Immune Suppression by Mycotoxin Polycontamination: The Current Challenge

The natural or innate immunity is present in all animals even before any interaction with pathogens. It is a sequence of processes allowing the body to fight against any foreign body. It is non-specific, local and fast. On the other hand the acquired immunity that is built up on the long-term after encounters with different infectious agents, allow better efficacy against pathogens already fought.

Innate immunity is composed of physical barriers (separating the external environment from the inside, such as the skin, mucous membranes, digestive and respiratory tracts) and of the inflammatory response. Inflammation is usually a beneficial process: its goal is to eliminate the pathogenic agent and repair tissue damage. However, the inflammatory reaction sometimes exceeds its objectives with deleterious effects as a result. This can be due to the aggressiveness of the pathogen, its persistence, abnormal regulation of the inflammatory process, or quantitative/qualitative abnormality of the cells involved in inflammation.

The causes of inflammation are many and varied: infectious agents, inert foreign substances, endotoxins... Inflammation begins with a response of "recognition" involving certain body cells (monocytes, macrophages, lymphocytes) or circulating proteins (antibodies, complement proteins...). This recognition phase is followed by the involvement of a whole sequence of cells and mediators which order of intervention is complex and variable. Some mediators, such as prostaglandins and cytokines are produced by different cell types, acting on several cell types and may control their own production by feedback control. This shows the complexity of the mechanisms of the inflammatory response.

Contamination of feed commodities by mycotoxins and bacteria is considered to be one of the most important negative factors in animal feed quality. Numerous researches have concluded that their absorption causes a decrease in performance including decreased growth rate and poor feed efficiency, and are promoting factors of many diseases (Pestka et al, 2007; Hanif et al., 2006). The gastrointestinal tract is the largest selective filter body and represents the first barrier against ingested chemicals, feed contaminants and other variety of toxins. 

Endotoxins are lipopolysaccharides (LPS) derived from the cell membranes of Gram-negative bacteria and are responsible for its organization and stability. Although endotoxins are linked within the bacterial cell wall, they are continuously liberated into the environment at cell death and during cell growth and division. Endotoxins are omnipresent (feed, drinking water, air, dust) as a part of a bacterial cell wall or as fragments of whole bacteria.

Additionally, the gastrointestinal tract is a large reservoir of both gram-positive and gram-negative bacteria, of which the gram-negative bacteria are a source of endotoxin. Intestinal epithelium is therefore permanently exposed to GRAM- bacteria, which are able to directly deposit their toxic and pro-inflammatory constituents at the intestinal epithelial apical surface. These can stimulate localized or systemic inflammation via the activation of pattern recognition receptors.

Endotoxins and inflammation can also regulate intestinal epithelial function by altering integrity, nutrient transport and utilization. Moreover, luminal endotoxins can enter circulation, via two routes. The first one is via nonspecific paracellular transport through epithelial cell tight junctions. Paracellular transport of endotoxins occurs through dissociation of tight junction protein complexes resulting in reduced intestinal barrier integrity, which can be a result of enteric disease, inflammation, or environmental and metabolic stress. The second one is transcellular transport through lipid raft membrane domains involving receptor-mediated endocytosis. Transcellular transport, via specialized membrane regions rich in glycolipids, sphingolipids, cholesterol and saturated fatty acids is a result of raft recruitment of endotoxin-related signaling proteins leading to endotoxin signaling and endocytosis. Both transport routes and the sensitivity to endotoxins may be altered by diet and environmental or metabolic stresses (Peskta et al, 2007).resulting in reduced intestinal barrier integrity, which can be a result of enteric disease, inflammation, or environmental and metabolic stress. The second one is transcellular transport through lipid raft membrane domains involving receptor-mediated endocytosis. Transcellular transport, via specialized membrane regions rich in glycolipids, sphingolipids, cholesterol and saturated fatty acids is a result of raft recruitment of endotoxin-related signaling proteins leading to endotoxin signaling and endocytosis. Both transport routes and the sensitivity to endotoxins may be altered by diet and environmental or metabolic stresses (Peskta et al, 2007).

Endotoxins do not act directly against cells or organs but through activation of the immune system, especially through monocytes and macrophages, with the release of a range of pro-inflammatory mediators, such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-1.These pro-inflammatory cytokines have both local and systemic effects (inflammation, fever and reduction in feed intake). Moreover this chain reaction leads to an increase of suppressors of cytokine signaling which have a negative action on GH-induced gene expression in liver. In short, endotoxins impact the production of IGF1 and alleviate its many actions of growth hormone (stimulation of cell replication, cell differentiation and the synthesis of cellular products) that have impact on productivity (growth, milk production, fertility). 

The Food and Agricultural Organization (FAO) estimates that mycotoxins, secondary metabolites produced by fungi, contaminate 25% of the world’s agricultural commodities. The presence of mycotoxins alters the quality of agricultural products resulting in economic losses estimated in billions of dollars annually worldwide. The consumption of food and feed contaminated by mycotoxins is a potential health hazard for both humans and animals. Mycotoxins are one of the most immunosuppressive factors coming from feed (Surai and Dvorska, 2005), having each of them a different impact on the immune system. Some of the most toxic or most common types of mycotoxins and their effects are described below.

T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. A publication by Oswald et al. evaluates the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like Receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM).

Macrophages play a key role in the immune inflammatory response being the first cells to be exposed to microorganisms. These cells are considered as sentinel cells against infectious pathogens and involved in phagocytosis, antigen presentation, production of antimicrobial effector molecules and release of cytokines and chemokines that in turn contribute to immune cell recruitment and activation.

The recognition of microorganisms by macrophages occurs through a major receptor family expressed in distinct cell subsets and tissues called the TLR. They are able to bind to a wide range of motifs derived from bacteria (LPS, peptidoglycan), parasites, fungi and viruses (ssRNA, dsRNA, CpG DNA).

Interaction of TLR with its specific ligands leads to the induction of various inflammatory cytokines such as IL-1β and TNF-α and reactive nitrogen species. This pro-inflammatory response is the first line of defence against infections.

A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1β, TNF- α , nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. These results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases (Oswald et al, 2012).

Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species.DON suppresses normal immune response to pathogens and simultaneously induces autoimmune like effects and upregulates or downregulates critical functions associated with activated macrophages. It is interesting that experimental dysregulation of IgA persisted up to four months after a discrete period of dietary DON exposure.

DON rapidly induces multi-organ expression of pro-inflammatory cytokines, and this is followed by up-regulation of several suppressors of cytokine signaling (SOCS), some of which are capable of impairing growth hormone (GH) signaling. DON induces hepatic insulin-like growth factor acid-labile subunit (IGFALS) mRNA upregulation. This effect co-occurs with robust hepatic suppressors of cytokine signaling 3 upregulation. So, oral DON exposure perturbs GH axis by suppressing two clinically relevant growth-related proteins, IGFALS and IGF1.

Moreover, DON has proved to have effects on the intestinal epithelium integrity. First of all it damages the epithelial cells, decreasing the villi length and thus the surface of absorption, resulting in poor nutrient absorption. It impairs the barrier function of the intestine by two mechanisms, one decreasing the intestinal expression of claudin proteins and activating MAP-Kinases that regulate tight junction proteins, and the other decreasing trans-epithelial electrical resistance (TEER). This results in an increased risk of trans-epithelial passage of both bacteria and endotoxins into the systemic system (Pinton et al, 2010).

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides and F. proliferatum, common contaminants of maize. It impairs both specific (B and T lymphocyte) and non specific (T lymphocyte, macrophage) immune functions. It causes both stimulation and suppression of responses to foreign antigens, decreases total immunoglobulins, IgG and macrophage phagocyte activity and also decreases the intestinal expression of IL-8. As IL-8 is implicated in the recruitment of neutrophils during inflammatory response, the decreased IL-8 production could lead to an impaired recruitment of neuthrophils and so it is associated with an increased susceptibility to enteric infection (Oswald et al, 2006). 

The response of affected animals to exposure to more than one mycotoxin can be the same as the response predicted from the summation of the response to each mycotoxin individually (additive), less than the predicted response from each toxin individually (antagonistic), or more tan the predicted summation of the responses from each individual mycotoxin (synergistic). The chance for multiple mycotoxin contamination of feedstuffs is relatively high and can occur for different reasons. Numerous species of Aspergillus, Penicillium and Fusarium are known to simultaneously produce more than one mycotoxin. When conditions are favorable for the growth of a single mould species, multiple mycotoxins could be produced in the suspect feedstuff. If a feedstuff is stored under specific environmental conditions that are conducive for growth and mycotoxin production by one mould, these same environmental conditions could also permit the growth and mycotoxin production by another moulds in the feedstuff. This, in turn, would lead to multiple mycotoxin contamination of the same commodity with the mycotoxins originating from different moulds. Furthermore, some animal feeds are manufactured from ingredients originating from diverse geographical locations. An ingredient from one locale could be contaminated with one mycotoxin while the other ingredient may have undergone spoilage by a totally different mould and be contaminated by a different mycotoxin. When both ingredients are used in the manufacture of a single animal feed, multiple mycotoxin contamination will result.

As previously stated, when multiple mycotoxins contaminate a single feed, the possibility exists for one mycotoxin to act in a synergistic fashion with the other such that the final response of the affected animal is unique and not typical of either mycotoxin. When a synergistic response is present, a rapid and accurate diagnosis of the problem is difficult. One example of synergy is between DON and FB1. We have seen the effects of Don on the intestinal epithelium, decreasing the villi height and impairing the barrier function of the intestine. Fumonisin reduces epithelial cells proliferation at intestinal level, so the damage provoked by DON is not repaired as there is less cell proliferation, making that low contamination level of DON in presence of Fumonisin has a big impact on digestive disorders.

As knowledge increases on mycotoxin and endotoxin action mechanisms, we realize that their effect is far more complex and wide than expected. Regardless of if they act via downregulation of the inflammatory immune response, or by setting-up uncontrolled and unnecessary inflammation, it is clear that limiting the entrance of toxins into the body is vital in achieving good health and performance in livestock production. 

The use of a wide spectrum toxin binder is a key factor to alleviate the effects described above. Demonstration of the effectiveness of a potential mycotoxin detoxifying agent in contaminated feed is often primarily conducted in in vitro conditions. Classical in vitro systems used for that purpose are simple but very far from the natural in vivo conditions. Important factors in relation to the digestion and the fate of feed compounds during passage through the gastrointestinal tract are the composition and pH of gastric and intestinal contents, the gastrointestinal transit conditions and the activity of bio-chemicals (enzymes) and of the intestinal microflora in the gastrointestinal tract. The activities of those factors through the gastrointestinal tract are dynamic processes, therefore, these processes cannot be simulated in static in vitro models but the real challenge is to test on dynamic models. To demonstrate in the most reproducible and reliable conditions the efficacy in vitro of a sequestrant/chelator material, the TNO TIM-1 in vitro dynamic gastrointestinal model can be used.

The TNO in vitro gastrointestinal model simulates in high degree the successive dynamic processes in the stomach and small intestine (TIM 1) and in the large intestine (TIM 2). These models are unique tools to study the fate of compounds during passage through the gastrointestinal tract. The studies for testing mycotoxins detoxifying agents are performed in the TIM-1 system, the TNO dynamic, multi-compartmental system of the stomach and small intestine. This computer-controlled model simulates the successive dynamic conditions in the gastric compartment and in the three successive compartments of the small intestine. In this system the gastrointestinal conditions were simulated digestive conditions of the pig after the intake of a pig feed.

In 2004, Döll et al, showed that zearalenone is not that difficult to bind comparing to other Fusarium mycotoxins. Thus, the challenge for a toxin binder is to reduce the absorption of mycotoxins like trichothecenes and fumonisins. Dr. Giussepina Avantaggiato, from CNR Institute of Sciences of Food Production (ISPA) in Italy has run several trials using this system to evaluate the efficacy of several commercial binding agents and substances potentially useful as chelating agents (Avantaggiato et al., 2003; 2004 and 2007). One of the trials carried out in 2004 investigated in vitro screening of 14 adsorbent materials. Commercial products used to detoxify Fusarium mycotoxins were tested in the pH range of 3-8 for deoxynivalenol (DON) and nivalenol (NIV) -binding ability. Only activated carbon was shown to be effective with binding capacities of 84 to 95% for DON and 60 to 63% for NIV, calculated from the adsorption isotherms. The study then used the dynamic laboratory model (TIM) to evaluate the small-intestinal absorption of DON and NIV and the efficacy of activated carbon in reducing the relevant absorption. The in vitro intestinal absorptions of DON and NIV were 51% and 21% respectively. Most absorption occurred in the jejunal compartment for both mycotoxins. The inclusion of activated carbon resulted in a significant reduction in the intestinal mycotoxin absorption. At 2% inclusion level, the absorption with respect to the intake was lowered from 51% to 28% for DON and from 21% to 12% for NIV. Using information from previous studies it can be concluded that activated carbon appears to be one of the most effective mycotoxin adsorbents. All other commercial products showed poor efficacy in their capacity of binding Fusarium mycotoxins. In practice, the use of activated carbon in animal production has some limitations. High concentrations of activated carbons (> 0.5%, w/w) should be avoided in order to minimise the risk of nutrient adsorption as well as an impairment of the caloric/nutritional value of the feed (NOSB, 2002; Ramos et al., 1996a; Ramos et al., 1996b). 

Specific technologies can modify clay structure at the nano scale, increasing the interlayer space and thus improving its adsorption capacity for larger molecules. This modification can be done by using natural agents such as algal polysaccharides.

Ulvans, polyanionic polysaccharides present in green algae, are sulphated xylorhamno-glucoronans. They are formed by a succession of disaccharides composed of an uronic acid (glucuronic acid or iduronic acid) and sulphated rhamnose. They interact with montmorillonite via silanol groups on the edges of the layers and compensation cations in the interlayer space of montmorillonite. The presence of ulvans in the interlayer space of the montmorillonite increases the accessible adsorptive surface and the number and types of adsorption sites, resulting in a matrix similar to the structure of activated charcoal. The adsorption of mycotoxins in this innovative material is a complex mechanism involving CEC (cation exchange capacity) and surface area of montmorillonite, the polyanionic structure of ulvans and the “microtubular” structure formed in the interlayer space, allowing ionic and hydrophobic interactions with mycotoxins (Demais et al, 2006).

When testing this new material using the TIM-1 system in TNO, results were even better than those obtained with activated charcoal for big mycotoxins such as DON and Fumonisin. In addition, the use of this product did not impair the bioaccessibility of nutrients. 

It is predicted that there will be 9000 million inhabitants on planet Earth in year 2050. Population feeding is a challenge not only from the quantitative point of view but also from qualitative point of view. To grant healthy food, free of potential harmful agents and the quantity to feed the population is a challenge that demands changes and efforts from all participants in food chain. Globalization and international trade enable the movement of raw matters worldwide to the consumption site. Climate change, storage conditions and livestock industrialization have increased the concentration and distribution of toxins in feed and food. To avoid the presence of mycotoxins and endotoxins in feed supply is becoming almost impossible and this has a deep impact in animal health and performance. A tool to decrease the chances of these toxins to enter in the animal organism is more than needed. The use of wide spectrum toxin binders in feed and optimum farm management practice is the only possible method to reduce toxins effects and their impact in the food chain. This will reduce problems associated with animal performance and human health. 

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Avantaggiato G, Havenaar R., Visconti A. 2004. Evaluation of the intestinal absorption of deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding efficacy of activated carbon and other adsorbent materials. Food and Chemical Toxicology, 42 pag. 817-824.

Avantaggiato G., Havenaar R., Visconti A. 2007. Assessment of the multi-mycotoxin-binding efficacy of a carbon/aluminosilicate-based product in an in vitro gastrointestinal model. Journal of Agricultural and Food Chemistry, 55 pag. 4810-4819.

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Hanif, N.Q., Naseem, M., Khatoon, S. and Malik, N. 2006. Prevalence of mycotoxins in poultry rations. Pak J Sci Indust Res, 49: 120-124

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