Journal of Food Quality
Volume 2017 (2017), Article ID 4067989, 7 pages
https://doi.org/10.1155/2017/4067989

Research Article

Seasonally Feed-Related Aflatoxins B₁ and M₁ Spread in Semiarid Industrial Dairy Herd and Its Deteriorating Impacts on Food and Immunity

Saideh Mozafari,¹ Mohammad Mohsenzadeh,¹ and Jalil Mehrzad2,3

¹Department of Food Hygiene and Aquaculture, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Azadi Square, Mashhad 1793, Iran
²Department of Pathobiology, Faculty of Veterinary Medicine and Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
³Section of Immunology, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Correspondence should be addressed to Mohammad Mohsenzadeh; mohsenza@um.ac.ir and Jalil Mehrzad; mehrzad@ut.ac.ir

Received 28 April 2017; Accepted 28 June 2017; Published 1 October 2017

Academic Editor: Alejandro Hernández

Copyright © 2017 Saideh Mozafari et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

To comparatively determine the levels of aflatoxin (AF) B₁ in feedstuffs and of AFM₁ in milk from semiarid industrial cattle farms in northeastern Iran during four seasons and to elucidate the effects of mixed AFB₁ and AFM₁ on bovine granulocytes, 72 feedstuffs (concentrate, silage, and totally mixed ration (TMR)) and 200 bulk milk samples were simultaneously collected for ELISA-based AFs detection. Isolated blood and milk neutrophils (/treatment) were also preincubated with mix of 10 ng/ml AFB₁ and 10 ng/ml AFM₁ for 12 h; the impact was assessed on neutrophils functions. AFB₁ levels in feedstuffs averaged 28 μg/kg (4–127 μg/kg), with TMR maximal ( μg/kg), concentrate ( μg/kg), and silage ( μg/kg). The levels of AFB₁ and AFM₁ in feedstuffs and milk averaged , , , and  μg/kg and , , , and  ppb μg/kg in winter (maximal), autumn, spring, and summer, respectively. Mix of AFB₁ and AFM₁ weakened various functions of granulocytes. It adds new reason why during winter semiarid raised food-producing animals show more immune-incompetence.

1. Introduction

Aflatoxins (AFs) are highly carcinogenic and immunotoxic secondary metabolites produced mainly by Aspergillus (A) flavus and A. parasiticus in feed/foods [1–4]. Environmentally, AFs exposure is inevitably rising [1, 5, 6], and their seasonal spread and impacts in semiarid industrial dairy farms’ mi(a)croenvironment remain unaddressed. Inappropriate environmental condition leading to the formation of aflatoxigenic molds varies and could be maximal during harsh seasons [7–10].

By contaminating agricultural commodities, AFs alarmingly cause immunodeficiency and cancer risks in mammals [2, 3, 5, 11]. Unfortunately, pivotally food animals increasingly consume aflatoxigenic mold-contaminated feed, worldwide [5, 7, 10]. Advanced analytical methods on AFB₁/M₁ detection have revealed that even in Europe AFs routinely circulate in the body of food-producing animals and humans through feed/foods [12, 13]. Predominantly hydroxylated AFB₁ metabolite (AFM₁) is as carcinogen as AFB₁ and bioaccumulates everywhere (meat, milk, etc.) in vivo [5, 14, 15].

It is worrying reality that AFs levels in feedstuffs can be far higher than what we apparently measure. Inextricable link of various cancers to environmentally relevant levels of AFs, existence of masked AFs, and limited analytical capabilities for detection of AFs in agricultural commodities, sera, and dairy products have rendered the aspects of AFs’ detection and impact the focus of most concern [1–3, 5].

With EU’s tolerable levels for AFB₁ in feed (2–8 μg/kg) and AFM₁ in milk (0.05 μg/kg) (the EU, 2010) and Iran’s permissible AFB₁ in feed (5–20 μg/kg) and foods (5–15 μg/kg) and AFM₁ in milk [(0.5 μg/kg, kg is equal to liter in case for milk) (Anonymous, 2002) (due to contaminated milk, the risk for lactating mammals is much higher compared to nonlactating ones) and globalization, routine monitoring of AFs is more urgent [16]]. Due to highly carcinogenic and immunosuppressive nature of AFs [even permissible level of AFB₁ is hazardous for immune cells/molecules in humans and animals [2–4]] and the lack of information on the immunotoxic influence of mixed AFB₁ and AFM₁ we aimed to (1) determine the seasonal levels of AFB₁ in various feedstuffs and of AFM₁ in bulk milk obtained from industrial cattle farms in agroecologically and geopolitically important semiarid northeastern Iran and (2) examine the in vitro effects of environmentally relevant levels of mix of AFB₁ and AFM₁ on different functions (free radicals or ROS production, phagocytic and killing capacity, and necrosis) of blood-and-udder’s key innate immune cells, neutrophils (PMN), using luminometry, flow cytometry, and bactericidal assays for two major vertebrates’ superbugs (i.e., Staphylococcus (S.) aureus and Escherichia (E.) coli).

2. Materials and Methods

Cluster random 72 feedstuffs (silage, concentrate, and totally mixed ration, TMR) and bulk milk () samples were simultaneously seasonally obtained from industrial cattle farms (see Table 1 and Figure 1; farms) in sterile cold condition accordingly (ISO 6497 2002 [10]). Much lower detected levels of AFB₁ and AFM₁ were eventually used for in vitro effects of mix of AFB₁ and AFM₁ on isolated bovine blood and milk PMN.

Table 1: Cluster random sampling and widespread contamination of aflatoxin B₁ in feedstuffs and aflatoxin M₁ in bulk milk during spring, summer, autumn, and winter in thestudy area (see also Figure 1).

Type of feed/milk
Sampling period
Samples,
≥Iran’s permissible level, (%)

Concentrate
April-May
6
4 (66.6)

July-August
6
3 (50.0)

November
6
5 (83.3)

February
6
6 (100.0)

Total
24
18 (75.0)

Corn silage
April-May
6
4 (66.6)

July-August
6
3 (50.0)

November
6
4 (66.6)

February
6
4 (66.6)

Total
24
15 (62.5)

TMR
April-May
6
5 (83.3)

July-August
6
4 (66.6)

November
6
5 (83.3)

February
6
6 (100.0)

Total
24
19 (79.2)

Bulk milk
April-May
49
39 (83.3)

July-August
51
34 (66.6)

November
49
39 (83.3)

February
51
44 (100.0)

Total
200
156 (78.0)

Figure 1: Upper panel, map of the study area showing many intensively large scaled industrial cattle farming in part of semiarid northeastern Iran, where feed and milk samples were seasonally obtained. Lower panel (a), chemical structure of aflatoxins (AFs) B₁ (C₁₇H₁₂O₆) and M₁ (C₁₇H₁₂O₇) routinely found in animal feed and milk. AFM₁ is derivative of hydroxy AFB₁ and predominant secondary metabolite of AFB₁. One oxygen atom more in AFM₁ leads to a polar lipophobic toxin. Middle, aflatoxin B₁ levels in different types of cattle feed ( feedstuffs), and lower, aflatoxin M₁ levels in bulk milk () during four seasons (in -axes, kg is equal to liter). Different letters with their symbols (i.e., #, ¥, and  for concentrate, silage, and TMR/totally mixed ration, resp.) represent significant difference between their respective treatments ( versus control).

Indirect ELISA were performed according to the test kits for feedstuffs (AFB₁ ELISA, EuroProxima, Beijerinckweg, The Netherlands) and bulk milk (Ridascreen® AFM₁, R-Biopharm, Germany), by measuring absorbance at 450 nm with ELISA reader (ELx 800, BioTek Instruments, USA). The lower detection limit for AFB₁ and AFM₁ was 0.05–0.1 μg/kg.

For in vitro effects of AFs on blood and milk PMN a group of 12 physiologically healthy dairy cows were used for PMN isolation and analyses, accordingly [2, 17].

AFB₁ and AFM₁ were obtained from Sigma and prepared according to Mehrzad et al. [2], with some modification (i.e., AFM₁ was directly dissolved in DPBS); they were then separately but simultaneously added, at a final concentration of 0 (control) and 20 ng/ml (10 ng/ml AFM₁ plus 10 ng/ml AFB₁) to the PMN cultured in complete RPMI 1640 medium (12 h, 37°C, 95% humidity, 5% CO₂). PMN were then washed and chemiluminescence (CL), flow cytometry-based neutrophils phagocytosis, and necrosis were measured according to the reported procedure [2, 3].

To evaluate the effect of AFs on superoxide anion () production and myeloperoxidase (MPO) activity of blood PMN, production was measured in post-AFs treated PMN by converting to nM of cytochrome reduced using the extinction coefficient = 2.1 × 10⁴ M−1 cm⁻¹ as described [18]. Post-AFs treated PMN MPO activity was measured based on the oxidation of ortho-dianisidine (0.8 mmol/l) of supernatant of sonicated PMN extract containing 0.1 mmol/l of added H₂O₂ using microtiter plate spectrophotometer at 450 nm (Multiskan Plus Type 314, Labsystems, Helsinki, Finland). The analyses of phagocytosis and killing of E. coli and S. aureus were done accordingly [2, 17].

Statistical analysis was performed using SPSS version 19 and SAS Version 9.1 (SAS Institute Inc., Cary, NC) software. The Chi-square and Fisher exact tests were used to assess the possible differences in AFs spread in feeds and milk. To compare the measured parameters of two groups, analysis of variance was used; all data were presented as means ± SEM and hypothesis testing was done at the 5% significance level.

3. Results

Of 72 feed samples, more than 52 (average 72.2%) were contaminated with far above permissible AFB₁ in feed (Table 1), from which the levels of AFB₁ contamination in concentrate, corn, silage, and TMR were 75%, 62.5%, and 79.2%, respectively. The intensity of AFs spread was higher in autumn-winter (Table 1 and Figure 1 B). AFB₁ level in feedstuffs ranged from 4 to 127 μg/kg with average of 28 μg/kg. Among feedstuffs TMR showed maximal level of AFB₁ ( μg/kg) followed by concentrate ( μg/kg) and corn silage ( μg/kg). Maximal level observed in winter followed by autumn, spring, and summer averaged , , , and  μg/kg, respectively (Figure 1 B). Similarly, bulk milk AFM₁ level ranged between , , , and  μg/kg, in winter, autumn, spring, and summer, respectively, ((Figure 1 C) in all cases, kg is equal to liter).

The PMN phagocytosis-(in)dependent CL/(non)particle-stimulated luminol-enhanced CL (Figures 2(a1) and 2(a2)) and the flow cytometry-based phagocytosis assay of blood (Figure 2(c1)) and milk (Figure 2(c2)) PMN consistently revealed a significant decrease in phagocytic activity and killing capacity by AFs-exposed PMN. Among PMN stimulated with PMA, latex beads, or Pansorbin®, the AUC for the mix of AFs-exposed blood and milk PMN were, respectively, 16, 38, and 30% and 18, 37, and 31% lower than control PMN. Further, in the AFs treated blood PMN stimulated with PMA, latex, and Pansorbin was slightly decreased (data not shown). Interestingly, the luminol-dependent CL arising from added hypochlorite (HClO) was significantly higher in mix of AFB₁- and AFM₁-treated group (Figure 2(b), upper panel insert). Furthermore, while low levels of mix of AFB₁ and AFM₁ showed little effects on blood PMN necrosis (Figure 2(c)), it decreased both the production of () by PMN (Figure 2(d)) and the MPO activity () of PMN (Figure 2(e)). Unlike post-AFs treated necrotic PMN, neutrophils exposed to mix of AFB₁ and AFM₁ were less efficient to phagocytose and kill S. aureus and E. coli (see Figures 2(a) and 2(b)).

Figure 2: Upper panel, luminol-dependant chemiluminescence (CL) in blood (a1) and milk (a2) neutrophils incubated with(out) 20 ng/ml of mix of aflatoxins (AFs) B₁ and M₁ for 12 h and stimulated with PMA, latex beads, and opsonized Pansorbin; values are expressed as the area under the curve (AUC) of relative light units (RLU)/s of 1000 viable neutrophils during 30 minutes. The inserted images representatively confirm the high purity and similar light microscopic appearance of neutrophils in both groups. Effect of above-mentioned level of mix of AFs on luminol-dependent CL from added hypochlorite (HClO) (b), necrosis (c), production (d), and myeloperoxidase (MPO) activity (e) in neutrophils. Lower panel, mix of aflatoxins (AFs) B₁ and M₁ weakens phagocytotic activity and killing capacity of blood neutrophils. Effect of 20 ng/ml of mix of AFB₁ and AFM₁ (non)treated for 12 h on the phagocytosis and killing activity of neutrophils against E. coli (a), S. aureus (b), and phagocytosis of 1 μm fluorescent microparticles by blood (c1) and milk (c2) PMN. Values are means ± SEM of 8 (; versus control).

4. Discussion

The level which is alarmingly higher than the permissible levels of AFs in feedstuffs and milk (5 μg/kg for feed AFB₁ and 5 ng/kg for milk AFM₁) is largely attributed to climate conditions and inappropriate management of grasses and grains during harvest, transportation, drying and mixing stages [7, 19]. For example, improper processing/storage condition of higher dry matter corn silage likely boosts AFB₁ production [6, 7, 10], due mainly to weakening anaerobic bacterial mediated lactic acid production, increasing mi(a)croenvironment pH, thereby boosting aflatoxigenic fungal growth. Owing to being categorized as group-1 carcinogens, many countries and regulatory agencies have harshly imposed tolerable limits on AFs. For example, the European Commission has set tolerable limit for AFB₁ and total AFs (B₁, B₂, G₁, and G₂) 2–8 μg/kg and 4–15 μg/kg, respectively, in crops such as nuts and grains (EU, 2010); furthermore, Iran has set the tolerable limit for AFB₁ for feedstuffs and foods 5–20 μg/kg and 5–15 μg/kg, respectively (Anonymous, 2002); also, maximal tolerable for milk AFM₁ in EU and Iran is 0.05 and 0.5 μg/kg, respectively. As such, routine harsher monitoring of AFs levels in different feedstuffs is urgent in semiarid northeastern Iran. For the detection of AFB₁ and AFM₁, herein we used ELISA with sensitivity and specificity of above 98% with acceptable recovery rate, LOD/LOQ, and precision; nevertheless it is worth simultaneously confirming their quantification with some more advanced analytical tool like HPLC or LC/MS/MS as done by others [12, 13].

Often, in droughty and poor crop years semiarid northeastern Iran, cows are normally more exposed to mold/AFs-contaminated feed, and so are warm-wet weather, delayed harvest, snow cover conditions, infrequent daily preparation/provision of TMR, and so on [6]. Also, in Iran almost all agricultural commodities are sent into commercial channels with zero pasture-based cattle farming; further, key component of animal feed, corn, is mainly imported and thus not fresh; in contrast, in developed countries ~30% feeds are retained for on-farm use, efficiently lessening AFB₁ exposure. Mean (minimum, maximum) relative temperature (°C) and absolute humidity (%) in our study area during April-May, July-August, November, and February were 21.9 (15.1, 28.7), 28.1 (20.4, 35.7), 9.6 (3.9, 15.4), and 5.1 (−3, 11.6) and 45 (25, 66), 25 (11.5, 37.5), 63.5 (42, 84), and 64 (40, 87), respectively, potentially easing fungal growth and AFs spread in feed and milk (Figure 1), thereby worsening feed/food quality in autumn-winter.

During harsh seasons more contaminated moldy feeds are often used in feedlots; analytical incapability to detect very low (invisible) levels of AFs and lack of AFs toxicity data in cows [5] exacerbate the complexity of AFs exposure in food animals and humans.

Among various feedstuffs analyzed in central and northeastern Iran, concentrate was maximally contaminated to both aflatoxigenic molds and AFB₁ [9, 10]. Though a tiny amount of feed AFB₁ appears in milk of AFB₁-consumed dairy cattle, AFB₁ simply transmit into milk as AFM₁ with high cancer risks for consumers, especially neonates and children, who consume far more milk and dairy products. AFM₁ is hardly degradable by pasteurization procedure [5, 15]; nonetheless, fermentation could lead to biodegradation of AFs in fermented dairy products and thus less toxicity.

The background of the selected doses examined here was (1) in line with the first part of this study on AFB₁ and AFM₁ quantification, (2) our current understanding on the metabolism and toxicodynamics of AFB₁, and (3) from researchers [2–5, 20] in bovine, porcine, and human in vitro models. Researchers have even used higher doses of AFs [21]. Although our knowledge on interaction(s) of AFs effects in animals, especially food/dairy-producing ruminants, is limited, nevertheless it is worth mentioning the point of intake of AFB₁ with the diet and its transfer to milk in high yielding lactating cows. Given that a cow with average daily milk production of 50 litter is consuming ~40 kg TMR per day and ~50% of AFB₁ in contaminated TMR goes to the blood with a tenth (mainly converted to AFM₁) to milk [20, 22], the assumed AFB₁ and AFM₁ in blood/milk can accumulatively reach above our ex vivo tested levels.

The inhibiting effect of AFs on the killing capacity of PMN for E. coli and S. aureus was manifested mainly by reduction of MPO activity and intracellular ROS production; this further confirms the negative effects of mix of AFB₁ and AFM₁ on the innate immunity; that is why AFB₁-exposed dairy cows are more susceptible to environmental infections, specially mastitis and metritis (unpublished data). Nevertheless, we know little on how mix of AFB₁ and AFM₁ behaves in the phagosome and lysosome, where extremely large amounts of oxidants and granule constituents, through MPO-H₂O₂-HOCl system, are released [2, 17, 23]. What happened on different stages of PMN necrosis and apoptosis remained further investigation.

In short, a relatively high level of feed and milk AFs with more pronounced AFB₁ spread in cereal crops during winter and considering the economic, health, and cancer importance of AFs, percussion measures, hugely requires boosting food/health quality and lowering the risk of AFs exposure in farm animals and human in the study region.

Additional Points

Practical Applications. Poor harvesting, drying, transportation, and storage of feed/agricultural commodities lead to mold growth and particularly AFs formation in feedstuffs and milk and thus many animal and public health risks. Feedstuffs and bulk milk were sampled from various industrial dairy farms during 4 seasons in semiarid northeastern Iran for AFB₁ and AFM₁ detection and their ex vivo effect on peripheral granulocytes. Alarmingly, maximal immunotoxic levels of AFB₁ and AFM₁ in feedstuffs and milk occurred in winter followed by autumn, spring, and summer. More controls of feed materials would be needed to prevent an undesirable contamination of feed, foods, and milk/dairy products.

Conflicts of Interest

No potential conflicts of interest were reported by the authors.

Acknowledgments

The authors gratefully acknowledge the financial support from Ferdowsi University of Mashhad (no. 17842).

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