Aflatoxin M1 in milk
While all bulk milk samples (n=72; 100%) showed contaminations with AFM1, 55 samples (76%) had levels below 81 ngkg−1. The remaining 17 samples (24%) with higher contaminations could potentially be below or above the standard limit of 100 ngkg-1. Twenty-one samples (29%) were contaminated below 50 ngkg−1. The real contaminations could probably be lower than the detected values due to the method of prediction (ELISA). Less AFM1 values are usually obtained with HPLC method compared with ELISA (Alvarado et al., 2017). A meta-analysis on Iranian studies (Khaneghahi-Abyaneh, et al., 2019) showed means of 59.19 and 35.23 ngkg−1 with ELISA (55 studies; 9224 samples) and HPLC (18 studies; 2606 samples) methods, respectively. Some Iranian studies have reported AFM1 contamination in UHT and pasteurized milk. Mashak et al. (2016) detected AFM1 (15 to 140 ngkg-1; HPLC method) in all (100%; n=30) samples, 66.7% (n=20) having levels below 50 ngkg−1, 20% (n=6) above 100 ngkg−1 and none over 500 ngkg−1. Tajik et al., (2016; ELISA method), found AFM1 (75.8±9.2 ngkg−1) in 77.7% of samples (n=280/360), 36% (n=130) showing levels below 50 ngkg−1 and 2.2% (n=8) exceeding 500 ngkg−1. Heshmati and Milani (2010; ELISA method) found AFM1 in 55.2% (n=116/210) of samples, with 21.9% (n=46) having levels below 50 ngkg−1 and none exceeding 500 ngkg−1. Based on a meta-analysis of 70 Iranian studies (Hamzeh-pour et al., 2020), 64% of raw milk samples were contaminated to AFM1 (mean 39.7 ngkg−1), 75% of which below 50 ngkg−1 and 9% above 500 ngkg−1. Contaminations above 50 ngkg−1 have been reported from other countries (Škrbić et al., 2013; Tsakiris et al., 2013). Škrbić et al. (2013) reported a mean of 300 ngkg−1 AFM1 in commercial milk samples from Serbia.
Seasonal differences in milk AFM1 levels have been reported with higher contaminations in cold seasons (Khaneghahi-Abyaneh, et al., 2019; Mozaffari Nejad et al., 2019; De Roma et al., 2017; Mahmoudi and Norian, 2015; Bahrami et al., 2016; Heshmati and Milani, 2010; Kamkar, 2005). In the present study, although the estimated milk contaminations in winter (61.25±28.91 ngkg−1) and summer (54.20±25.51 ngkg−1) were not statistically different (P=0.279), the contaminations above 81 ngkg−1 or the median (70 ngkg−1) were mostly detected in winter and the chance of contaminations above median was 5.33 times in winter. Interestingly, the AFB1 contamination of feeds, as the main source of contamination, was significantly higher in summer than in winter (P<0.002). As the main source of milk contamination, the AFB1 contaminations of feeds have been reported to be higher in winter (Bahrami et al., 2016; Dimitrieska-Stojković et al., 2015; Mahmoudi & Norian, 2015; Simas et al., 2007). In the present study, however, the AFB1 contaminations of all rations and feed ingredients were significantly higher in summer than in winter (see below).
Aflatoxin B1 in Feed ingredients and TMRs
Ninety percent of the examined ingredients (n=207/230) were positive for AFB1, 10% (n=23/230) had non-detectable values and 13.9% (n=32/230) had values above 101.25 ngkg−1. All ingredients exhibited higher contaminations in summer (P<0.05). The non-detectable AFB1 values were mostly seen in winter (n=17/23) and the values above 101.25 ngkg−1 were detected mainly in summer (n=27/32). These differences could be due to the synergistic effects of ambient temperature and feed moisture on behavior of mycotoxigenic fungi in summer, which is affected by climatic changes at any stage of production chain (Paterson and Lima, 2010; Magan et al., 2011; Guchi, 2015; Alvarado et al., 2017).
Iranian studies focusing on AFB1 contamination of dairy feeds are not abundant. Bahrami et al. (2016) examined 160 samples of corn silage, alfalfa hay, straw and barley (n=40 each), detected AFB1 in 82.5% of samples with levels above 5,000 ngkg−1 (Iranian standard) in 65% of corn silage and 10% of straw samples. Different prevalence rates have been reported for aflatoxin contamination of feeds from various countries such as China (42%; Zheng et al., 2013), Costa Rica (33%; Chavarría et al., 2015), Portugal (22%; Abrunhosa et al., 2016), Tanzania (65%, Mohammed et al., 2016) and Turkey (61.7%, Kocasari et al., 2013; 34.9% Sahin et al., 2013; 26.3%, Sahin et al., 2016). A global study covering 2009 to 2011 (Rodrigues and Naehrer, 2012) and 4,627 feed samples reported aflatoxin contamination in 33% of samples (mean: 21,000 ngkg−1). A survey of world reports on 1,113 feed samples from 44 countries between 2012 and 2015 (Kovalsky et al., 2016) reported that only a few samples from Africa and Europe exhibited levels exceeding 20,000 ngkg−1, which is the maximum permitted level for some non-dairy animals (Kunsagi et al., 2011; ISIRI, 2016). Different prevalence rates among various studies might be due to climatic variations and different detection methods. Ghali et al. (2008, 2009) detected aflatoxins in 76.4% and 62% of sorghum samples with ELISA and HPLC methods, respectively.
Consistent with the feed ingredients, the TMRs also had higher contaminations in summer. The total daily AFB1 contents of bunk TMRs were 537.05±558.79 ng in winter and 1375.50±905.02 ng in summer. It is implied from some evidences that these levels were low enough to result in milk AFM1 levels below 50 ngkg−1. About 0.3 to 6.2% of AFB1 is secreted as AFM1 to milk (Becker-Algeri et al., 2016; FAO/WHO, 2002) and in cows producing >30 kg milk/day, about 6% of AFB1 may be transformed to AFM1 (Britzi et al., 2013). In the present study, with milk production of 30.5kg/cow/day and assuming 6% bio-transformation, the AFM1 content of milk could range from 1.05-2.7 ngkg−1 (totally 32 to 82.5 ng). However, the estimated AFM1 levels were 61.25±28.91 ngkg−1 in winter and 54.20±25.51 ngkg−1 in summer. Thus, the true contaminations of the ingredients could be higher than the detected levels. As the AFB1 content of TMR samples taken from bunks were higher than those calculated from the contaminations of separate ingredients, the contamination could be increased during processing. Common devices such as mills and mixing wagons (Pinotti et al., 2016) and remainders of contaminated feeds in wagons, preparation areas and bunks may increase the level of aflatoxin in the ration. The difference could also be due to the small quantities of samples taken from huge volumes of feeds. The lack of correlation between feed AFB1 and milk AFM1 could be due to variations in daily intake of AFB1.
Seasonal variations in AFB1 in feeds and AFM1 in milk
With elevated dietary AFB1 levels and high chance of TMR contaminations above median (716 ng/kg; OD=5.57; P=0.002) in summer, higher levels of AFM1 in milk would be expected, but the findings were opposite. The reason could be the affection of liver functions by heat stress in summer (Bernabucci et al., 2010; Gallo et al, 2015; Marchese et al., 2018; Min et al., 2021). Transformation of AFB1 to AFM1 happens mainly in the liver by the action of cytochrome P450 enzymes (Marchese et al., 2018; Min et al., 2021), involved in a vast variety of reactions. Compromised liver functions and reduced hepatic enzyme activities have been observed in heat stressed cows (Bernabucci et al., 2010; Fan et al., 2018). Compromised liver activities may be long lasting as it was observed in mid lactation cows (Mohebbi-Fani et al., 2020). Lower dry matter intakes in summer could also have a role in this finding. So, non-dietary factors affecting the metabolism of AFB1 may fade the effect of higher intakes of toxin in summer. Paradoxically, if the general health of the cows is improved, the AFM1 contamination of milk would potentially be elevated. Thus, the most logic way to control the aflatoxin contamination in milk is to control the critical contaminants of the rations.
Critical feed contaminants
The AFB1 content of all ration ingredients showed some correlations with AFB1 content of TMRs. The strongest correlations were seen for grain mix and corn silage, which had the highest proportions in the rations. However, the impact of all other ingredients should also be taken into account. Beet pulp also could be an important source of contamination (strong correlations) regardless of its low incorporation or absence in rations. Alfalfa hay also could be an important contaminant, despite that its AFB1 contents were not correlated with those of the total rations. It is incorporated in nearly all rations in considerable amounts in Iranian farms and may be substituted for corn silage in many conditions. The co-occurrence and the synergistic effects of various mycotoxins even at relatively low levels (Kovalskey et al., 2016) should be kept in mind. In addition to controlling the AFB1 contaminations of feed ingredients, tainting of feedstuffs during processing should also be controlled. Intensifying the controlling measures in summer, with probable higher contaminations of feedstuffs, may reduce the overall milk contamination. Responses to these efforts may be rapid as AFM1 enters the milk 12-24 hours after consumption of AFB1 and drops to non-detectable levels about 72 hours of removal of AFB1 from ration (Pettersson, 1998). Based on the results of this study, a great majority of milk produced in the studied farms could have AFM1 contaminations below the ISIRI limit (100 ngkg−1). Contaminations below 50 ngkg−1 appear to be achievable and affordable in the studied farms.