Currently the industry is aware of mycotoxins and their detrimental effects on animals. Hence in practice, toxin binders are applied and seem to solve the problem. But what happens if the animal is simultaneously facing a bacterial challenge, are toxin binders still sufficient under these circumstances?
Global Product Manager
Impextraco
WHAT ARE ENDOTOXINS AND MYCOTOXINS?
Gram-negative bacteria, such as E. coli and Salmonella, have a double membrane with an intermediate peptidoglycan layer. Lipopolysaccharides (LPS), also known as endotoxins, are components of the outer membrane of Gram-negative bacteria and responsible for cell integrity, stability and protection from the environment. LPS consist of 3 parts, namely an O antigen (polysaccharide), a core oligosaccharide and a lipid A domain for anchoring in the bacterial cell membrane structure. Due to the existence of the gut microbiome, endotoxins are naturally present in the gastro-intestinal tract (GIT) of animals. They are released upon bacterial replication or lysis and toxic to production animals as they significantly impact immune status, but also gut barrier permeability. Certainly, when gram-negative bacteria overload the GIT, they can become very harmful. However, certain immune cells produce enzymes that detoxify endotoxins before they can have these negative effects. Moreover, when LPS enter the bloodstream, it can cause endotoxemia and in severe cases septic shock and heart failures. It is one of the most important reasons why bacterial translocation to the bloodstream is often detrimental both for humans and for farm animals.
On the other hand, mycotoxins are toxic metabolites produced by different species of mold. The most known species are Fusarium, Aspergillus and Penicillium. These molds contaminate crops globally before and after harvest. Mycotoxins are chemically and heat stable, so they will survive feed processing and will end up in the final feed. Therefore, close monitoring is important as well as protecting the animals from their harmful effects. In practice, monitoring is performed via mycotoxin predictive models as well as feed analysis via both precise or rapid techniques. Simultaneously, animal feed is supplemented with anti-mycotoxin additives to prevent negative effects and performance losses. Common additives are clay minerals, alone or in combination with functional ingredients to improve gut health, immune response and organ functioning.
SIMILARITIES IN EFFECTS OF ENDOTOXINS AND MYCOTOXINS
Looking more in depth at the effects in the animal, we discover some similarities between mycotoxin and endotoxin contamination. Endotoxins are known to be highly inflammatory both in the intestine and in the peripheral tissues. Actually, LPS are known antigens or in more difficult terms pathogen-associated-microbial-patterns (PAMP). PAMP are parts of bacteria that are recognized by receptors on immune cells and epithelial cells, which generates an immune response. More specifically, LPS is a ligand for Toll-like receptor 4 by its lipid domain. This receptor is mainly found on membranes of antigen presenting cells, for example monocytes, macrophages, and dendritic cells. The role of these immune cells is to initiate an innate immune response and activate different lymphocytes. After binding of LPS to these immune cells, a cascading event triggers production of pro-inflammatory cytokines and mediation of an inflammatory response. Prolonged and chronic exposure eventually leads to gut inflammation, intestinal damage and increased gut permeability. This leaves the animal exposed and vulnerable to systemic infections. Bacterial translocation through the gut barrier can lead to septic shock due to prolonged systemic inflammation.
Similarly, mycotoxins are toxic for the intestinal epithelial cells when ingested by animals and change the immune status of the animal. Mycotoxins are damaging the gut epithelium and hereby reducing the gut barrier function and gut integrity. In contrast to endotoxins, they have no antigenic properties, so cannot be recognized directly by antigen presenting cells. This means that they do not trigger inflammation directly. However, Toll-like Receptors on immune cells also recognize so called Damage-associated-molecular patterns (DAMP’s), which are released by damaged or dying host cells. Thus, by destroying the gut barrier and increasing the gut permeability, mycotoxins trigger an immune response. In addition, mycotoxins can enter the bloodstream and destroy immune cells leading to a lower pool of immune cells, i.e. immunosuppression. This leaves the animal vulnerable to secondary diseases. Therefore, we can conclude that both endotoxins and mycotoxins change the immune response and weaken the animals leading to detrimental performance losses.
COMBAT WITH CLAY MINERALS
In practice, clay minerals are routinely supplemented to the feed to adsorb mycotoxins and wash them away from the intestinal tract. The interaction of mycotoxins with the toxin binder is dependent on the polarity of the toxin and the surface charge of the clay. In an acidic environment, there is a higher surface charge and thus adsorption of mycotoxins is highest in the gastric stomach of the animal. At intestinal pH, which is closer to neutral, the negative charge disappears and there is a risk of releasing the toxin from the clay surface, namely desorption. The release is thus dependent on the strength and stability of the binding. Polarity is an important factor, but the structure and molecular size of the toxin is also of importance. For example, aflatoxins are polar, small and 2-D in structure. Therefore, they have a high binding percentage as aflatoxins fit between the layers of clay minerals. In contrast, fumonisins are polar and very big in molecular size. This implies that the binding site is limited towards the exterior of the clay mineral and not in between the layered sheets. Hereby fumonisins show more desorption as the molecule is not trapped by the binder (figure 1). Furthermore, it is possible to enhance the binding efficiency of less polar mycotoxins by modifying the clay’s surface. In addition, research has shown that clay minerals could bind LPS (figure 1). However, gram-negative bacteria in the intestine reside close to the epithelial barrier and hence the contact time between LPS and the clay mineral might not be sufficient to prevent epithelial damage. For this reason, it is more important to lower pathogenic counts in the gut microbiome instead of combatting their endotoxins with toxin binders.
As toxin binders have limitations, effective anti-mycotoxin solutions should be based on supporting the animal’s health by preventing damage to organs, the gut barrier and modulating the immune system. In that way, it is not important which mycotoxin is ingested by the animal. When the gut barrier and liver are functioning properly, the animal’s immunity and performance can simply not be compromised by the toxin. Therefore, Impextraco believes that effective mycotoxin solutions should be evaluated on real animal trials (in vivo trials), and more specifically on research on the immune status and gut health in the presence of a specific mycotoxin challenge. With the aid of biomarkers, many gut, organ and immune parameters can be evaluated nowadays.
IN VIVO RESULTS
Our solution, Elitox®, was evaluated in vivo during a fumonisin contamination. In total 480 Cobb® male broilers were raised until 39 days of age. The chicks were divided over 24 pens so that the average initial body weight in each pen was identical at the start of the trial. The animals were fed ad libitum with a starter, a grower and a finisher feed between day 1-14, day 15-32, day 33-39, respectively. There were 3 treatments and 8 replicates per treatment. The first group received a basal corn-soybean meal diet, while the second group received the same diet which was contaminated with 100 ppm fumonisins in the grower phase from day 21-28. The third group received the contaminated diet supplemented with Elitox® at a 2 kg/T inclusion rate. The aim of the trial was to evaluate the impact of a severe and acute fumonisin contamination on the immunity of the broilers using flow cytometry measurements. Per treatment, 8 blood samples were collected and analyzed.
Flow cytometry detects and quantifies the proportion of different subsets of immune cells based on cell membrane markers, i.e. the presence or absence of CD4 and CD8 receptors. As such you receive a picture of the immune status of the bird, which allows to detect changes in immune response. In the following results, the impact of fumonisin contamination on the bird’s immune response is demonstrated. On day 28, directly after the acute exposure to fumonisins, a clear impact on immune cells was noticed. In the contaminated group, the total pool of different subsets of immune cells was clearly lowered indicating immunosuppression (figure 2). This can be explained by the toxic effects of fumonisins towards circulating immune cells. In fact, fumonisins resemble cell membrane components and hence interfere with the building blocks of membranes of immune cells. Hereby different immune cells are targeted and destroyed by the toxin. Nevertheless, Elitox® prevented this reduction in immune cells and thus protected the birds from the negative effects of fumonisins. As we have shown in figure 1 that commonly used clay minerals are limited in fumonisins binding, immune supporting agents are therefore very important tools to combat mycotoxicosis effectively.
Looking closer to different subsets of immune cells, the mucosal helper lymphocytes, cytotoxic lymphocytes as well as peripheral blood helper lymphocytes were restored by the anti-mycotoxin solution. This shows that the prevention of the mycotoxin related symptoms is both at a gut level as on a systemic level. The role of helper lymphocytes is to organize the adaptive immune response and to activate other lymphocytes. Furthermore, cytotoxic lymphocytes are important in cellular immunity and eliminating invading pathogens.
AN ADDITIONAL LPS CHALLENGE
As discussed above endotoxins can exert inflammatory effects on the gut barrier and hence change the immune status of the animal. Thus, based on the above-mentioned in vivo results, an experiment was designed to investigate synergies between LPS and mycotoxins on broiler’s immune response. In total 84 animals were divided over 3 treatments with 4 replicates each. A basal corn-soybean meal diet was formulated as a control diet, while aflatoxins (315 ppb) and T2-toxin (858 ppb) were added to the other 2 treatments. The third treatment additionally received Elitox® at 2 kg/T inclusion in the final feed. On day 21, two animals per cage were injected with 100ml LPS from E. coli 24 hours before blood sampling. 24 hours post LPS injection blood was sampled and submitted for flow cytometry analysis to determine circulating lymphocytes, the inflammation response and pro-inflammatory cytokines (IL-6). In contrast to the fumonisin trial (figure 2), an increase in the mucosal helper lymphocytes and cytotoxic lymphocytes was observed. This shows that mycotoxins in combination with a bacterial challenge trigger an inflammatory response instead of immunosuppression. This finding was also confirmed by elevated levels of pro-inflammatory cytokines, for example Interleukin-6 (figure 3).
In conclusion, it is noted that a combination of LPS and mycotoxins triggers an inflammation response and consequently shows there is a synergy between both endotoxins and mycotoxins. In practice, animals are continuously exposed to various challenges and all of them should be eliminated to avoid gut inflammation and performance losses. Our trial data demonstrated that a single mycotoxin challenge leads to an immune suppression, which makes the animal vulnerable to secondary challenges, but not yet impacts performance. Once an additional challenge, for example endotoxins of bacterial origin is added, an inflammatory reaction arises leading to subsequent performance losses. Since efficient binding is limited to a few classes of mycotoxins, it is evident that effective mycotoxin solutions should focus on safeguarding animal health, i.e. by immunomodulation. Additionally, they contribute to tackling different challenges, like gram-negative bacteria with their inflammatory endotoxins.
About Arno Duchateau
Arno Duchateau is a master in bioscience engineering who graduated in 2019 at the University of Leuven (Belgium). Since 2020, he worked as Global Product Manager in the specialty feed additives division at Impextraco focusing on gut health and mycotoxin solutions. Here, he provides technical support to business partners, customers and colleagues as well as managing different product brands from development to marketing and sales.
