Introduction

Animal intake of mycotoxin-contaminated feed can, depending on time and level of exposure, result in acute or chronic toxicity. The outcome of mycotoxin toxicity can involve histological changes, as well as mutagenic, cytotoxic, teratogenic, carcinogenic, estrogenic, hematotoxic, and immunotoxic effects (CAST, 2003). Pigs are highly sensitive to mycotoxins and its exposure to these substances can decrease growth performance and feed intake, reduce immune resistance, and increase rates of infectious diseases and reproductive disorders (KANORA; MAES, 2009; BRYDEN, 2012). Further, production costs associated with the need for therapeutic drug administration can also increase.

Among mycotoxins producers, there is relevant concern about fungi species of Fusarium genera, that produce zearalenone (ZEA) and fumonisin (FB), and Aspergillus genera, producers of aflatoxin (AF) (BRYDEN, 2012). Based on worldwide surveys, Grenier and Applegate (2013) categorized experimental mycotoxin doses as realistic, occasional, or unrealistic according to the contamination levels found under most field conditions. However, animals are rarely exposed to a single type of mycotoxin and simultaneous multiple contamination has become a major health concern. This is because mycotoxin combinations could act synergistically to produce toxic effects (SPEIJERS; SPEIJERS, 2004; KLARIC et al., 2012).

Some feed additives have been evaluated as possible approaches to reduce the toxic effects of mycotoxins in animal production (JUAN-JUAN et al., 2010; YANG et al., 2012; FU et al., 2013). Clay minerals (JIANG et al., 2010a), enzymes (JARD et al., 2011), antioxidant vitamins (SORRENTI et al., 2013), natural biopolymers, and extracts (JUAN-JUAN et al., 2010; QUILES et al., 2015) have all been demonstrated to minimize the toxic effects of mycotoxins. However, reports on the use of anti-mycotoxin additives blended into contaminated diets are scarce (MACHADO JÚNIOR et al., 2014).

The toxic effects of mycotoxins in pigs, based on realistic doses that occur under most field conditions or on multi-contamination in the diet, are shown in only few studies (DILKIN et al., 2003; GRENIER et al., 2011; BRACARENSE et al., 2012). The extensive range of exposure levels and mycotoxin types associated with multi-contaminations increases the importance of such investigations as a means to protect the safety of food for human and animal consumption. Therefore, the aim of this study was to evaluate the combined effect of ZEA, FB, and AF, and the efficacy of an anti-mycotoxin additive blended in piglet's diet on growth performance, mononuclear immune cells, serum biochemical profile, organ weights and histological changes, and immunohistochemistry.

Material and Methods

Animals and experimental design

The experimental procedure was carried out in the animal facility at Pontifícia Universidade Católica do Paraná (PUCPR, Curitiba, Brazil) in accordance with guidelines of their Ethics Committee on the Use of Animals (CEUA-PUCPR, Protocol 0908). A total of 48 weanling piglets (24 castrated male and 24 female piglets), with an initial average body weight of 6.88 ± 0.89 Kg, and at 21 d of age were assigned in a randomized block design to three treatments and eight replicates per treatment, during a short-term experimental period of 21 d. The experimental unit comprised two animals (male/male or female/female) receiving feed and water ad libitum. However, to evaluate the treatment effects on weight of the reproductive tract, each female piglet was considered as an experimental unit; keeping 8 replicates/treatment.

Experimental diets

The formulated diets for this study were isocaloric and isonitrogenous (Table 1) to assure or exceed the nutrient required for piglets, based in Rostagno et al. (2011). High-performance liquid chromatography (HPLC) analysis characterized the treatments as the following contamination: T1 - basal diet, no detected mycotoxins, T2 - multi-contaminated basal diet, 88.5 ppb ZEA, 3,994 ppb total FB (2,577 ppb FB₁ + 1,417 ppb FB₂), and 107.5 ppb total AF (82.0 ppb AFB₁+ 1.5 ppb AFB₂ + 24.0 ppb AFG₁), and T3 - same mycotoxin levels of T2 plus an anti-mycotoxin additive. Only purified AFB₁ was artificially incorporated into the T2 and T3. All the other mycotoxins were naturally present in raw material of the T2 and T3. The anti-mycotoxin additive incorporated into T3 is a mixture of natural biopolymer and herbal extracts, enzymes, vitamin C, and hydrated sodium calcium aluminum silicates.

The diets with mycotoxin combinations present lower doses of ZEA and FB than recommended for EU guideline, while present doses of AF above of the regulatory levels (GUERRE, 2016).

Animal growth performance

The growth performance of each individual piglet was recorded on d21. Average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency rate [Gain:Feed (G:F)] were determined.

Flow cytometry and serum biochemistry

Blood samples were taken via the jugular vein on d21 from the heaviest pig of each pen (a total of 24 animals, 8 replicates/treatment), independently of sex. Then, an 8-mL aliquot from each sample was aseptically collected in separate tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA, Becton Dickinson, New Jersey, USA) for analysis by flow cytometry (FERNANDES FILHO et al., 2013). Another 8-mL aliquot from each sample was collected in a tube without anticoagulant. This sample was taken for serum biochemistry analyses (JIANG et al., 2010a).

A volume of 180 µL of the cell pellet (about 10⁶ cells) was used to that the specific monoclonal antibodies bind to the following respective recognized cell surface antigen: double positive T lymphocytes (CD4⁺/CD8⁺), helper T lymphocytes (CD4⁺/CD8^-), cytotoxic T lymphocytes (CD4^-/CD8⁺), ratio between CD4⁺/CD8^-:CD4^-/CD8⁺ (CD4:CD8), B lymphocytes (B cell subset), monocytes/granulocytes (CD172a), for 30 min at room temperature. The monoclonal antibodies were acquired from Southern Biotech (Birmingham, AL, USA) and data were analyzed with CellQuest Pro software (Becton Dickson, Franklin Lakes, NJ, USA).

Blood aliquots fated to serum biochemistry analyses, collected as aforementioned (a total of 24 animals, 8 replicates/treatment), evaluated the liver function through the determination of aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), and alkaline phosphatase (ALP) activities. Serum concentrations of albumin, creatinine, total protein, and urea were also determined. All serum parameters were measured by a semi-automatic biochemistry analyzer (Quick Lab II Drake, Belo Horizonte, MG - Brazil).

Organ weights, histology changes and immunohistochemical analysis

One day after the end of the experiment, 24 animals (12 female and 12 male piglets) with the highest body weight from each pen were slaughtered to register the relative organ weights (liver, kidney, and lung) (n=8). The other 12 female piglets were also slaughtered to evaluate the relative weight of reproductive tract (ovary, uterus, and vagina-vestibule) (a total of 24 female piglets, n=8). The relative organ weights were calculated according to the final body weight of the piglets.

After the organ weight measurements, samples of the liver, kidney, lung, jejunum, ileum, uterus, and ovary were collected, fixed in 10% neutral buffered formalin solution, serially exposed to graded alcohol concentration, and embedded in paraffin. Sections of 4-µm thickness were obtained and stained with hematoxylin-eosin. Except for the intestine sections, the collected tissues were evaluated by light microscopy. The severity of damage was classified as no lesion, mild (< 25% to low extent), moderate (26-50% to intermediate extent), or severe (> 51% to large extent).

Sections from the liver and jejunum were subjected to immunohistochemical analysis. The tissue microarray (TMA) technique was applied on the paraffin blocked tissue (BAURAKIADES et al., 2014) and sections were examined for expression of COX-2 (monoclonal antibody, clone SP21, Spring Bioscience, Pleasanton, CA, USA) and TNF-α (monoclonal antibody, clone TA20, Santa Cruz Biotechnology, Heidelberg, Germany). The percentage of marked fields was observed with the aid of the Image Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD, USA) and ZEN 2.0 Image software (Blue ed., Carl Zeiss Microscopy GmbH, 2011) for counting marked cells.

Statistical analysis

Data were analyzed using the Statgraphics^® 4.1 software program. The data were subjected to ANOVA and Tukey's test to account for multiple comparisons. P values<0.05 were considered statistically significant.

Results and Discussion

Mycotoxins can cause serious adverse effects on animal growth performance. In the present study, multi-mycotoxin treatment did not affect the feed consumption or weight gain (Table 2). A deleterious effect on piglet's growth performance was caused by the combination of 30,000 ppb FB₁ (high level) and 50 ppb AFB₁ (DILKIN et al., 2003). At the present, the short co-exposure levels of ZEA, AF and FB required to affect piglet's growth performance remain unclear. However, our results enable state that in the period and dose tested, the multi-mycotoxin diet was not deleterious to the body weight gain, feed consumption or feed conversion.

The absence of toxicity associated with the multi-mycotoxin treatment rendered it impossible to evaluate the efficacy of an anti-mycotoxin additive. The mixed compounds of the anti-mycotoxin additive could reduce mycotoxin effects by acting differently on each mycotoxin present in the diet. The variety of mechanisms of action is an important feature for an efficient anti-mycotoxins additive due the occurrence of multi-mycotoxins in the feed and different molecular structure of each mycotoxin.

Clay minerals and natural polymers are mycotoxin binders that reduce intestinal absorption of mycotoxins by acting as adsorbents or sequestering (JUAN-JUAN et al., 2010). However, the different molecular structure of FB (GRENIER et al., 2017), for example, and other mycotoxins became difficult its adsorption or sequestration. Thus, the presence of enzymes in anti-mycotoxins additives can degrade or transform no adsorbed mycotoxins in less toxic metabolites (DEVREESE et al., 2013). Other compounds such as herbal extracts and antioxidant agents could promoter intestinal protection like improved immune response, cell turnover and a role against the deleterious effect of the oxidative stress induced by mycotoxins (SORRENTI et al., 2013).

Nevertheless, we can state that the anti-mycotoxin additive did not affect the feed intake or induce toxic response in weanling piglets. Also, it is important highlight that although the multi-contaminated diets present lower doses of ZEA and FB than recommended for EU guideline for monocontamination, while present doses of AF above of the regulatory levels (GUERRE, 2016). Nevertheless, since in combinations, the mycotoxins were not toxic for young piglets.

Recurrent exposure to mycotoxins could also impair piglet's health by affecting intestinal inflammatory response (GRENIER; APPLEGATE, 2013) and tight junctions, increasing gut permeability and translocation of pathogenic agents (BASSO et al., 2013). Hence the liver could be the following targeted organ of mycotoxins, since it is the major detoxifying organ, or yet to be target of translocated pathogenic agents. The combination of deoxynivalenol (DON) and FB₁ pronouncedly reduced the expression of cytokines in spleen of piglets when compared to the group that received mono-mycotoxin (GRENIER et al., 2011). Thereof, we investigated COX-2 and TNF-α expression on liver and jejunum for being proinflammatory immediate-early genes induced at sites of inflammation (WANG; DUBOIS, 2010). However, multi-mycotoxin treatment did not affect COX-2 or TNF-α expression (Figure 1). Higher doses in short-term exposure studies are most likely necessary to promote intestinal inflammation.

After intestinal absorption, mycotoxins are differentially distributed to tissue and organs (VOSS et al., 2001; ZOLLNER et al., 2002; FU et al., 2013). Consequently, toxic mono-contamination effects of ZEA, FB, and AF can increase relative organ weights and induce histopathological changes (CAST, 2003). However, there was no treatment effect on relative weight of liver, kidney, and lung (Table 2). In addition, the histopathological changes observed in these organs ranged from mild to moderate and no contrasting lesions were associated with multi-mycotoxin intake (Table 3).

Between the tested mycotoxins, only ZEA and its metabolites can bind to the estrogenic receptor present in the reproductive tract and cause an increase in estrogenic activity (GAJECKA et al., 2011, 2012; MUKHERJEE et al., 2014). One typical signal in female piglets is the enlargement of the reproductive tract (JIANG et al., 2010b, 2011; WANG et al., 2012). Alterations from the treatments on relative weights of reproductive tract (Table 2) was not detected. Information on the effect of low doses of ZEA in combination with other mycotoxins provided to female piglets, in short exposure, was not available until this study. However, different results could be attributed to differences in mycotoxins, dose levels, and exposure lengths.

Mycotoxins are reported to promote liver lesions, which can change liver enzymes and protein serum concentration in piglets (THIEU et al., 2008; JIANG et al., 2011). In our study, dietary exposure to ZEA, FB, and AF, at the tested levels, did not change the activity of the AST, GGT, or ALP enzymes (Table 2). Only the total protein concentration was increased (P<0.05) after d21 in piglets exposed to the multi-mycotoxin treatment (T2) compared to that in the control group (T1) (Table 2). However, the observed variation in total protein concentration found in this study was within the normal limit (4.0 to 5.8 g/dL) of pathogen-free 6-week old piglets (COOPER et al., 2014). This suggests that there was no effect on liver function. In piglets, DON and FB in combination had less effect on biochemistry parameters than mono-contaminations and the authors, after to evaluate other studies testing the same mycotoxins, suggested an opposite effect of these two mycotoxins (GRENIER et al., 2011). At the present, it is unclear whether other co-contaminations in low doses could present pronounced effect on biochemical parameters in piglets. To better of our knowledgement, further experiments based on realistic doses (GRENIER; APPLEGATE, 2013), different mycotoxin combinations, or experimental periods of exposure in piglets should be considered; particularly as such studies will be most relevant to commercial livestock.

The mononuclear cells are predominant in chronic immune process and mediate cytokines release and humoral response. A decreased expression of IL-6 mRNA in piglets exposed to 5,900 ppb FB has been associated with defective antigen presentation, impaired lymphocyte activation, and decreased IgG response (GRENIER et al., 2011). ZEA alone has been reported to reduce the humoral immunity response mediated by B cells (MARIN et al., 2011). Aflatoxin B₁ has been reported to decrease cell-mediated immunity (MEISSONNIER et al., 2008). The toxic effect of FB on the immune response has been shown to occur through inhibition of ceramide synthesis, as indicated by sphingoid base accumulation (MACHADO JÚNIOR et al., 2014). This mechanism can inhibit lymphocyte development and affect production of cytokines. Although the mechanism by which ZEA and AF affect the immune system remains unclear, the in vitro cytotoxic effect of α-zearalenol can be enhanced by FB₁ (LUONGO et al., 2006). However, after 21 d, levels of circulating mononuclear cells, measured by flow cytometry, were not affected by the multi-mycotoxin treatment (Table 2). Taken together, these data could be of utility in determining legal limits for multi-contaminant levels of mycotoxins in swine feedstuffs.

Conclusions

During 21 days after weaning, a multi-contaminated diet containing 88.5 ppb ZEA, 3,994 ppb FB, and 107.5 ppb AF did not exert negative effects on piglets' health, and the efficacy of the anti-mycotoxin additive was not posed due the absence of toxicity associated with the multi-mycotoxin treatment.

Acknowledgments

The authors thank the Impextraco Latin America^® for financing and the Pontifícia Universidade Católica do Paraná (PUCPR) for supporting this study. Antonio Diego Brandão Melo also would like to thank for the support by a grant from CAPES, Brazil.

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