating dairy cows. The main causes are the climatic conditions, especially humidity and temperature of the region. More precaution should be taken on hygiene controls to avoid mycotoxin contamination. In conclusion, the present study shows that the incidence of AFB1 in feedstuff can be a potential problem for animal health. For this reason feed should be inspected and controlled routinely for AFs, as to see whether regulation on aflatoxins is fulfilled.

Acknowledgements This work was supported by Tunisian Ministry of higher Education and Scientific Research (Unit of Immunology, Environmental Microbiology and Cancerology) and Higher Institute of Biotechnology of Beja (Animal biotechnology Dept.). I thank the work team of the Research Unit, Toxicology and Environment (Urgent Medical Aid Centre of Tunis).

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Natural occurrence of aflatoxins (B₁ and M₁) in feed, plasma and raw milk of lactating dairy cows in Beja, Tunisia, using ELISA.

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal co...
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