Introduction
Emerging and modified mycotoxins have received great interest after clinical signs were observed in animals that did not correlate with the levels of mycotoxins detected in their feed. In this scenario, it is important to consider that natural contamination of raw materials and feed often involves ‘multi-contamination’ by different mycotoxins, including emerging and modified mycotoxins that are not routinely detected in standard control analyses.
Information on the toxicity of emerging mycotoxins is limited. According to the latest EFSA publication (2014) focusing on beauvericin and enniatins, few studies have evaluated the acute toxicity of these mycotoxins in vivo. One study in mice evaluating the effect of beauvericin is mentioned.
However, different in vitro studies have evaluated the toxicity of these mycotoxins in poultry. The summary table shows the different studies that provide insights into the known mechanism of action of these emerging mycotoxins.
Mechanisms of action of emerging mycotoxins
1. Oxidative stress
Considering the intracellular redox balance, several studies have reported an increase in reactive oxygen species (ROS) levels in various cell cultures. This increase of ROS has been accompanied by an increase in lipid peroxidation products such as MDA or TBARS (biomarkers of oxidative stress) and a decrease in antioxidant enzymes such as glutathione. Thus, emerging mycotoxins have been shown to induce oxidative stress at the cellular level.
Nevertheless, EFSA (2014) reported a study in which a reduction of ROS was observed in human cells treated with hydrogen peroxide (Dornetshuber et al., 2009), suggesting that more information is needed on the redox balance and emerging mycotoxins in different cell lines, and even, considering the animal species.
2. Cytotoxicity
The cytotoxicity of emerging mycotoxins has been evaluated in different in vitro studies. However, focusing on avian cell lines, the study by Dombrik-Kurtzmann (2003), which evaluated the effect of beauvericin on turkey lymphocytes, is noteworthy. DNA fragmentation and subsequent apoptosis were observed. Thus, there is in vitro evidence of the cytotoxicity of emerging mycotoxins in poultry.
Indeed, potent cytotoxic activity by inducing apoptosis has been described. According to EFSA (2014), apoptosis is induced mainly by two pathways:
- Increased intracytoplasmic calcium levels that stimulate calcium-dependent endonucleases.
- They intercalate with DNA, paving the way for endonuclease activity.
In addition, several authors have attributed the cytotoxicity of beauvericin and enniatins to their ionophore properties.
3. Intestinal integrity
Given that cytotoxicity has been observed in various cell lines and knowing that the intestinal barrier is the first contact of animals with mycotoxins after ingestion, it is of great interest to know the effect of beauvericin and enniatins on intestinal cells. However, the effect of emerging mycotoxins on the intestinal barrier has been little studied to the date.
Springer et al. (2016) evaluated the effect of beauvericin and enniatins in the transmembrane electrical resistance of intestinal epithelial cells, specifically in the jejunum, as this is an important region for mycotoxins absorption. Increased intestinal permeability was observed in association with decreased expression of tight junctions. In particular, enniatin B showed the strongest effect, followed by beauvericin, enniatin B1, enniatin A and enniatin A1. Among the enniatins, an additive effect was described; this was not observed in combination with DON. In contrast, Albonico et al. (2016) did not describe an effect of beauvericin on intestinal permeability or pro-inflammatory cytokines in case of single contamination, but when presented together with fumonisin B1 or DON. The difference between studies may be due to the cell culture used as well as the concentrations of the mycotoxins. However, the studies as a whole demonstrate that emerging mycotoxins have an impact on gut integrity and underline the importance of being aware of multi-contaminations by mycotoxins.
4. Contextualization of in vivo studies
According to EFSA (2014), the available studies on the toxicity of emerging mycotoxins in poultry were conducted on broilers, laying hens and turkeys exposed to a multi-contamination of Fusarium mycotoxins, including beauvericin and enniatins. In general, the natural source of mycotoxins for the studies was maize and no effects on production parameters, carcass yield and relative organ weights such as liver, spleen, bursa of Fabricius, heart, etc. were observed. Furthermore, no concentrations of beauvericin and enniatins were detected in products for animal consumption (muscle tissue or egg).
Contamination levels that do not cause adverse effects on the productive parameters and physiology of poultry according to EFSA (2014) are shown below:
Table 1. Levels (ppb in feed) with no observed adverse effects1.
| Broiler chickens | Laying hens | Turkeys | ||
|---|---|---|---|---|
| Beauvericin | 12600 ppb | 8930 ppb | 2480 ppb | |
| Enniatins | B | 12720 ppb | 11230 ppb | – |
| B1 | 4060 ppb | 3060 ppb | – | |
1 In multi-contamination with other Fusarium mycotoxins.
Considering live weight and consumption, levels are shown according to live weight of the animal/d.
Table 2. Levels (µg/kg BW/day) with no observed adverse effects1.
| Broiler chickens | Laying hens | Turkeys | ||
|---|---|---|---|---|
| Beauvericin | 1220 µg/kg PV/día | 536 µg/kg PV/día | 136 µg/kg PV/día | |
| Eniatins | B | 763 µg/kg PV/día | 674 µg/kg PV/día | – |
| B1 | 244 µg/kg PV/día | 216 µg/kg PV/día | – | |
1In multi-contamination with other Fusarium mycotoxins.
It should be noted that these values are presented in accordance with the maximum levels assessed for the mycotoxins in the studies reviewed. The absence of negative effects on poultry may be due to the low bioavailability and rapid elimination of these mycotoxins in the body (Fraeyman et al., 2018).
Recent studies have demonstrated the effects of emerging mycotoxins on the intestinal barrier and the productive parameters. The reduction in crypt depth observed in broilers challenged with enniatin may be due to the inhibitory effect on enterocyte proliferation exerted by the mycotoxin (Fraeyman et al., 2018). Along the same lines, Santos and van Eerden (2021) observed an increase in the villus height:crypt depth (VH:CD) ratio of the ileum of 14-day-old broiler chicks that was attributed to a reduction in intestinal cell proliferation, without an immediate effect on villus height. At 28 days, a reduction in villus height in jejunum and VH:CD ratio in jejunum and ileum (with increased crypt depth) was observed. Thus, the reduced surface area for nutrient absorption is a possible explanation for the poorer performance observed. On the other hand, growth and feed efficiency are compromised as poultry expends more energy to restore the epithelium. These results have been observed in broilers challenged after ingestion of a multi-contaminated diet with mycotoxins such as beauvericin, enniatins, DON and metabolites.
Table 3. Summary of in vitro and in vivo studies of the effect of emerging mycotoxins in poultry1.
| Reference | Study | Mycotoxins | Effects | |
|---|---|---|---|---|
| Prosperini et al. (2013) reference from EFSA (2014) | In vitro | Caco-2 cells | beauvericin | ↑ROS, MDA, GSSG ↓ GSH ↑ apoptosis |
| Prosperini et al. (2013) reference from EFSA (2014) | In vitro | Caco-2 cells | enniatin | ↑ ROS |
| Mallebrera et al. (2014) reference from EFSA (2014) | In vitro | CHO-K1 cells | beauvericin | ↑Lipid peroxidation ↓ glutathione |
| Dornetshuber et al. (2009) reference from EFSA (2014) | In vitro | Human promyelocytic leukaemia cells (HL60) and human cervical carcinoma cells (KB-3-1) | beauvericin enniatin | ↓ ROS when cells are treated with H2O2 |
| Dombrik-Kurtzmann (2003) reference from EFSA (2014) | In vitro | Turkey lymphocytes | beauvericin 8-50 µmolar | Fragmentation ADN apoptosis |
| Albonico et al. (2016) | In vitro | Caco-2 cells | beauvericin 1,5 µmolar beauvericin 1,5 µmolar + fumonisin B1 1,5 µmolar beauvericin 1,5 µmolar + DON 1,5 µmolar | No effect in TEER, pro-inflammatory cytokines ↓TEER ↑ pro-inflammatory cytokines (IL-8) |
| Vesonder et al. (1999) reference from EFSA (2014) | In vivo | Pekin ducks 7 d | <100 mg/kg BW beauvericin (purified mycotoxin) | No acute toxicity |
| Leitgeb et al. (1999, 2003) reference from EFSA (2014) | In vivo | Broiler chickens 37 d | 800 ppb beauvericin 5600 ppb DON 700 ppb 15-acetilDON 600 ppb ZEN 500 ppb moniliformin 300 ppb NVI (maize naturally contaminated) | No negative effect in BWG, FCR, weight of liver No differences in meat quality, blood parameters |
| Leitgeb et al. (1999, 2003) reference from EFSA (2014) | In vivo | Turkey 11 weeks (growing period) | 2480 ppb beauvericin 1200 ppb DON 300 ppb 15-acetilDON 200 ppb ZEN 3000 ppb moniliformin 200 ppb NVI (maize naturally contaminated) | No differences in BW, FCR No differences in relative weight of cooked carcass, spleen, heart, bursa of Fabricius, liver No differences in blood parameters |
| Zollitsch et al. (2003) reference from EFSA (2014) | In vivo | Broiler chickens 37 d | 4200, 8400, 12600 ppb beauvericin 900, 1800, 2700 ppb moniliformin (maize naturally contaminated) | No differences in BWG, FCR No differences in carcass characteristics or chemistry composition No residues of beauvericin or moniliformin in the carcass or in the intestinal organs |
| Leitgeb et al. (2003) reference from EFSA (2014) | In vivo | Turkey 84 d | ≤ 2480 ppb beauvericin ≤ 2350 ppb moniliformin | No negative effects in BWG, FCR No differences in physiological parameters |
| Ivanova et al. (2014) reference from EFSA (2014) | In vivo | Broiler chickens | 12700 ppb enniatin B | No adverse effects |
| Ivanova et al. (2014) reference from EFSA (2014) | In vivo | Laying hens | 11200 ppb enniatin B | No adverse effects |
| CODA-CERVA (2011/2012) reference from EFSA (2014) | In vivo | Broiler chickens | 12720 ppb enniatin B 4060 ppb enniatin B1 10310 pb beauvericin DON 3-acetyl-, 15-acetyl-, de-epoxy-DON ZEN, α-, β-zearalenol T2 HT2 | No adverse effects |
| CODA-CERVA (2011/2012) reference from EFSA (2014) | In vivo | Laying hens | 11230 ppb enniatin B 3600 ppb enniatin B1 8930 ppb beauvericin | No adverse effects |
| Fraeyman et al. (2018) | In vivo | Broiler chickens 21 d | 2352 ppb enniatin B vs 135 ppb (control) | Liver: no effect on histopathology, limited transfer Plasma: limited transfer Duodenum: reduces CD, suggests inhibitory effect on duodenal enterocyte proliferation Jejunum, ileum: no effect on VH, CD, VH/CD |
| Callebaut et al. (2012) | In vivo | Broiler chickens 14 d | 28 ppb enniatin A 440, 491 ppb enniatin A1 11233, 12716 ppb enniatin B 3599, 4057 ppb enniatin B1 8926 10313 ppb beauvericin | No effect on growth, fattening |
| Springler et al. 2016 | In vitro | IPEC-J2 cells | enniatin A1 enniatin B enniatin B1 beauvericin | Strongest effect: enniatin B > beauvericin > enniatin B1 > enniatin A > enniatin A1: ↓trans-epithelial electrical resistance (TEER) Additive effect between enniatins No additive effect with DON |
| Santos and van Eerden (2021) | In vivo | Broiler chickens 35 d | 0-14 d: 2060 vs 878 ppb DON 132 vs 99 ppb DON-3-Glucoside 28 vs 90 ppb enniatin B 13 vs 16 ppb enniatin B1 10 vs 0 ppb alternariol 14-28 d: 2360 vs 941 ppb DON 1670 vs 851 ppb DON-3-Glucoside 0 vs 18 ppb ZEN 40 vs 60 ppb enniatin B 9,5 vs 16 ppb enniatin B1 4,2 vs 3,5 ppb alternariol 28-35 d: 57,3 vs 57,3 ppb DON 8,4 vs 8,4 ppb enniatin B 13 vs 16 ppb enniatin B1 6,1 vs 6,1 beauvericin | ↓BWG, ↑FCR (no 28-35d, with a marginally contaminated diet) ↑ VH:CD ileum 14 ↓ VH:CD jejunum 28d, ileum 28d ↓VH: jejunum 28 d, ↑ CD ileum 28d ↑ ileum globe cells 14d Marker of intestinal lesion: deficiency in jejunum (not in ileum)Intestinal viscosity: increased in duodenum 14d |
1 BW: body weight; BWG: body weight gain; CD: crypt depth; FCR: food conversion ratio; GSSG: glutathione disulfide; GSH: reduced glutathione; MDA: malondialdehyde; ROS: reactive oxygen species; TEER: transepithelial/transendothelial electrical resistance; VH: villus height.
Conclusion
These results reinforce once again the idea of having a global vision of the mycotoxin problem, including emerging mycotoxins. Recognizing their presence and impact on animal production is essential to address the challenges they pose effectively.
