Lack of Toxic Interaction between Fusariotoxins in Broiler Chickens Fed throughout Their Life at the Highest Level Tolerated in the European Union

Fusarium mycotoxins (FUS) occur frequently in poultry diets, and regulatory limits are laid down in several countries. However, the limits were established for exposure to a single mycotoxin, whereas multiple contamination is more realistic, and different studies have demonstrated that it is not possible to predict interactions between mycotoxins. The purpose of this study was thus to compare the toxic effect of deoxynivalenol (DON), fumonisins (FB) and zearalenone (ZON), alone and in combination on broiler chickens, at the maximum tolerated level established by the EU for poultry feed. Experimental corn-soybean diets incorporated ground cultured toxigenic Fusarium strains. One feed was formulated for chickens 0 to 10 days old and another for chickens 11 to 35 days old. The control diets were mycotoxin free, the DON diets contained 5 mg DON/kg, the FB diet contained 20 mg FB1 + FB2/kg, and the ZON diet contained 0.5 mg ZON/kg. The DONFBZON diet contained 5, 20, and 0.5 mg/kg of DON, FB1 + FB2, and ZON, respectively. Diets were distributed ad libitum to 70 broilers (male Ross PM3) separated into five groups of 14 chickens each reared in individual cages from one to 35 days of age. On day 35, after a starvation period of 8 h, a blood sample was collected, and all the animals were killed and autopsied. No difference between groups that could be attributed to FUS was observed in performances, the relative weight of organs, biochemistry, histopathology, intestinal morphometry, variables of oxidative damage, and markers of testicle toxicity. A significant increase in sphinganine and in the sphinganine to sphingosine ratio was observed in broilers fed FB. Taken together, these results suggest that the regulatory guidelines established for single contamination of broiler chickens fed with DON, FB, and ZON can also be used in the case of multiple contamination with these toxins.


Diets and Performances
shows the expected and measured levels of FUS in the diets. Although traces of DON, FB, and ZON were found in the control diets, their levels were always <100 µg/kg and were considered not significant. Measured concentrations of DON, FB, and ZON in the contaminated diets differed between 10% and 20% from the expected levels. The biggest difference was found for DON in the DONFBZON diet. In all the diets, no unexpected FUS, other than DON, FB, and ZON, were detected or were <20 µg/kg. All other mycotoxins were below the limit of quantitation.  1 As described in Material and Methods, 10 experimental diets were formulated to provide respective total protein and metabolizable energy of 22% and 2880 kcal/kg for chickens from 0 to 10 days of age and 19.5% and 3050 kcal/kg for chickens from 11 to 35 days of age. Expected and measured concentrations of fusariotoxins (µg/kg) in the diets contaminated by deoxynivalenol alone (DON), fumonisins alone (FB), zearalenone alone (ZON) and a mixture of the three toxins (DONFBZON). 2 Sum of FB1 + FB2. 3 Concentrations of fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively.
Neither mortality nor any signs of mycotoxicosis were observed in any of the chickens during the 35-days study. The effects of feeding FUS on performances at the two ages are detailed in Table 2. From 0 to 10 days, no differences among groups were observed in body weight (BW), feed consumption (FC), daily weight gain (DWG), and feed conversion ratio (FCR). By contrast, a significant increase in FC was observed in chickens fed the ZON and DONFBZON diets from 11 to 35 days, but had no consequence for BW and FCR. Over the entire period (0 to 35 days), the only significant difference observed among the groups was in FC, but no group differed significantly from the control group (data not shown).

Relative Organ Weights and Histopathology
Although some signs of runny noses, puddle hearts, and signs of reactivity in the caeca tonsils were found in some chickens, these signs were not more marked in one group than in another. The effects of FUS on the relative weights (RW) of liver, kidney, intestine, caecum, gizzard, and hearth are shown in Table 2. Although some differences in the RW of the liver were found among groups, no group differed from the control group. No significant difference in the RW of pancreas, spleen, proventriculus, duodenum, jejunum, ileum, caeca tonsils, and Fabricius bursa was observed among groups ( Table 2). The color of the liver and caeca tonsils did not differ among groups.
Histopathological examination of the hepatic and renal samples revealed discrete non-specific inflammatory lesions with no difference in frequency or intensity among groups. Lymphoid hyperplasia with signs of typhlitis was observed in the caeca mucosa. These lesions varied greatly depending on the section examined and were not more frequent in one group than in another. Characterization of intestinal morphometry of duodenum, jejunum, and ileum revealed no significant differences in the size of the villus and crypts in the duodenum and ileum among groups whereas a small increase in crypt depth of the jejunum was observed in broilers fed the diet containing FUS (Table 3). Moreover, no significant difference in the surface area and the villus to crypt ratio of the duodenum and ileum was found among groups (data not shown). By contrast, in the jejunum, the surface area of the crypt increased in broilers fed FUS whereas the villus to crypt ratio decreased and the surface area of the villus was not affected (Table 3). No significant difference in the number of goblet cells in the duodenum, jejunum, and ileum was found among groups.

Serum Biochemistry and Hematology
The effect of FUS on biochemistry and hematology are presented in Table 4. No statistical difference in proteins, cholesterol, IgA and Hb concentrations was found among groups. In the same way, no effect was observed on LDH, ALP, and ALT activities or on the number of erythrocytes and leucocytes. Only a small but significant increase in the concentration of uric acid was observed in chickens fed with ZON alone.  1 Values were obtained from 14 animals per group aged 35 days and are expressed as mean ± SD. One-way ANOVA was performed to compare groups. When a significant difference was observed (* p < 0.05), means were compared (Kruskall-Wallis). Different letters in the same row identify statistically different groups (p < 0.05). 2

Oxidative Markers and Antioxidant Enzyme Activity in Plasma and Liver
Different variables were investigated to identify oxidative damage to broiler chickens (Table 5). MDA and TGlu contents in plasma, and MDA, TGlu, and GSSG contents in the liver remained unchanged after exposure to FUS. The GSH/GSSG ratio in the liver was also unaffected by the toxins (data not shown). Investigation of the activities of most of the enzymes involved in defense against oxidative damage, SOD, CAT, GsPx, and GsRed, both in plasma and liver, failed to reveal any effect of the FUS, alone or in association.

Testis Toxicity
The weight of the testis was similar in all the groups but the ZON group presented a 25% increase in weight compared to the control group ( Table 6). The diameter of the seminiferous tubule in the ZON group was also 17% larger than in the controls, whereas a 21% decrease was observed in the DONFBZON group. Cell death observed by ISEL staining failed to reveal any significant difference among groups (data not shown). The activity of the cleaved caspase 3 was similar in the five groups (Table 6).  3 Results are expressed in the following units: Testis weight, mg; seminiferous tubules diameter, µm; caspase 3 activity, relative luminescence units (RLU)/mg protein; deleted in azoospermia-like (DAZL), ng/mg protein; proliferating cell nuclear antigen (PCNA), ng/mg protein; interferon gamma (IFN-γ), pg/mg protein; interleukin 1 beta (Il1β), pg/µg proteins; total antioxidant capacity (TAC), trolox equivalent antioxidant capacity/mg proteins; catalase (CAT) activity, in pmol/min/mL/µg proteins; cAMP, µMolar/mg proteins; testosterone, ng/µg proteins.
As revealed by VASA staining, germ cells were present in the testis ( Figure 1). The number of immature germ cells estimated by intra-testis Dazl content was higher in the FB group than in the other groups. This increase was associated with higher PCNA content, a marker of cell proliferation, in the FB group. As revealed by VASA staining, germ cells were present in the testis ( Figure 1). The number of immature germ cells estimated by intra-testis Dazl content was higher in the FB group than in the other groups. This increase was associated with higher PCNA content, a marker of cell proliferation, in the FB group.
Inflammation markers (IFN-γ and IL1β) and oxidative stress measured by TAC and catalase activity were not affected by exposure to mycotoxins. Despite the fact that testosterone and cAMP production was measured in an immature testis (before puberty, 35 days post-hatching), no effect was observed.

Sphinganine to Sphingosine Ratio
Free Sa and So were quantified in the liver and the Sa to So ratio was calculated ( Figure 2). No significant difference in So concentration was observed among groups whereas the Sa level and the Sa to So ratio increased in broilers fed with the FB and DONFBZON diets (ANOVA, p < 0.05). Complementary comparison of means (Kruskall-Wallis) revealed no significant difference in the Sa level in the liver between broilers fed the FB and DONFBZON diets, whereas the Sa/So ratio was significantly higher in chicken fed the DONFBZON diet than in chickens fed the FB diet (p < 0.05). This result was surprising because the total amount of FB1 + FB2 was only slightly higher in the FB diet than in the DONFBZON diet, 23.13 and 19.23 mg FB1 + FB2/kg, respectively (Table 1). No significant difference in Sa and So contents or in the Sa/So ratio in the liver was observed between chickens fed the control diet and chickens fed the DON and ZON diets (data not shown). Inflammation markers (IFN-γ and IL1β) and oxidative stress measured by TAC and catalase activity were not affected by exposure to mycotoxins. Despite the fact that testosterone and cAMP production was measured in an immature testis (before puberty, 35 days post-hatching), no effect was observed.

Sphinganine to Sphingosine Ratio
Free Sa and So were quantified in the liver and the Sa to So ratio was calculated (Figure 2). No significant difference in So concentration was observed among groups whereas the Sa level and the Sa to So ratio increased in broilers fed with the FB and DONFBZON diets (ANOVA, p < 0.05). Complementary comparison of means (Kruskall-Wallis) revealed no significant difference in the Sa level in the liver between broilers fed the FB and DONFBZON diets, whereas the Sa/So ratio was significantly higher in chicken fed the DONFBZON diet than in chickens fed the FB diet (p < 0.05). This result was surprising because the total amount of FB1 + FB2 was only slightly higher in the FB diet than in the DONFBZON diet, 23.13 and 19.23 mg FB1 + FB2/kg, respectively (Table 1). No significant difference in Sa and So contents or in the Sa/So ratio in the liver was observed between chickens fed the control diet and chickens fed the DON and ZON diets (data not shown).

Discussion
In this study, a single exposure to DON, ZON, and FB from 0 to 35 days did not lead to any alteration in the performances of the broiler chickens or have a significant effect on organ weight, histopathology or biochemistry. These results are not surprising because single exposure involved FUS concentrations in diets that are equal to or below regulatory limits in poultry diets and these species are known to tolerate FUS [2,11,[23][24][25]. Moreover, only a weak effect of FUS was found on intestinal morphometry of duodenum, jejunum, and on the number of goblet cells, in agreement with a long term study conducted in slow-growing chickens fed 2 to 10 mg DON/kg diet [26]. Conversely, some studies reported that DON altered the small intestinal morphology and the length of the villi in the jejunum in broilers [27,28] and that FB reduced the length of the villi and the crypt depth of the ileum [29]. Differences between studies could be due to the way the toxins were added to the diet or to external factors. Indeed, studies carried out to evaluate the effects of mycotoxins can be conducted by naturally contaminated feedstuffs, culture material or pure crystalline toxins. The response from the different sources is an important contributor to the susceptibility in the model (i.e., naturally contaminated material is often more toxic in research environments presumably because other lesser-known or unknown compounds may contribute to their effects). In this study, the objective was to compare single-and multiple-dose effects of DON, ZON, and FB with the maximum recommended in poultry feed in Europe. The use of mycotoxins obtained from fungal culture extracts appeared the best way to obtain contaminated feeds with the desired exposure scenarios. Indeed, the culture material, although partially purified, still contains the metabolites that are formed in the synthesis of the final mycotoxin. Moreover, it is reasonable to assume that the mix of the extract with the feed during its manufacture results in little variation in mycotoxins bioavailability, at least compared to the administration of toxins by oral gavage [30]. With regard to external factors, this study was carried out under conditions aimed at optimizing the measurement of the effects of toxins on healthy animals. For this reason, breeding in individual cages was preferred to breeding in parquet floors. In general, farming management can change the performance and response of animals to various factors, including stress. Similarly, concomitant exposure to infectious or parasitic agents may alter the response of animals to mycotoxins, as observed in broiler chickens fed DON and FB at a not toxic level that amplified the severity of coccidiosis [19].
Only a few investigations have been conducted on oxidative stress in the course of FUS exposure in broiler chickens. One study reported that 7.54 mg DON/kg feed down-regulated heme-oxygenase and upregulated xanthine oxidoreductase mRNA in the liver [31]. Another study reported that 100 mg FB1/kg of feed increased hepatic MDA levels and CAT activity in chickens [32]. Moreover, the intake of contaminated feed containing DON and ZON in combination was reported to significantly reduce the activity of glutathione peroxidase and to increase the level of MDA in liver tissue [33]. In the present study, several variables were measured in plasma and liver to reveal oxidative stress, but none was significantly changed by FUS exposure.
Effects of FUS on reproductive function are usually not reported in avian species, except with very high levels of ZON [11,34]. The weights of testes were significantly reduced in broilers fed 200 to 400 mg ZON/kg [35]. In mammals, the most toxic effect of FUS other than ZON on reproductive function has been mild-to-moderate lesions of testes with Sertoli cell degeneration and impaired spermatogenesis observed in rabbits fed 0.13 to 5 mg FB1/kg diet for 175 days [36,37]. Studies conducted in mice suggested that DON may have an adverse effect on the epididymal weight at 10 mg/kg of feed for 90 days with slight changes in relative testis weight and spermatid counts, but no histological changes [38]. In the present study, no significant difference was observed in the variables measured to reveal toxicity of FUS in broiler testes except a decrease in the diameter of the seminiferous tubule and a decrease in catalase activity in the DONFBZON group that are difficult to interpret. All these results confirm that broilers are less sensitive to the toxic reproductive effects of FUS than mammals, even when a mixture of toxins was used.
Using biomarkers of effects is a good way to reveal the effect of mycotoxins on health at a level of exposure lower than one that is toxic. Alteration of sphingolipid metabolism has been known for several years to be the best biomarker of FB exposure in most animal species, including poultry [9,11]. In the present study, a significant increase in the level of Sa in liver concomitant with an increase in the Sa/So ratio was observed in the FB-treated group compared to the control group. This result is in agreement with previous data obtained with higher levels of FB in broiler chicken diets, and with data obtained in plasma using the same dose [29,38]. Together, the lack of histopathological damage to the liver and the lack of increase in LDH activity in plasma confirmed the alteration of sphingolipid metabolism occurred at a level of FB that is not hepatotoxic in broiler chickens.
Taken together, these results confirm that single exposure of broiler chickens from 1 to 35 days of age to FUS concentrations in diets that are equal to or below regulatory limits for poultry feed had no deleterious effect on health. Interestingly, no additive, synergistic or antagonist effect on the variables measured was observed in broilers exposed to the diet containing a mixture of DON, FB, and ZON at similar levels as those used in formulation trials conducted with a single toxin. Although previous studies on chickens revealed interactions between FUS, no study was conducted using EU regulatory limits. Concerning sphingolipids in the liver, the Sa/So ratio was significantly higher in chickens fed the DONFBZON diet than in chickens fed the diet containing FB alone, whereas the measured concentration of FB1 + FB2 was higher in the FB diet than in the DONFBZON diet. The apparent synergistic effect of FUS on the Sa/So ratio should be interpreted with caution and should take the effects on Sa and So and not only the effects on the Sa/So ratio into account. Indeed, previous studies on ducks and turkeys with low doses of FB demonstrated that the level of FB1 in the diet is always correlated with an increase in Sa content in the liver. By contrast, the effects of low doses of FB1 on So content are less pronounced and vary with the study [39][40][41]. Moreover, most studies on avian species have shown that toxic levels of FB in diets are responsible for the liberation of LDH by the hepatocytes in plasma and for an increase in LDH activity in this medium [41][42][43][44], which was not the case in our chickens fed the DONFBZON diet.
In conclusion, this study demonstrated for the first time the lack of strong interactions between DON, FB and ZON fed alone or in combination to broiler chickens from 1 to 35 days in age on the variables measured. Together, the lack of effect of these toxins on performances, organ weight, biochemistry, histopathology, variables of oxidative damage and markers of testis toxicity suggest that the regulatory guidelines established for single contamination of broiler chickens fed DON, FB and ZON can be used in the case of multiple contamination with these toxins.

Fusariotoxin Production and Experimental Diets
All the experimental diets containing mycotoxins incorporated ground cultured toxigenic Fusarium strains. Briefly, fumonisins were produced on crushed corn at 25 • C using F. verticillioides strain L12. Deoxynivalenol was produced by growing F. graminearum strain I159 on wheat at 23 • C, and zearalenone was obtained by culturing F. graminearum strain I171 on rice at 21 • C. After four weeks of growth, the cultured Fusarium species were dried at 90 • C for 3 h, ground and sieved through a 0.6 mm mesh. The concentrations of mycotoxins in the powders were measured by HPLC-MSMS.
Experimental corn-soybean diets were used to best meet the nutritional needs of the animals. One feed was formulated for 0 to 10 days of age (CP = 22%, ME = 2880 kcal/kg) and another for 11 to 35 days of age (CP = 19.5% and ME = 3050 kcal/kg). Powders containing the mycotoxins were incorporated to obtain 10 different experimental diets ( Table 1). The control diets (Control) were free of mycotoxins. The deoxynivalenol (DON), fumonisin (FB), and zearalenone (ZON) diets were made by incorporating powdered cultured materials to obtain 5 mg DON/kg, 20 mg FB1 + FB2/kg, and 0.5 mg ZON/kg, respectively. The three toxins were added in the diet that contained the mixture of fusariotoxins (DONFBZON) at concentrations of 5, 20 and 0.5 mg/kg for DON, FB1 + FB2, and ZON, respectively. The concentrations of mycotoxins in the different diets was checked by HPLC-MSMS.

Animal Husbandry and Sample Collection
All experimental procedures with animals were in accordance with the French National Guidelines for the care and use of animals for research purposes. The experimental protocol was approved by the French Ministry of Higher Education and Research and registered under number 02032.01. Seventy broilers (male Ross PM3) were reared in individual cages in an experimental station (ARVALIS-Institut du vegetal, Villerable, France). Each experimental diet was distributed to 14 broilers ad libitum throughout the experiment along with ad libitum access to water. Individual body weight and feed consumption were measured weekly. On the 35th day of age, after a starvation period of 8 h, a blood sample was collected. Samples were centrifuged and serum was collected and stored at −80 • C until analysis. The animals were stunned by electrocution and killed by exsanguination. Autopsies were performed on all the animals to investigate macroscopic lesions and to examine the color of the liver and cecum tonsils. The heart, liver, spleen, thymus, pancreas, kidneys, and testicles were collected and weighed. The intestine was emptied, then the gizzard, proventriculus, duodenum, jejunum, ileum, and caeca (including caeca tonsils) were isolated and weighed. The length of the duodenum, jejunum and ileum segments was measured. The color of the liver and cecum tonsils was measured with a chromameter (Konica Minolta, Europe). Samples of liver, kidney, spleen, duodenum, jejunum, ileum, caecum, caeca tonsils, and Fabricius bursa were placed in 10% formaldehyde for microscopic examination. The remaining liver and testicles were stored at −80 • C until analysis.

Biochemistry, Hematology, and Histopathology
Plasma concentrations of lactate dehydrogenase (LDH) EC 1.1.1.27, alkaline phosphatase (ALP) EC 3.1.3.1, and alanine aminotransferase (ALT) EC 2.6.1.2 were analyzed using a clinical chemistry KONELAB 20 analyzer (Fisher Scientific SAS, Illkirch, France) and are expressed in UI/L according to international guidelines. Proteins, cholesterol, and uric acid were measured according to the manufacturer's instructions and are expressed in g/L or mmol/L of plasma. Immunoglobulin A (IgA) assays were performed using the chicken IgA Elisa kit following the manufacturer's instructions (Bethyl Laboratories Inc., Montgomery, TX, USA). Hemoglobin (Hb) and erythrocytes contents were measured using Hycel Celly analyzer (EUROCELL Diagnostics, Rennes, France) and are expressed in mg/L and in 10 3 number of cells/mL, respectively. The white blood cell count was done manually in Malassez cells, and the results are expressed in 10 3 number of cells/mL. Except for the testes, fixed tissues were trimmed, embedded in paraffin, and 4 µm sections cut with a microtome, and stained with hematoxylin, eosin, and saffron (HES). All the tissues from the control and treated groups were examined microscopically. Fixed testes were processed in paraffin and serially sectioned at 7 µm. The diameter of the seminiferous tubules was measured with round or nearly round tubules from each testicular section stained with Meyer's hematoxylin (Sigma, l'Isle d'Abeau Chesnes, France). At least 40 measurements of the diameter of the transverse sections of seminiferous tubules were measured using an ocular measuring device. Four different chickens were analyzed per treatment.

Immunohistochemistry and Detection of Apoptosis
Testes embedded in paraffin were serially sectioned at 7 µm. Deparaffinized sections were hydrated, microwaved for 5 min in an antigen unmasking solution (Vector Laboratories, Inc., AbCys, Paris, France), and left to cool to room temperature. The sections were washed in a PBS bath for 5 min and immersed in peroxidase blocking reagent at room temperature for 10 min to quench endogenous peroxidase activity (DAKO Cytomation, Dako, Ely, UK). After two PBS baths for 5 min, nonspecific background was prevented by incubation in 5% lamb serum/PBS for 30 min. Finally, the sections were incubated with PBS containing primary antibody against chicken Vasa overnight at 4 • C [46]. The following day, after two PBS baths for 5 min, sections were incubated for 30 min at room temperature with "ready to use" labeled Polymer-HRP anti-rabbit (DAKO Cytomation, Dako, Ely, UK). Finally, substrate containing the sequence asp-glu-val-asp, and after cleavage by caspases, a luciferin substrate is liberated and is used by luciferase to generate light. The amount of luminescence is proportional to the amount of caspase activity present in the cell lysate. Luminescence was measured in relative light units and normalized to 100,000 seminiferous tubule cells.
The chicken proliferating cell nuclear antigen (PCNA) was measured to analyze cell proliferation, whereas when azoospermia-like protein (DAZL) is deleted, this is a marker of spermatogonia used to reveal the immature germ cell content. Interleukin 1β (Ilβ), and interferon-γ (IFN-γ) concentrations were quantified to assess inflammatory response in the testis. All standards and samples were assayed according to the manufacturer's recommendations (Cusabio, Eurobio, Courtaboeuf, France).
Total antioxidant capacity (TAC) and catalase activity were measured by the Cayman's antioxidant assay kit according to the instruction manual (Cayman, Interchim, Montluçon, France). Assay plates were red by using an ELISA plate reader at 750 nm for TAC and at 540 nm for catalase activity. The TAC assay is based on the ability of antioxidants (vitamins, proteins, lipids, glutathione, uric acid, etc.) to inhibit the oxidation of 2,2 -azino-bis,3-ethylbenzthiazoline-6-sulphonic acid. Total antioxidant capacity is expressed as Trolox equivalent antioxidant capacity per mg of testicular protein extract.
The concentration of cAMP was measured using the cAMP-Glo assay as recommended by the manufacturer (Promega, Madison, WI, USA). The concentration of testosterone was assessed by radioimmunoassay in duplicate as previously described [51]. The sensitivity test was 15 pg/tube and intra-assay coefficients of variation of 5.3%.

Determination of the Sphinganine to Sphingosine Ratio
Calculation of the sphinganine (Sa) to sphingosine (So) ratio requires prior determination of free Sa and free So contents (Riley et al., 1994a). Briefly, 0.2 nmol of C 20 sphinganine (Matreya, Inc., Pleasant Gap, PA, USA) used as internal standard were added to 100 µL of plasma or S9. Sphingolipids were extracted by alkaline methanolic-chloroform and the chloroform phase was washed twice with alkaline water. Extracts were dried under nitrogen flow, then suspended in 20 µL methanol, and sonicated for 10 min. Derivatization of sphingolipid was performed with ortho-phthalaldehyde before injection using automate (ICS M2200, Toulouse, France). The HPLC system is composed of an ICS M2200 HPLC autosampler (ICS, Toulouse, France) that delivers methanol-water (90:10, v/v) to a Prontosil C18 cartridge equipped with a C18 pre-column filter (Bischoff, Leonberg, Germany). A fluorescence detector (FD-500 Shimazu, Kyoto, Japan) was used for detection, the excitation wavelength was 335 nm, and the emission wavelength 440 nm. Mean retention times were 12, 17 and 29 min for sphingosine, sphinganine and C 20 sphinganine, respectively. Concentrations were calculated by linear regression from standard solutions that were injected daily. C 20 sphinganine was used as an internal standard to monitor the extraction rate the sphingolipids in the samples.

Statistical Analysis
Data on all response variables are reported as means ± SD or SEM and were the subject of one-way analysis of variance (ANOVA) after a normality test (Shapiro-Wilk). When a significant difference was observed (p < 0.05), the difference between means was determined by individual comparison of means (Kruskall-Wallis). Statistically different groups (p < 0.05) are identified by a different letter. All statistical analyses were conducted by XLSTAT Biomed. Funding: This study was supported by CASDAR grants (project 2012-2015 MYCOVOL). The authors would like to thank the RMT Quasaprove for supporting the project.

Conflicts of Interest:
The authors declare no conflict of interest.