Presentación porciFORUM 2023 - Panagiotis Tassis

Micotoxinas

Enemigas infiltradas de la salud y productividad porcina

Panagiotis Tassis

ProfesorAdjuntodeMedicinayReproducciónPorcinaClínicade AnimalesdeProducción,FacultaddeMedicinaVeterinariadela UniversidadAristótelesdeTesalónica

Mycotoxins as a global swine feed risk

• Mycotoxins are secondary metabolites of certain fungi species that can be found in grains worldwide.

• They are produced before (fungi as plant pathogens), or after harvest of grains, or even during storage (fungi growing saprophytically). (Bryden, 2012).

• The most important mycotoxin producing fungi belong to the genera: Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps

Risk factors for mycotoxin contamination of swine feedstuff

Biological factors: Susceptible crop Compatible toxigenic fungi

Environmental factors:

Temperature

Distribution / Processing

Humans

Animals

Moisture

Mechanical injury of grains

Insect/Bird damage –infestation

Fungus

Storage factors:

Temperature

Moisture

Detection

Harvesting factors:

Temperature

Moisture

Crop maturity

Detection

Susceptible grains: Corn, Wheat, Oats, Barley, Sorghum, Cottonseed, Rye etc

Global feed survey 2009-2019: 74.821 samples from 100 countries

Ø Mycotoxin contamination of grains is a global phenomenon

Ø Final feed samples showed significant levels of mycotoxin contamination

Ø Majority of samples contaminated at levels < guidance /maximum levels set by the EU

Ø Climate change may increase the severity of “mycotoxin contamination of grains” phenomenon

Ø Concurrent exposure to >1 mycotoxins in grains is the “rule” not the “exception”

Concurrent contamination of grains by >1 mycotoxins because:

- Final swine feed consists of more than one type of grains

- Most fungi can produce more than one mycotoxin

- Swine feedstuff can be contaminated by >1 fungi strain

• Modified forms and metabolites toxic potency?

• Emerging mycotoxins menace?

Results: 2020, In Europe the most prevalent mycotoxins globally are the Fusarium mycotoxins DON (70% of corn samples) followed by ZEN and FB

Mycotoxins effects on swine health and performance

The pig is the domestic species showing the highest susceptibility to multiple mycotoxins, for instance, deoxynivalenol, zearalenone and fumonisins (Holanda and Kim, 2021)

Mycotoxins effects on swine health and performance

Basic AFB1 effects on health and performance

AFs are metabolized by the liver cytochrome P450 enzymes to a reactive epoxide intermediate which becomes more carcinogenic, or they are hydroxylated and the less harmful aflatoxins M1 and M2 are produced (Yu et al.

2012)à The liver has a central role in AFB1 detoxification

Aflatoxins: inhibit RNA polymerase transcription of DNA to mRNA in the nucleus, reducing cell protein synthesisà increasing cell toxicity and death.

AFB1: higher toxicity and carcinogenic effects than other aflatoxins.

AFB1 may suppress antigen-presenting cells by altering the function of dendritic cells and eventually reducing T-cell proliferation and differentiation.

Chronic AFB1 exposure: immune suppression, hepatic damage, impaired growth, and may interact with the DNA, leading to neoplasia development.

Effects of AFs in liver and kidneysà influence cholesterol synthesis and, later, vitamin D activation, as well as the calcium and phosphorus balance. Ponchon

Mycotoxins effects on swine health and performance

Basic DON effects on health and performance

• DON binds to the ribosome, inhibits protein (60S ribosomal unit) and nucleic acid synthesis and triggers ribotoxic stress leading to the activation of kinases, MAPKs and their downstream signaling pathways (Pierron et al., 2016; Croubels, 2022)

• DON toxicosis: Gastrointestinal signs: abdominal discomfort, diarrhea [due to inhibition of sodium-glucose dependent transporter (SGLT-1) activity], vomiting, anorexia, reduced weight gain (Ensley and Radke, 2019).

• DON: impairs intestinal barrier integrity and function (nutrient absorption), intestinal histomorphology, disrupts local intestinal immune response, triggers and potentiates intestinal inflammation (Grenier and Applegate 2013; Pierron et al., 2016).

Reduced DOM-1 toxicity

Evidence of oocyte maturation and embryo development impairment along with reduction of feed intake are main reasons behind the DON-induced detrimental reproductive effects in pigs (Lu et al., 2018).

DON show direct dose-dependent effects on granulosa cell steroidogenesis and proliferation, with a direct ovarian effect that could impact reproductive performance in swine (Ranzenigo et al. 2008; Yang et al., 2019).

Oocytes from gilts are more sensitive than those from sows when exposed to DON during in vitro maturation (Schoevers et al., 2021)

Short-term exposure to DON at relatively high doses (3 and 5.5mg/kg diet for swine) can impair reproduction and fetal development in animals (Yu et al., 2017).

Mycotoxins effects on swine health and performance

Basic ZEN effects on health and performance

ZEN absorption: Jejunum (70 - 85%) and ileum (15 - 30%)

(Avantaggiato et al., 2003)

Bioavailability: Uptake of ZEN single oral dose = Approximately

80-85% is present in blood (Zinedine et al., 2007)

Implied that most of the ZEN present in blood is available for cellular uptake and activation of ERs (Metzler et al., 2010)

Phase I biotransformation of ZEN

Other modified and masked forms, including derivatives conjugated with glucose, sulfate and glucuronic acid, occur during phase II metabolism

Dänicke and Winkler 2015; Liu and Applegate, 2020; Kinkade et al., 2021

ZEN (resorcyclic acid lactone) and metabolites effects at cellular level:

è Can cross cell membranes (e.g. uterine cells) binding to the cytosolic E2 (E2: estradiol 17b) receptorsà Forms a ZEN-E2 receptor complex (ZEN-E2R)à

è Transfer of the complex into the cell nucleus (è Inhibition of the formation of a

hormone–receptor complex) and binds to specific nuclear E2 receptorsà

è Activation of the gene responsible for mRNA synthesis (normally generated by E2)à

increase in RNA polymerase activity, synthesis of uterine estrogen-induced proteinà

è estrogen-like effects that induce anabolic and reproduction activity.

Everett et al. 1987; Malekinejad et al., 2006; Lorenz et al. 2018

Pigs, as more responsive species produce ~5-fold more α-ZEL than β-ZEL

ZEN: reproductive disorders, increased oxidative stress, decreased the nutrient digestibility while reducing growth rate.

Young gilts: 1–5 ppm of ZENà edema and hyperemia of the vulva, even vaginal and rectal prolapse

Cyclic sows: nymphomania, pseudopregnancy, ovarian atrophy and changes in the endometrium

Increased mean weight of the uteri + accelerated development of the ovaries in post-weaning piglets, possibly leading to subsequent reproductive disorders

Inhibition of FSH release and secretion --> suppression of follicle maturation at the preovulatory stage.

a- and β- ZEL : Reduced oocyte maturation rate (α>β-ZEL)

a-ZEL: Inhibition of progesterone production in pigs

Mycotoxins effects on swine health and performance

Basic FBs effects on health and performance

FBs structurally resemble the long-chain base backbones of the sphingoid bases, such as sphingosine (So) and sphinganine (Sa) and inhibit the enzyme ceramide synthase involved in conversion of Sa to So and their conversion to complex sphingolipids.

FBs:

Acute, fatal pulmonary edema (high dosages);

Hepatotoxicosis with icterus and liver necrosis (subacute exposure).

Impairment of the intestinal barrier integrity and reduced growth rate;

Possibly chronic pulmonary effects and immunosuppresion;

Histological lesions of massive pulmonary interlobular edema;

Liver apoptosis and bile retention;

Serum increases in AST, GGT, bilirubin, and cholesterol are characteristic

FB effects on intestinal homeostasis

Intestinal villous fusion and atrophy è intestinal absorption of nutrients.

Impairment of local immune response: Decrease of intestinal IL-8 and intestinal expression of IL-12p40

Goblet cells (mucous producing cells) density. Tight junction proteins (mainly occludin and E-cadherin).

Bacterial translocation to other organs and proliferation of intestinal opportunistic bacteria (due to increased intercellular spaces and intestinal permeability).

«Transepithelial Electrical Resistance» (TEER)

Mycotoxins effects on swine health and performance

Basic OTA effects on health and performance

OTA is a nephrotoxic mycotoxin and a well-known potent inhibitor of protein synthesis that causes renal toxicity and possesses carcinogenic, teratogenic, immunotoxic and possibly neurotoxic properties.

Acute ochratoxicosis in pigs: kidney disease (nephropathy)

Chronic ochratoxicosis: first signs of reductions of feed consumption and WG at the level of 1–1.4 mg OTA/kg of feed (Boudergue et al., 2009).

Dehydration, diarrhea, polyuria and polydipsia can be observed (Szczech et al.,1973).

OTA induces toxicity in piglet kidneys and livers by reducing antioxidant parameters and causing oxidative stress, with histopathological alterations (Zhang et al., 2016).

Mycotoxins effects on swine health and performance

Basic T-2 effects on health and performance

T-2 toxin inhibits protein, RNA and DNA synthesis, induces apoptosis and necrosis in some cell types and lipid peroxidation, which threaten the cell membrane integrity.

T-2 toxin interacts with the peptidyl transferase of the 60S ribosomal subunit inhibiting the formation of the new peptide-bond formation.

T-2 toxin is able to cause lipid peroxidation with the production of ROS because of its amphophilic nature that allows the molecule to be incorporated into the bilayer membranes.

(Jaradat et al. 2005; Tiemann et al. 2006; Bouaziz et al. 2006, 2008; Chaudhari et al. 2009)

The immune system is one of the main targets of T-2 toxin showing an immunomodulatory activity: Stimulation at low doses or inhibition at high doses

T-2 toxin perturbs the maturation of the antigen-presenting cells by altering lymphocyte proliferation antibody levels leading to an increased susceptibility to infectious diseases.

T-2 toxin showed a local irritant effect and caused serious hemorrhagic inflammation due to a damaging effect exerted on the blood vessel walls with hemorrhagic diathesis. • Other findings: necrosis and ulceration in the digestive tract, anorexia, leucopenia, inhibition of erythropoiesis; reproductive and teratogenic effects, altering the cytoskeleton of porcine oocytes, inhibitory effects on IGF-I and FSH-induced steroid production in porcine granulosa cells..

(Caloni et al. 2009; Meissonier et al. 2008; Zhang et al. 2016; Devreese et al. 2013; Adhikari et al. 2017)

Mycotoxins effects on immunity and vaccination efficacy

The health and economic implication of the effects of mycotoxins on the immune defense system of pigs is significant. Three major outcomes have been described (Pierron et al., 2016):

- Increased susceptibility to infectious diseases

- Reactivation of chronic infections

- Decreased vaccination efficacy

As observed in vivo, trichothecenes can be stimulatory in some leukocyte models but inhibitory in others; paradoxically, these activities sometimes cooccur.

• Immune cells (macrophages, B and T lymphocytes and natural killer (NK) cells) are sensitive to DON, 3-Ac-DON and 15-Ac-DON, and dose-dependent immunostimulatory/inflammatory or immunosuppressive effects can be observed.

• Differential inflammatory gene expression and DON-induced apoptosis are mechanisms that play a significant role on those immune effects.

(Pestka et al. 2004; Zhou et al. 2005; Pestka 2008, 2010a, 2010b;; Maresca et al. 2013)

Low levels of DON have been shown to increase susceptibility to viral infections (Savard et al., 2014).

DON naturally contaminated feed has been shown to decrease porcine reproductive and PRRSV specific antibody responses following experimental infection (Savard et al., 2015).

Detrimental effects on vaccine efficacy has been shown also for T2 (Meissonnier et al. 2008), FB1 (Taranu et al., 2005) and OTA (Stoev et al., 2012)

Rückner et al., 2022: exposure of pigs to DON at subtoxic levels (approx. 1 or 2 mg DON/kg feed) for a period of 4 weeks deteriorates the efficacy of vaccination against clinical signs of PRRS.

After PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects. (Pierron et al., 2023): Reduced IFN-γ producing lymphocytes and similar viral loads in the lung of pigs receiving DON (2.81 ppm) as the non-vaccinated groups

Interaction between mycotoxins in vivo and in vitro

The effect of a mixture of mycotoxins cannot be predicted solely on the basis of the effect of the individual compounds.

In vitro studies (reviewed by Alasane-Krembi et al., 2017):

Aflatoxins (B1, B2, M1, and M2): Synergistic toxic effect,

AFB1 + FB1: Antagonistic carcinogenic effect but a Synergistic immunotoxic effect.

AFB1 + trichothecenes: Synergistic or additive effects.

Porcine kidney cells: AFB1 + DON = Synergistic cytotoxic damage to incubated cells

In vitro studies:

• DON + acetylated forms (intestinal porcine cells): All mixtures showed a higher inhibitory effect than the single mycotoxins à DON + DON at low, intermediate, and high doses= antagonistic, additive, and synergic effects, respectively

Intestinal porcine cells: DON + FB1 + ZEN = Additive cell toxicity

Intestinal porcine cells: DON +ZEN = Synergic inhibitory effect on cell

proliferation

Ex vivo studies (jejunal porcine explants): strong (2–14-fold increase)

Synergic effect of DON and NIV regarding inflammatory cytokine expression.

(Alassane-Kpembi et al., 2013, 2015, 2017a, 2017b; Lei et al., 2013

In vivo studies

• AFs + DON: Liver damage and impairment of immune functionà decreased growth

• DON + FB1: reduced the apparent ileal digestibility of nutrients in feeds in newly weaned pigs

• DON + ZEN: Additive or synergistic effects for parameters of immune function

AFs + FBs: Synergistic effect in reducing the feed intake and weight gain in pigs.

Afs + DON: Synergistic effect in reducing cholesterol and glucose and in increasing white blood cells + additive effect on creatine phosphokinase reduction + a less than additive effect on reducing weight gain.

DON + FBs: Synergistic effect on decreasing weight gain and increasing hepatic enzymes, + Additive effect for reducing feed intake.

Grenier and Oswald, 2012; Chaytor et al., 2011; Weaver et al., 2013; Dabrowski et al., 2016; Holanda et al., 2020

Masked and emerging mycotoxins as threats to swine health and performance

Masked or modified mycotoxins are biologically modified phase II metabolites formed by plant defense mechanisms through glucosylation catalyzed by uridine diphosphate –glucosyltransferases (Zhang et al., 2020).

The term “modified mycotoxin”: denotes all forms derived from mycotoxins, regardless of their origin including compounds formed by reactions throughout processing, by microorganisms, or by the metabolism of plants (Rychlik et al., 2014; Berthiller et al., 2013).

Kouri et al. 2003; Bertero et al. 2018; Zhang et al. 2020

Common modified mycotoxins: deoxynivalenol-3-glucoside (D3G), DON (DON3S) and DON (DON15S) (formed through the plant defense mechanism); ZEN-16glucoside (ZEN-16G), ZEN-14G, ZEN-14-Sulfate, 3-ADON and 15-ADON….

Absorption and hydrolysis of 3-ADON and 15-ADON to DON as well as extensive pre-systemic hydrolysis of D3G to DON were observed in swine

ZEN-14G, ZEN-16G, and ZEN-14-sulfate were hydrolyzed to ZEN and converted to other unidentified metabolites along the digestion of pigs and excreted in the urine and feces.

Broekaert et al. 2015, 2017; Binder et al. 2017

Fusarium emerging mycotoxins: Beauvericin (BEA), enniatins (ENN), moniliformin (MON)

Toxic effects of BEA are due to its molecular structure= gives to the toxin its ionophoric properties leading to the capacity to act as an ion carrier throughout the cytoplasmic membrane.

BEA may inhibit ovarian function à detrimental to reproductive performance in pigs.

ENN (ENNB: most important): incorporates into the lipid bilayers of cell membranes acting as selective pores and increasing the permeability for alkali cations. Rapid metabolization of ENNs = explains their low in vivo toxicity.

MON (100 mg MON/kg feed): BW and ADFI reductions, alteration of haematologic and serum biochemical values, and increases in heart weight and hydropericardium (Harvey et al., 2001).

Tonshin et al., 2010; Gruber-Dorninger et al., 2016; Prosperini et al., 2017

A population of F. graminearum in the Midwest United States: Producing the compound 3ANX (also called NX-2) and its deacetylated form NX (also called NX-3)

• NX activates TNFR1, TNFR2 and IL-10 signaling pathways.

TNFR1 promotes inflammation: Increased TNF, IL1B and IL6.

TNFR2 targets anti-inflammatory effects: Promotes stability of regulatory T cells (Treg) lymphocytes (Yang et al., 2018; Pierron et al., 2022).

DON, 3ANX, NX: targeted genes implicated in inflammation, immune response, cell proliferation, differentiation, apoptosis, and growth. Several of these genes are linked to NF-kB activity (Hayden and Ghosh, 2014; Pierron et al., 2022).

Novel findings on mycotoxins diagnostics and alleviation techniques on the field

Urine is a good candidate to monitor DON, ZEN, OTA, CIT exposure

Serum: monitor DON, OTA and CIT (Tkaczyk et al., 2021).

Micro RNAs: Small (approximately 22 nucleotides long), non-coding RNAs act as negative regulators of gene expression. MicroRNA regulates the toxicological mechanism of four mycotoxins (AFB1, OTA, DON, ZEN) in vivo and in vitro (Chen et al. 2022).

Segura-Wang et al. (2021): The response of microRNAs to DON holds remarkable potential for biomarker applications: Serum microRNA response to DON increased over time and declined after removal of contaminated diets.

Feed additives:

• Inorganic Compounds phyllosilocates of the clay mineral group (smectite, hydrated sodium aluminosilicates (HSCAS), and specifically bentonite or montmorillonite + tectosilicates like zeolites, or activated charcoal).

• Aluminosilicates: comparatively lower adsorbability to mycotoxins, but it is considerably enhanced for the hydrated sodium calcium form, particularly for AFs; as well as in association with organic compounds

• Bentonites show low adsorbability to DON (3.2%) in vitro in comparison to other mycotoxins, such as AFs (92.5%), due to the higher polarity of AFs

Galvano et al., 1997, 1998; Huwig et al., 2001; Kong et al., 2014; Sabater-Vilar et al., 2007; Yiannikouris et al., 2006, 2013

Feed additives:

• Diatomaceous earth has an intermediate adsorbability to mycotoxins, though it is amid the inorganic materials with the highest adsorbability to Fusarium

mycotoxins

• Yeast with cell all comprised of glucans, mannans, and chitin. The use of inactivated yeast may enable yeast cell wall interaction with enterocytes and with mycotoxins, similar to using yeast culture or yeast cell wall extract, improving intestinal health and reducing mycotoxin toxicity (Holanda and Kim, 2021).

Yeast cell wall, and more particularly β-D-glucans, have robust adsorbability to AFB1 and ZEN but with restricted efficiency to DON.

• Saccharomyces cerevisiae strains: 65% average detoxification of AFB1 and 72% of FB1 after 24h of incubation.

• Yeast- and algae-derived β-glucans can show higher adsorbing abilities than mineral adsorbents, especially under alkaline pH for DON and ZEN

• Bacteria: Lactobacillus species: 60% average detoxification of AFB1, 70% of FBs and 30% of DON after 24h of incubation

• Microbial genera with DON-detoxifying capacity: Eubacteria, Anaerofilum, Collinsella, Bacillus, and Clostridiales

• Streptococcus and Enterococcus (gram+): effective up to 33% toward DON in corn silage. (Niderkorn et al. 2007; Yu et al. 2010; Franco et al. 2011; Chlebicz et al. 2020)

Lactobacillus helveticus: could adsorb 55%, and heat-inactivated Lactobacillus plantarum could adsorb up to 71% of DON in liquid media

• DON: small chemical structure and low polarityà difficult to find compounds with strong adsorbability to DON which could mitigate its toxic effects

•

Phytobiotics, Antioxidants and Algae derivatives: Milk thistle, or rosemary etc.

Biodegradation and Biotransformation products:

• Eubacterium sp. strain BBSH 797: detoxification of trichothecenes

• Yeast strain Trichosporon mycotoxinivorants: able to degrade OTA and ZEN

• Aspergillus, Rhyzopus and Penicillium spp.: Particularly effective for OTA removal

• Yeast Exophiala spinifera: capable of degrading FB1.

Duvick et al., 2001; Schatzmayr et al., 2006; Binder et al., 2007; Abrunhosa et al., 2010; Gacern et al., 2020; He et al., 2022

Mycotoxin-detoxifying agents containing multiple components to mitigate the toxic effects of mycotoxins has shown more benefits in comparison to those with single components

The best way to evaluate mycotoxin binders is with in vivo experiments.

Mycotoxins effects on swine reproduction

DON and ZEN alone or in combination: Parent toxins and metabolites

Mycotoxins effects on swine reproduction

DON and ZEN alone or in combination on boar semen in vitro

Immotile spermatozoa

DON effect: p = 0.001

ZEN effect : p < 0.001

DON+ZEN effect : p < 0.001

Interaction: Less than additive:

DON + ZEN vs. ZEN: p > 0.05

DON + ZEN vs. DON: p < 0.05

DON vs. ZEN: p < 0.05

Tassis et al., 2020

Progressive

DON effect : p=0,056

ZEN effect : p < 0.001

DON+ZEN effect : p < 0.001

Interaction: Less than additive

Means of Progressive motile measurements with 95%CL for each time period

motile spermatozoa

Rapid motile spermatozoa

DON effect : p = 0.063 (4h)

ZEN effect : p < 0.001

DON+ZEN effect : p < 0.001

Interaction: 0 h: Synergy type 1 (Potentiation)

1–4 h: Less than additive

DON + ZEN vs. DON: p < 0.05

DON vs. ZEN: p < 0.05.

DON + ZEN vs. ZEN: 0 h: p < 0.05; 1–4 h: p > 0.05.

DON and ZEN either alone or in combination can be considered as harmful to boar semen characteristics in vitro.

Exposure to DON alone negatively affected two important CASA parameters (i.e., immotile and progressive motile spermatozoa), along with sperm morphology and viability, suggesting a toxic effect on boar semen.

A negative effect of a lower ZEN concentration (i.e. 62.8μΜ; in comparison to previous findings) was detected for most investigated parameters.

DON and ZEN interaction showed a mixed picture that included predominantly less than additive effects, as well as synergism in less parameters.

Mycotoxins effects on swine reproduction

Only P values < 0.05 (treatment Vs. DMSO groups) are presented Tassis et al., 2022

Only P values < 0.05 (treatment Vs. DMSO groups) are presented

Modification of DON towards DOM-1 or ZEN towards HZEN can be considered as a detoxification procedure as proved by the absence of in vitro effects on boar semen.

Conclusions – Take home message

• Mycotoxins threat in swine feed remains a global issue that could intensify due to global warming phenomena.

• Mycotoxins can affect health, reproductive activity and performance of swine with a significant financial impact for the Farmer.

• Attention to vague-long term clinical symptoms, as well as symptoms related to recent feed alterations should be evaluated carefully.

• On site affordable diagnosis solutions are a significant part of the counteracting strategy and should be further evaluated. Proper sampling procedures are mandatory.

• When selecting an adsorbent material please specify the mycotoxin intented first with feed analysis and evaluate the in vivo results of the product.

• Bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity (e.g. DONàDOM-a; ZENàHZEN etc.)

Regular screening of feed on farm is strongly advised. When evaluating effects of in vivo studies of under field conditions: Diets naturally contaminated with DON may impair pig growth at 0.6 mg/kg of diet, whereas for diets that are artificially contaminated with purified deoxynivalenol, growth impairment is observed at 1.8 mg/kg of diet (Dersjant-Li et al., 2003).

• The toxic effect is stronger when mycotoxins are co-contaminants, even if the levels are below the guidelines