By Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition

Most grains used in feed are susceptible to mycotoxin contamination, causing severe economic losses all along feed value chains. As skyrocketing raw material prices force producers to include a higher proportion of economical cereal byproducts in the feed, the risks of mycotoxin contamination likely increase. In this article, we review why mycotoxins cause the damage they do – and how effective toxin-mitigating solutions prevent this damage.

Mycotoxin contamination of cereal byproducts requires solutions

Cereal byproducts may become more important feed ingredients as grain prices increase. But also from a sustainability point of view and considering population growth, using cereal byproducts in animal feed makes  a lot of sense. Dried distiller’s grains with solubles (DDGS) are a good example of how byproducts from food processing industries can become high-quality animal feed.

Figure 1: Byproducts are a crucial protein source (data from FEFAC Feed & Food 2021 report)

Still, research on what happens to mycotoxins during food processing shows that mycotoxins are concentrated into fractions that are commonly used as animal feed (cf. Pinotti et al., 2016 + link to article IH+MC ). To safeguard animal health and performance when feeding lower-quality cereals, it is essential to monitor mycotoxin risks through regular testing and to use toxin-mitigating solutions.

Problematic effects of mycotoxins on the intestinal epithelium

Most mycotoxins are absorbed in the proximal part of the gastrointestinal tract. This absorption can be high, as in the case of aflatoxins (ca. 90%), but also very limited, as in the case of fumonisins (< 1%); moreover, it depends on the species. Importantly, a significant portion of unabsorbed toxins remains within the lumen of the gastrointestinal tract.

Importantly, studies based on realistic mycotoxin challenges (e.g., Burel et al., 2013) show that the mycotoxin levels necessary to trigger damaging processes are lower than the levels reported as safe by EFSA, the Food Safety Agency of the European Union. The ultimate consequences range from diminished nutrient absorption to inflammatory responses and pathogenic disorders in the animal (Figure 2).

Figure 2: Mycotoxins’ impact on the GIT and consequences for monogastric animals

1.  Alteration of the intestinal barrier‘s morphology and functionality

   Several studies indicate that mycotoxins such as aflatoxin B1, DON, fumonisin B1, ochratoxin A, and T2, can increase the permeability of the intestinal epithelium of poultry and swine (e.g. Pinton & Oswald, 2014). This is mostly a consequence of the inhibition of protein synthesis.

   As a result, there is an increase in the passage of antigens into the bloodstream (e.g., bacteria, viruses, and toxins). This increases the animal’s susceptibility to infectious enteric diseases. Moreover, the damage that mycotoxins cause to the intestinal barrier entails that they are also being absorbed at a higher rate.

2. Impaired immune function in the intestine

   The intestine is a very active immune site, where several immuno-regulatory mechanisms simultaneously defend the body from harmful agents. Immune cells are affected by mycotoxins through the initiation of apoptosis, the inhibition or stimulation of cytokines, and the induction of oxidative stress.

   For poultry production, one of the most severe enteric problems of bacterial origin is necrotic enteritis, which is caused by Clostridium perfringens toxins. Any agent capable of disrupting the gastrointestinal epithelium – e.g. mycotoxins such as DON, T2, and ochratoxin – promotes the development of necrotic enteritis.

3. Alteration of the intestinal microflora

   Recent studies on the effect of various mycotoxins on the intestinal microbiota show that DON and other trichothecenes favor the colonization of coliform bacteria in pigs. DON and ochratoxin A also induce a greater invasion of Salmonella and their translocation to the bloodstream and vital organs in birds and pigs – even at non-cytotoxic concentrations.

   It is known that fumonisin B1 may induce changes in the balance of sphingolipids at the cellular level, including for gastrointestinal cells. This facilitates the adhesion of pathogenic bacteria, increases in their populations, and prolongs infections, as has been shown for the case of E. coli. The colonization of the intestine of food-producing animals by pathogenic strains of E. coli and Salmonella also poses a risk for human health.

4. Interaction with bacterial toxins

   When mycotoxins induce changes in the intestinal microbiota, this can lead to an increase in the endotoxin concentration in the intestinal lumen. Endotoxins promote the release of several cytokines that induce an enhanced immune response, causing inflammation, thus reducing feed consumption and animal performance, damage to vital organs, sepsis, and death of the animals in some cases.

   The synergy between mycotoxins and endotoxins can result in an overstimulation of the immune system. The interaction between endotoxins and estrogenic agents such as zearalenone, for example, generates chronic inflammation and autoimmune disorders because immune cells have estrogen receptors, which are stimulated by the mycotoxin.

Increased mycotoxin risks through byproducts? Invest in mitigation solutions

To prevent the detrimental consequences of mycotoxins on animal health and performance, proactive solutions are needed that support the intestinal epithelium’s digestive and immune functionality and help maintain a balanced microbiome in the GIT. As the current market conditions will likely engender a long-term shift towards the inclusion of more cereal byproducts in animal diets, this becomes even more important.

Trial data shows that EW Nutrition’s toxin-mitigating solution SOLIS MAX provides effective protection against feedborne mycotoxins. The synergistic combination of ingredients in SOLIS MAX mycotoxins from damaging the animals’ gastrointestinal tract and entering the blood stream:

In-vitro study shows SOLIS MAX’ strong mitigation effects against wide range of mycotoxins

Animal feed is often contaminated with two or more mycotoxins, making it important for an anti-mycotoxin agent to be effective against a wide range of different mycotoxins. A dose response evaluation of SOLIS MAX was conducted a at an independent laboratory in Spain, for inclusion levels of 0.10%, 0.15%, and 0.20% (equivalent to 1 kg, 1.5 kb, and 2 kg per ton of feed). A phosphate buffer solution at pH 7 was prepared to simulate intestinal conditions in which a portion of the mycotoxins may be released from the binder (desorption).

Each mycotoxin was tested separately by adding a challenge to buffer solutions, incubating for one hour at 41°C, to establish the base line (see table). At the same time a solution with the toxin challenge and SOLIS MAX was prepared, incubated, and analyzed for the residual mycotoxin. All analyses were carried out by high performance liquid chromatography (HPLC) with standard detectors.

Figure 3: SOLIS MAX adsorption capacity against different mycotoxins (%)

The results demonstrate that SOLIS MAX is a very effective solution against the most common mycotoxins found in raw materials and animal feed, showing clear dose-response effects.

Mycotoxin risk management for better animal feed

A healthy gastrointestinal tract is crucial to animals’ overall health: it ensures that nutrients are optimally absorbed, it provides effective protection against pathogens through its immune function, and it is key to maintaining a well-balanced microflora. Even at levels considered safe by the European Union, mycotoxins can compromise different intestinal functions, resulting in lower productivity and susceptibility to disease.

The globalized feed trade, which spreads mycotoxins beyond their geographical origin, climate change and raw material market pressures only escalates the problem. On top of rigorous testing, producers should mitigate unavoidable mycotoxin exposures through the use of solutions such as SOLIS MAX – for stronger animal health, welfare, and productivity.

References

Antonissen, Gunther, An Martel, Frank Pasmans, Richard Ducatelle, Elin Verbrugghe, Virginie Vandenbroucke, Shaoji Li, Freddy Haesebrouck, Filip Van Immerseel, and Siska Croubels. “The Impact of Fusarium Mycotoxins on Human and Animal Host Susceptibility to Infectious Diseases.” Toxins 6, no. 2 (January 28, 2014): 430–52. https://doi.org/10.3390/toxins6020430.

Burel, Christine, Mael Tanguy, Philippe Guerre, Eric Boilletot, Roland Cariolet, Marilyne Queguiner, Gilbert Postollec, et al. “Effect of Low Dose of Fumonisins on Pig Health: Immune Status, Intestinal Microbiota and Sensitivity to Salmonella.” Toxins 5, no. 4 (April 23, 2013): 841–64. https://doi.org/10.3390/toxins5040841.

Burton, Emily J., Dawn V. Scholey, and Peter E. Williams. “Use of Cereal Crops for Food and Fuel – Characterization of a Novel Bioethanol Coproduct for Use in Meat Poultry Diets.” Food and Energy Security 2, no. 3 (September 19, 2013): 197–206. https://doi.org/10.1002/fes3.30.

Ghareeb, Khaled, Wageha A. Awad, Josef Böhm, and Qendrim Zebeli. “Impacts of the Feed Contaminant Deoxynivalenol on the Intestine of Monogastric Animals: Poultry and Swine.” Journal of Applied Toxicology 35, no. 4 (October 28, 2014): 327–37. https://doi.org/10.1002/jat.3083.

Mani, V., T. E. Weber, L. H. Baumgard, and N. K. Gabler. “Growth and Development Symposium: Endotoxin, Inflammation, and Intestinal Function in livestock1,2.” Journal of Animal Science 90, no. 5 (May 1, 2012): 1452–65. https://doi.org/10.2527/jas.2011-4627.

Obremski, K. “The Effect of in Vivo Exposure to Zearalenone on Cytokine Secretion by Th1 and Th2 Lymphocytes in Porcine Peyer’s Patches after in Vitro Stimulation with LPS.” Polish Journal of Veterinary Sciences 17, no. 4 (2014): 625–32. https://doi.org/10.2478/pjvs-2014-0093.

Oswald, I. P., C. Desautels, J. Laffitte, S. Fournout, S. Y. Peres, M. Odin, P. Le Bars, J. Le Bars, and J. M. Fairbrother. “Mycotoxin Fumonisin B1 Increases Intestinal Colonization by Pathogenic Escherichia Coli in Pigs.” Applied and Environmental Microbiology 69, no. 10 (2003): 5870–74. https://doi.org/10.1128/aem.69.10.5870-5874.2003.

Pinotti, Luciano, Matteo Ottoboni, Carlotta Giromini, Vittorio Dell’Orto, and Federica Cheli. “Mycotoxin Contamination in the EU Feed Supply Chain: A Focus on Cereal Byproducts.” Toxins 8, no. 2 (February 15, 2016): 45. https://doi.org/10.3390/toxins8020045.

Pinton, Philippe, and Isabelle Oswald. “Effect of Deoxynivalenol and Other Type B Trichothecenes on the Intestine: A Review.” Toxins 6, no. 5 (May 21, 2014): 1615–43. https://doi.org/10.3390/toxins6051615.

By Marisabel Caballero, Global Technical Manager Poultry, and Dr. Inge Heinzl, Editor, EW Nutrition

Consistently rising feed prices compel feed producers to resort to alternative feed ingredients. By-products of milling and ethanol distillation would be good options. The following article shows what should be paid attention to when using these feeds.

Keeping high-quality animal protein affordable requires cost-efficient alternatives

For a high percentage of consumers, the price of food products is one of the most decisive purchase factors; however, quality and sustainable use of resources are also of high importance. So, to comply with market requirements, meat producers must find cost-efficient and sustainable sources of feed ingredients. Feed prices already increased during the COVID-19 pandemic. Shortage of workforce and high shipping costs led to discontinuity in the supply chain, long delivery times, and increased costs for certain raw materials. Due to the Ukrainian crisis, there is no improvement to be seen. Alternatives must be considered more vigorously to compensate for this limited feed supply.

Grain by-products are an option

The use of grain by-products occurring at milling or ethanol production can cover a part of animal nutritional demands. Additionally, it contributes to sustainable usage of the available sources, as the remains of the production of human consumables are put back into the food chain.

However, increasing levels of by-products in the feed also have their sticking points. The raw materials grains or corn are often contaminated with mycotoxins, impacting the quality of this kind of feed.

Milling processes reduce mycotoxins in food

Before the whole process of milling, the grains are sorted and cleaned. Kernels with extensive mold growth, broken kernels, fine materials, and dust are removed.

When it comes to reducing mycotoxins by sorting and cleaning, the results vary a lot. They are influenced by several factors, including the initial condition of the grains, the type and level of contamination, and the type and efficiency of the cleaning process (Pinotti et al., 2016). The cleaning process has been shown to remove from 5 to 80 % of DON and NIV, 5 to 40 % of ZON (Schaarschmidt & Fauhl-Hassek, 2018), and 50 to 60 % of T2/HT2 contamination in wheat (Pascale et al., 2011). Debranning, the mechanical process by which the outer layers of wheat grains are removed, further reduces mycotoxin content in wheat grain from 15 to 80% of the initial contamination (Aureli et al., 2007; Rios et al., 2009). However, neither the cleaning and debranning nor the milling process include a step that destroys mycotoxins.

In white flour for human consumption, mycotoxin levels typically range from 50 to 70% of the wheat grain (Cheli et al., 2013).

The milling of maize shows a reduction factor of about 4 for aflatoxins and about 10 for zearalenone from the grain to the final human products. Contrarily, concentration triplicates for both aflatoxins and zearalenone in the case of the by-products such as germs, bran, and animal flour.

Milling processes concentrate mycotoxins in animal-feed fractions

The milling and pre-milling processes reduce the content of mycotoxins in products for human consumption, but what about the parts removed and normally used in animal feeds? Several studies (Tibola et al., 2015; Hoffmans et al., 2022) indicate that the concentration of mycotoxins is higher in the wheat fractions intended for animal feeds such as bran, flour shorts screenings, and middlings. However, their level in feedstuffs is variable and affected by several factors such as the type of mycotoxins, the level and extent of fungal contamination, and the complexity of the cereal processing technology.

Compared to the concentration in wheat grain, these concentrations in by-products may be up to 800 % but more typically range from 150 % to 340 % (Cheli et al., 2013). EW Nutrition’s worldwide mycotoxin survey shows a similar trend (Figures 1 and 2), in which DON levels are nine times higher in wheat midds than wheat grains, and fumonisin is eight times higher in wheat bran.

Figure 1 + 2: Mycotoxins levels in grain and by-products

Highest concentrations in germ and bran fraction

After corn milling, animal feed fractions such as germ and bran have a low yield ranging from 5 to 7 % and are mostly composed of the outer parts of the kernels; as a consequence, an important concentration of mycotoxins occurs in these fractions (Schollenberger et al., 2008). When taking corn grains as the base, the contamination of aflatoxins goes up to three times in corn germ and up to nine times in bran (Brera et al., 2006; Pietri et al., 2009). For fumonisins, a double concentration can be expected (Brera et al., 2004), and for zearalenone, up to four times (Brera et al., 2006). Recently, Park and collaborators (2018) evaluated the distribution of 12 mycotoxins during wet milling of corn and found higher concentrations in corn gluten feed and corn bran.

Milling is a crucial step in the post-production of rice, in which the husk and the bran layers are removed. Rice bran is a common ingredient for animal feeds, in which aflatoxin is a common contaminant. It is believed that most of the aflatoxin contamination in rice bran occurs due to non-optimal storage conditions (Takahashi et al., 1989); however, a concentration of the toxin during milling of stored paddy rice also occurs, and the levels can triplicate compared with the grains (Trucksess et al., 2011).

The concentration of mycotoxins in DDGS during the ethanol production

Destillers’ dried grains with solubles (DDGS), a by-product of ethanol production, is a valuable feed ingredient, particularly as a source of protein for ruminants and monogastric animals at a competitive price.

Also here, mycotoxin contamination raises concerns with regard to their use in animal feeds. Mycotoxins are not destroyed during the ethanol fermentation process or during the production of DDGS. Moreover, a concentration of DON, ZEA, and fumonisin from corn to DDGS of 2–3.5 times has been reported for industrial ethanol production (Bennett et al., 1981; Schaafsma et al., 2009; Bowers & Munkvold, 2014).

In summary, studies on the fate of mycotoxins during food processing have shown that mycotoxins are concentrated in the fractions commonly used as animal feed. Moreover, high variability in mycotoxin contamination of cereal by-products has been evidenced, representing barriers to an increased acceptance of several food by-products as feed ingredients.

Feed formulation: Consider the mycotoxin contamination in by-products

Higher inclusions of cereals have an impact on their safe use in feeds. To evaluate this impact, we can simulate two different scenarios with different inclusions of by-products:

Table 1: Different levels of by-products’ inclusion rates

*Risk Tool (masterrisktool.com)

In the first lower inclusion scenario, the risk for broilers in the starting phase considers the low inclusion of raw materials; the losses related to the contamination (without management) are mild. When increasing the levels of by-products, the risk category also increases. The losses are more important for the operation, ranging from gut barrier alterations with impaired production parameters to alterations in the immune response and increased susceptibility to disease.

Mycotoxins in by-products effective toxin risk management can help!

Given the pros of including cereal by-products in animal feeds, such as their saving potential and their link with sustainability of resources, their utilization is advisable; however, understanding how mycotoxin distribution and concentration change during grain processing is critical. Today’s knowledge is limited to a few mycotoxins in cereal milling.

Therefore, when considering using these by-products in the animal feed, we must bear in mind that:

• modified mycotoxins and mycotoxin co-contamination can be present, contributing to additive/synergistic effects on animal health.
• toxin risk management strategies, including analysis, risk evaluation, and risk mitigation must be pursued to prevent those undesired effects.

References:

Aureli, G., and M.G. D’Egidio. “Efficacy of Debranning on Lowering of Deoxynivalenol (DON) Level in Manufacturing Processes of Durum Wheat.” Tecnica Molit. 58 (2007): 729–33.

Bennett, G. A., A. A. Lagoda, O. L. Shotwell, and C. W. Hesseltine. “Utilization of Zearalenone- Contaminated Corn for Ethanol Production.” Journal of the American Oil Chemists’ Society 58, no. 11 (1981): 974–76. https://doi.org/10.1007/bf02659774.

Bowers, Erin, and Gary Munkvold. “Fumonisins in Conventional and Transgenic, Insect-Resistant Maize Intended for Fuel Ethanol Production: Implications for Fermentation Efficiency and DDGS Co-Product Quality.” Toxins 6, no. 9 (2014): 2804–25. https://doi.org/10.3390/toxins6092804.

Brera, Carlo, Carla Catano, Barbara de Santis, Francesca Debegnach, Marzia de Giacomo, Elena Pannunzi, and Marina Miraglia. “Effect of Industrial Processing on the Distribution of Aflatoxins and Zearalenone in Corn-Milling Fractions.” Journal of Agricultural and Food Chemistry 54, no. 14 (2006): 5014–19. https://doi.org/10.1021/jf060370s.

Brera,Carlo, Francesca, Debegnach, Silvana Grossi, and Marina Miraglia. “Effect of Industrial Processing on the Distribution of Fumonisin B1 in Dry Milling Corn Fractions.” Journal of Food Protection 67, no. 6 (2004): 1261–66. https://doi.org/10.4315/0362-028x-67.6.1261.

Cheli, Federica, Luciano Pinotti, Luciana Rossi, and Vittorio Dell’Orto. “Effect of Milling Procedures on Mycotoxin Distribution in Wheat Fractions: A Review.” LWT – Food Science and Technology 54, no. 2 (2013): 307–14. https://doi.org/10.1016/j.lwt.2013.05.040.

Park, Juhee, Dong-Ho Kim, Ji-Young Moon, Jin-Ah An, Young-Woo Kim, Soo-Hyun Chung, and Chan Lee. “Distribution Analysis of Twelve Mycotoxins in Corn and Corn-Derived Products by LC-MS/MS to Evaluate the Carry-over Ratio during Wet-Milling.” Toxins 10, no. 8 (2018): 319. https://doi.org/10.3390/toxins10080319.

Pascale, Michelangelo, Miriam Haidukowski, Veronica Maria Lattanzio, Marco Silvestri, Roberto Ranieri, and Angelo Visconti. “Distribution of T-2 and HT-2 Toxins in Milling Fractions of Durum Wheat.” Journal of Food Protection 74, no. 10 (2011): 1700–1707. https://doi.org/10.4315/0362-028x.jfp-11-149.

Pietri, A., M. Zanetti, and T. Bertuzzi. “Distribution of Aflatoxins and Fumonisins in Dry-Milled Maize Fractions.” Food Additives & Contaminants: Part A 26, no. 3 (2009): 372–80. https://doi.org/10.1080/02652030802441513.

Pinotti, Luciano, Matteo Ottoboni, Carlotta Giromini, Vittorio Dell’Orto, and Federica Cheli. “Mycotoxin Contamination in the EU Feed Supply Chain: A Focus on Cereal Byproducts.” Toxins 8, no. 2 (2016): 45. https://doi.org/10.3390/toxins8020045.

Ríos, G., L. Pinson-Gadais, J. Abecassis, N. Zakhia-Rozis, and V. Lullien-Pellerin. “Assessment of Dehulling Efficiency to Reduce Deoxynivalenol and Fusarium Level in Durum Wheat Grains.” Journal of Cereal Science 49, no. 3 (2009): 387–92. https://doi.org/10.1016/j.jcs.2009.01.003.

Schaafsma, Arthur W, Victor Limay-Rios, Diane E Paul, and J David Miller. “Mycotoxins in Fuel Ethanol Co-Products Derived from Maize: A Mass Balance for Deoxynivalenol.” Journal of the Science of Food and Agriculture 89, no. 9 (2009): 1574–80. https://doi.org/10.1002/jsfa.3626.

Schaarschmidt, Sara, and Carsten Fauhl-Hassek. “The Fate of Mycotoxins during the Processing of Wheat for Human Consumption.” Comprehensive Reviews in Food Science and Food Safety 17, no. 3 (2018): 556–93. https://doi.org/10.1111/1541-4337.12338.

Schollenberger, M., H.-M. Müller, M. Rüfle, S. Suchy, and W. Drochner. “Redistribution of 16FusariumToxins during Commercial Dry Milling of Maize.” Cereal Chemistry Journal 85, no. 4 (2008): 557–60. https://doi.org/10.1094/cchem-85-4-0557.

Takahashi, H., H. Yazaki, M. Manabe, S. Matsuura, and S. Kimura. “Distribution of Citrinin and Aflatoxins in Steamed Milled Rice Kernels Inoculated with Penicillium Citrinum and Aspergillus Flavus.” Mycotoxins 1990, no. 31 (1989): 49–53. https://doi.org/10.2520/myco1975.1990.49.

Trucksess, M.W., H.K. Abbas, C.M. Weaver, and W.T. Shier. “Distribution of Aflatoxins in Shelling and Milling Fractions of Naturally Contaminated Rice.” Food Additives & Contaminants: Part A 28, no. 8 (2011): 1076–82. https://doi.org/10.1080/19440049.2011.576441.

By Vinil Samraj Padmini, Global Category Manager Feed Quality, and Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition

Climate across the globe has changed, with rising atmospheric temperatures and carbon dioxide levels. This change favors the growth of toxigenic fungi in crops and thus increases the risk of mycotoxin contamination. When contaminating feed, mycotoxins exert adverse effects in animals and could be transferred into products such as milk and eggs.

95% of the samples were contaminated with at least one mycotoxin

EW Nutrition constantly analyzes feed and raw material samples for their mycotoxin contamination. We report challenges from the most common mycotoxins hindering animal health around the globe.

Worldwide, more than 4,000 analyses on more than 1,000 samples were performed between June – October of the present year. The samples covered grain and by-products commonly used in animal feed worldwide. Figure 1 shows the percentage of the samples tested for which a positive result was found, detailing the number of mycotoxins per sample.

The number of mycotoxins analyzed per sample can vary based on regional risk-evaluation, including weather conditions, raw material origin and past frequency of positives. However, a minimum number of samples per region is always analyzed for the full spectrum, in order to monitor and corroborate the risk level.

3 or more mycotoxins per sample

95% of the samples were contaminated with at least one mycotoxin. In Europe and Latin America, most samples were analyzed for up to five mycotoxins, and were found contaminated with at least two. In South Asia, three mycotoxins were regularly analyzed per sample and most samples were positive for two. Worldwide, it is common to find samples with 3 or more mycotoxins, indicating that, even in raw materials, poly-contamination is the rule.

Aflatoxin: Main concern for South Asia

From all samples tested positively for mycotoxin contamination, 55% were contaminated with Aflatoxins. In all regions, the maximum levels lay over the thresholds for dairy and poultry. In Europe, less than 20% of the samples were contaminated with Aflatoxin. In Europe and the USA, the average contamination is low, hence this toxin can hardly be considered an issue for animal production in those areas (Figure 2).

In South Asia, where high temperatures and humidity are prevalent, Aflatoxin was detected in more than 95% of the samples and the average contamination is over all thresholds. Management strategies, such as the use of mold inhibitors for stored grain and toxin binders in feed, are necessary in this area to keep animals healthy and productive.

Fumonisins: Main concern for LATAM, also global

Fumonisin was found in 70% of the samples globally and roughly in 90% of the samples coming from Latin America (figure 3). Moreover, in LATAM, more than 50% of the results have values over the threshold for dairy and swine, and 14% over the threshold for poultry, making it a great concern in the area. South Asia is the second concern area, with a high proportion of contaminated samples (80%) and 14% of them representing a danger for poultry production.

Deoxynivalenol (DON): Present worldwide

All across the regions, the maximum tested levels lay over the threshold for dairy, poultry, and swine. This trichothecene was found in more than 70% of the samples analyzed worldwide. In the United States, more than 75% of the positive tested samples showed a contamination with DON and the average of the positives exceeded the thresholds for swine and poultry.

The region with highest maximum values is LATAM, followed by South Asia, and the region with the highest frequency of positives in analyzed samples is Europe. Thus, it can be concluded that the worldwide frequency and levels in which DON is found represent a high risk for production animals.

Ruminants can tolerate 10–20 times more DON than, for example, pigs. The majority of ingested DON is converted into the less toxic de-epoxy DON, but the degradation rate is influenced by different factors such as the diet, where high starch decrease the process. Moreover, DON also has a detrimental effect on rumen microorganisms, impacting its fermentative capacity.

T2: A danger for poultry producers word-wide

Average levels of T2 were over the threshold for poultry in all regions, with a high presence (>70% of the analyzed samples) in Europe, the US & LATAM.

Zearalenone: 80% positive tests globally

More than 80% of all samples tested for this mycotoxin were found positive. The maximum contaminations lay over the thresholds for dairy and swine. These high levels found should not be ignored, considering feedstuffs for long living and reproduction animals.

A bad year for crops could be a bad year for production animals

The high mycotoxin contamination found so far in 2021 is partially explained by climate events, such as high temperature and humidity. Temperate zones such as Europe or parts of the USA tend to have higher contaminations compared with previous years.

Multiple mycotoxins co-occur, increasing their impact on animals. Certain combinations of mycotoxins are known to have synergistic or additive effects, aggravating their adverse effects.

To safeguard animal performance, it is important to continually strive for low levels of contamination and to manage the risk of mycotoxins through the use effective tools to measure, interpret, and manage the risk. MasterRisk can aid in the interpretation of mycotoxin risks, weighing in the animal species, age, purpose, as well as the mycotoxin exposure and interactions.

How lipopolysaccharides cause disease

LPS are rather large and structured chemical molecules with a weight of over 100,000 D. They are highly thermostable; boiling in water at 100°C for 30 minutes does not destabilize their structure. LPS consist of three chemically distinct sections: a) the innermost part, lipid A, consisting mostly of fatty acids; b) the core, which contains an oligosaccharide; and c) the outer section, a chain of polysaccharides called O-antigen (Figure 1).

Figure 1: Structure of an LPS

The toxicity of LPS is mainly caused by lipid A; however, both lipid A and O-antigen stimulate the immune system. This happens when the LPS pass the mucosa and enter the bloodstream or when they attack the leukocytes.

The intestinal mucosa is the physical immune barrier that protects the microvilli from external agents (bacteria, free LPS viruses, etc.). Despite its strength (the thickness, for example, amounts to ≈830 µm in the colon and ≈123 µm in the jejunum), vulnerable points exist (cf. Zachary 2017).

LPS can easily come into contact with the cells of the lamina propria (a layer of connective tissue underneath the epithelium) through the microfold (M) cells of the Peyer’s patches (which consist of gut-associated lymphoid tissue). The M cells are not covered by mucus and thus exposed.

Secondly, LPS can also pass through the mucosa, where they become entangled in this gelatinous structure. There, they come into contact with the lymphocytes or can reach the regional lymph nodes through the afferent lymphatic vessels.

Thirdly, LPS might affect the tight junctions, the multiprotein complexes that keep the enterocytes (cells that form the intestinal villi) cohesive. By destabilizing the protein structures and triggering enzymatic reactions that chemically degrade them, LPS can break the tight junctions, reaching the first capillaries and, consequently, the bloodstream.

The presence of endotoxins in the blood, endotoxemia, can trigger problematic immune responses in animals. An innate immune stimulation leads to an increase in the concentration of pro-inflammatory cytokines in the blood and, consequently, to an induced febrile response in the animal: heat production increases, while the available metabolic energy decreases.  As a result, performance suffers, and in the worst-case scenario, septic shock sets in. Furthermore, when LPS compromise intestinal integrity, the risk of secondary infections increases, and production performance may decline.

LPS’ modes of action

How does all of this happen? The physiological consequences of endotoxemia are quite complex. Simplified, the immune system response to LPS in the blood takes three forms:

• The stimulation of TLR4 (toll-like receptor 4) induces monocytes and macrophages to secrete critical pro-inflammatory cytokines, primarily interleukin (IL) IL-1β, IL-6, IL-8, and tumor necrotic factor (TNF) α and β. TLR4 is a structure on the cell membrane of mainly macrophages and leukocytes, which is activated by the LPS-binding protein (LBP).
• The complement cascade constitutes about 10% of plasma proteins and determines the chemotaxis and activation of leukocytes. It can form a membrane attack complex (MAC), which perforates the membranes of pathogenic cells, enabling lysis.
• The Hagemann factor, also known as coagulation factor XII: once stimulated by LPS, it initiates the formation of fibrin (through the intrinsic coagulation pathway), which might lead to thrombosis. The Hagemann factor directly stimulates the transformation of prekallikrein to kallikrein (enzymes involved in regulating blood pressure).

Figure 2: How LPS leads to endotoxemia – 3 modes of action

These three modes of action of inflammatory stimulation lead to important physiological reactions:

• Pro-inflammatory cytokines (see above) modulate the functional expression of other immune cell types during the inflammatory response;
• Metabolites of arachidonic acid (prostaglandins, leukotrienes, and lipoxins), intra- and extracellular messengers that influence the coagulation cascade;
• Synthesis in the blood of bradykinin, a peptide responsible for the typical symptoms of inflammation, such as swelling, redness, heat and pain;
• PAF (platelet-activating factor), which creates inflammatory effects through narrowing of the blood vessels and constriction of the airways, but also through the degranulation of leukocytes.

The symptoms of endotoxemia are:  hypotension, metabolic acidosis, hemoconcentration, intestinal hemorrhage, fever, activations of neutrophils and endothelial cells, and predisposition to thrombosis.

In case of a progression to septic shock, the following sequence takes place:

1) Reduction in blood pressure and increased heart rate (hemodynamic alterations)

2) Abnormalities in body temperature

3) Progressive hypoperfusion at the level of the microvascular system

4) Hypoxic damage to susceptible cells

Up to here, symptoms follow a (severe) endotoxemia pathogenesis. A septic shock furthermore entails:

5) Quantitative changes in blood levels of leukocytes and platelets

6) Disseminated intravascular coagulation (see Hageman factor)

7) Multi-organ failure

8) Death of animal

If an animal is continously challenged with endotoxins, experiences septic shock, or comes close to it, it risks developing LPS tolerance, also known as CARS (compensatory anti-inflammatory response syndrome). This syndrome essentially depresses the immune system to control its activity. The anti-inflammatory prerogative of CARS is not to interfere directly with the elimination of pathogens but to regulate the “excessive” inflammatory reaction in a hemostatic way. However, this regulation can be extremely dangerous as the syndrome involves a lack of homeostasis control, and an excessive depression of the immune system leaves the organism exposed to the actual pathogens.

Farm animal research on endotoxemia pathogenesis

Lipopolysaccharides are difficult to quantify in the intestine of a live animal. One way to evaluate a possible endotoxemia is to analyze biomarkers present in the bloodstream. The most important one is the LPS themselves, which can be detected in a blood sample taken from the animal via ELISA. Other biomarkers include pro-inflammatory interleukins, such as TNF α and β, IL-6 or IL-8, and fibrin and fibrinogen (though they are not specific to endotoxemia). It is vital to carry out a blood sample analysis to deduce a possible endotoxemia from symptoms and performance losses in the animal.

How the metabolic effects of endotoxemia depress performance

One of the biggest issues caused by endotoxemia is that animals reduce their feed intake and show a poor feed conversion rate (FCR). Why does this happen? The productive performance of farm animals (producing milk, eggs, or meat) requires energy. An animal also requires a certain baseline amount of energy for maintenance, that is, for all activities related to its survival. As a result of inflammation and all those physiological reactions mentioned above, endotoxemia leads to a feverish state. Maintenance needs to continue; hence, the energy required for producing heat will be diverted from the energy usually spent on producing milk, egg, meat, etc., and performance suffers.

The inflammation response can result in mitochondrial injury to the intestinal cells, which alter the cellular energy metabolism. This is reflected in changes to the levels in adenosine triphosphate (ATP), the energy “currency” of living cells. A study by Li et al. (2015) observed a respective reduction of 15% and 55% in the ATP levels of the jejunum and ileum of LPS-challenged broilers, compared to the unchallenged control group. This illustrates the extent to which animals lose energy while they experience (more or less severe) endotoxemia.

Figure 3: Reduction in ATP level in Jejunum and Ileum in broilers (adapted from Li et al., 2015)

A piglet study by Huntley, Nyachoti, and Patience (2017) took this idea further (Figure 4):  3 groups of 10 Yorkshire x Landrace pigs, weighing between 11 and 25 kg, were studied in metabolic cages and in respiratory chambers. This methodology allows for simultaneous measurement of oxygen consumption, CO₂ production, energy expenditure, physical activity, and feed/water intake. The study found that LPS-challenged pigs retained 15% less of the available metabolizable energy and showed 25% less nutrient deposition. These results show concrete metabolic consequences caused by the febrile response to endotoxemia we discussed above.

Figure 4: Retained Energy as % of ME intake and nutrient deposition of pigs in metabolic cages (adapted from Huntley, Nyachoti, and Patience, 2017)

Control treatment (CON) = Pigs fed by a basal diet
Immune system stimulation treatment (ISS) = Pigs given LPS (E. coli serotype 055:B5) injection

A loss of energy retained due to a reduction in available metabolizable energy leads to losses in performance as the amount of energy available for muscle production and fat storage will be lower. Furthermore, the decrease in feed intake creates a further energy deficit concerning production needs.

A trial carried out at the University of Illinois examined the effects of repeated injections of 400 μg E. coli LPS on chick performance from 11 to 22 days after hatching. The chicks were fed casein-based diets with graded levels of arginine. LPS administration reduced weight gain (P<0.05) and feed intake, and these effects tended to be worse at higher levels of arginine supplementation (Figure 5). The researchers hypothesize that, in response to endotoxin and elevated cytokine levels, macrophages use more arginine to produce nitric oxide, diverting it from protein production for muscle development.

Figure 5: Effects of LPS on feed intake and body weight gain in chicks fed graded level of arginine (based on Webel, Johnson, and Baker, 1998)

NC = negative control

This data on poultry complements the results for swine, again showing that endotoxin-induced energy losses quantifiably depress animal performance even in milder disease cases.

The way forward: Endotoxin mitigation

Animals suffering from endotoxemia are subject to severe metabolic dysfunctions. If they do not perish from septic shock, they are still likely to show performance losses. Moreover, they at great risk of immunosuppression caused by the immune system “overdrive.” Effective endotoxin mitigating agents can help to prevent these scenarios.

EW Nutrition’s Mastersecure Gold is not only a leading anti-mycotoxin agent; thanks to its specific components, it effectively binds bacterial toxins. An in vitro study conducted at the Hogeschool Utrecht laboratory (part of Utrecht University) evaluated the binding capacity of Mastersecure Gold on LPS compared to three different competitor products. All products were tested at two different inclusion rates. At an inclusion rate of 0.25%, only Mastersecure Gold reduced the toxin load on the solution by 37%. At 1% inclusion, Mastersecure Gold (noted as Mastersorb below) bound 75% of the toxin, while only one competitor product demonstrated any binding (10%).

Lipopolysaccharides are a constant challenge for animal production. The quantity of Gram-negative bacteria in an animal intestine is considerable; therefore, the danger of immune system over-stimulation through endotoxins cannot be taken lightly. Producers need to prioritize the maintenance of intestinal eubiosis in production animals proactively; for instance, through targeted gut health-enhancing additives based on phytomolecules and, possibly, organic acids.

Most importantly, the detrimental impact of LPS can be mitigated by using a high-performance agent such as Mastersecure Gold. To limit losses from an energy point of view yields positive results in terms of production levels and the prevention of secondary infections, preserving animal health and farms’ economic viability.

By Claudio Campanelli, EW Nutrition

References

Adib-Conquy, Minou, and Jean-Marc Cavaillon. “Compensatory Anti-Inflammatory Response Syndrome.” Thrombosis and Haemostasis 101, no. 01 (2009): 36–47. https://doi.org/10.1160/th08-07-0421.

Huntley, Nichole F., C. Martin Nyachoti, and John F. Patience. “Immune System Stimulation Increases Nursery Pig Maintenance Energy Requirements.” Iowa State University Animal Industry Report 14, no. 1 (2017). https://doi.org/10.31274/ans_air-180814-344.

Li, Jiaolong, Yongqing Hou, Dan Yi, Jun Zhang, Lei Wang, Hongyi Qiu, Binying Ding, and Joshua Gong. “Effects of Tributyrin on Intestinal Energy Status, Antioxidative Capacity and Immune Response to Lipopolysaccharide Challenge in Broilers.” Asian-Australasian Journal of Animal Sciences 28, no. 12 (2015): 1784–93. https://doi.org/10.5713/ajas.15.0286.

Mani, Venkatesh, James H Hollis, and Nicholas K Gabler. “Dietary Oil Composition Differentially Modulates Intestinal Endotoxin Transport and Postprandial Endotoxemia.” Nutrition & Metabolism 10, no. 1 (2013): 6. https://doi.org/10.1186/1743-7075-10-6.

Webel, D.M., R.W. Johnson, and D.H. Baker. “Lipopolysaccharide-Induced Reductions in Body Weight Gain and Feed Intake Do Not Reduce the Efficiency of Arginine Utilization for Whole-Body Protein Accretion in the Chick.” Poultry Science 77, no. 12 (1998): 1893–98. https://doi.org/10.1093/ps/77.12.1893.

Zachary, James F. “Chapter 4 – Mechanisms of Microbial Infections.” Essay. In Pathologic Basis of Veterinary Disease, 132–241. St Louis, MO: Mosby, 2017. https://doi.org/10.1016/B978-0-323-35775-3.00004-7.

Structure

Biochemically, endotoxins are lipopolysaccharides (LPS). They are composed of a relatively uniform lipid fraction (Lipid A) and a species-specific polysaccharides chain. Their toxicity is mainly due to the lipid A; the polysaccharide part modifies their activity. Unlike the bacteria, their endotoxins are very heat stable and resist sterilization. The names endotoxin and lipopolysaccharides are used synonymously with “endotoxin” emphasizing on the occurrence and biological activity and “lipopolysaccharide” on the chemical structure (Hurley, 1995).

General structure of Gram-negative lipopolysaccharides (according to Erridge et al., 2002)

Impact

Endotoxins belong to the so-called pyrogen-agents (they provoke fever), activating several immunocompetent cells’ signaling pathways. Early contact with endotoxins leads to activation and maturation of the acquired immune system. Braun-Fahrländer and co-workers (2002) found that children exposed to endotoxins had fewer problems with hay fever, atopic asthma, and atopic sensitization. This might be an explanation that in human populations, after the elevation of the hygiene standards, an increase of allergies could be observed.

Different animal species show different sensibilities to endotoxin infusions, e.g. (healthy) dogs, rats, mice, hens tolerate concentrations ≥1mg / kg body weight, whereas (healthy) ruminants, pigs, horses react very sensible already at concentrations <5μg / kg body weight (Olson et al., 1995 cited in Wilken, 2003).

Reasons for increased exposure of the organism to endotoxins

Endotoxins usually occur in the gut, as the microflora also contains gram-negative bacteria. The precondition for endotoxins to be harmful is their presence in the bloodstream. In the bloodstream, low levels of endotoxins can still be handled by the immune defense, higher levels can get critical. An increase of endotoxins in the organism results from higher input and/or lower clearance or detoxification rate.

Higher input of endotoxins into the organism

The “normal” small amounts of endotoxins arising in the gut due to regular bacterial activity and translocated to the organism have no negative impact as long as the liver performs its clearance function. Also, the endotoxins stored in the adipose tissue are not problematic. However, some factors can lead to a release of the endotoxins or translocation of endotoxins into the organism:

1.      Stress

Stress situations such as parturition, surgeries, injuries can lead to ischemia in the intestinal tract and translocation of endotoxins into the organism (Krüger, 1997). Other stress situations in animal production, such as high temperatures and high stocking densities, contribute to higher endotoxin levels in the bloodstream. Stress leads to a higher metabolic demand for water, sodium, and energy-rich substances. For a higher availability of these substances, the intestinal barrier’s permeability is increased, possibly leading to a higher translocation of bacteria and their toxins into the bloodstream.

Examples:

• Higher levels of endotoxins in pigs in an experimental study suffering from stress due to loading and transport, elevated temperatures (Seidler (1998) cited in Wilken (2003)).
• Marathon runners (Brock-Utne et al., 1988) and racing horses (Baker et al., 1988) also showed higher endotoxin concentrations in the blood proportional to the running stress; thus, trained horses showed lower concentrations than untrained.

2.      Lipolysis for energy mobilization

If endotoxins, due to continuous stress, consistently get into the bloodstream, they can be stored in the adipose tissue. The SR-B1 (Scavenger receptor B1, a membrane receptor belonging to the group of pattern recognition receptors) binds to lipids and the lipopolysaccharides, probably promoting the incorporation of LPS in chylomicrons. Transferred from the chylomicrons to other lipoproteins, the LPS finally arrives in the adipose tissue (Hersoug et al., 2016). The mobilization of energy by lipolysis e.g., during the beginning of lactation, for example, leads to a re-input of endotoxins into the bloodstream.

3.      Damage of the gut barrier

In normal conditions, due to bacterial activity, endotoxins are present in the gut. Damage of the gut barrier allows translocation of these endotoxins (and bacteria)  into the bloodstream.

4.      Destruction of Gram-negative bacteria

Another “source” for endotoxins is the destruction of the bacteria. This can be done on the one hand by the organism’s immune system or by treatment with bactericidal substances targeting gram- bacteria (Kastner, 2002). To prevent an increased release of endotoxins, in the case of Gram-negative bacteria, a treatment with bacteriostatic substances only inhibiting the growth and not destroying the bacteria, or with bactericidal in combination with LPS-binding agents, would be a better alternative (Brandenburg, 2014).

5.      Proliferation of gram-negative bacteria

As gram-negative bacteria also release small amounts of endotoxins when they grow, everything promoting their proliferation also leads to an increase of endotoxins:

Imbalanced feeding

High yielder cows e.g., are fed diets rich in starch, fat, and protein. Increased feeding of fat leads to a higher concentration of endotoxins in the organism, as the same “transporter” (scavenger receptor class B type 1, SR-BI) can be used (Hersoug et al., 2016) for the absorption of fat as well as for the absorption of endotoxins.

In a study with humans as representors of the monogastric species, Deopurkar and co-workers gave three different drinks (glucose – 100% carbohydrate, orange juice – 92% carbohydrate, and cream – 100% fat) to healthy participants. Only the cream drink increased the level of lipopolysaccharides in the plasma.

Infectious diseases

Infectious diseases like mastitis, metritis, and other infections caused by gram-bacteria such as E. coli, Salmonella, etc. can be regarded as sources of endotoxin release.

Decreased detoxification or degradation

Main responsible organ: the liver

Task: detoxification and degradation of translocated endotoxin. The liver produces substances such as lipopolysaccharide binding proteins (LBP) which are necessary for binding and neutralizing lipopolysaccharide structures.

During the post-partum period, the organism is in a catabolic phase, and lipolysis is remarkably increased for energy generation due to milk production. Increased lipolysis leads, as mentioned before, to a release of endotoxins out of the adipose tissue but also fatty degeneration of the liver. A fatty degenerated liver cannot bring the same performance in endotoxin clearance than a normal liver (Andersen, 2003; Andersen et al., 1996; Harte et al., 2010; Wilken, 2003).  In a study conducted by Andersen and co-workers (1996), they couldn’t achieve complete clearance of endotoxins in cows with fatty livers. The occurrence of hepatic lipidoses increases after parturition (Reid and Roberts, 1993; Wilken, 2003).

Also, other diseases of the liver influence endotoxin clearance in the liver. Hanslin and co-workers (2019) found an impaired endotoxin elimination in pigs with pre-existing systemic inflammatory response syndrome.

Relation between lipid metabolism and endotoxin metabolism (according to Fürll, 2000, cited in Wilken, 2003)

Issues caused by endotoxins

Endotoxins, on the one hand, can positively stimulate the immune system when occurring in small amounts (Sampath, 2018). According to McAleer and Vella (2008), lipopolysaccharides are used as natural adjuvants to strengthen immune reaction in case of vaccination by influencing CD4 T cell responses. On the other hand, they are involved in the development of severe issues like MMA-Complex (Pig Progress) or a septic shock (Sampath, 2018).

MMA Complex in sows

MMA in sows is a multi-factorial disease appearing shortly after farrowing (12 hours to three days), which is caused by different factors (pathogens such as E. coli, Klebsiella spps., Staph. spps. and Mycoplasma spps., but also stress, diet). MMA is also known as puerperal syndrome, puerperal septicemia, milk fever, or toxemia. The last name suggests that one of the factors intervening in the disease is bacterial endotoxins. During the perinatal phase, massive catabolism of fat takes place to support lactation. The sows often suffer from obstipation leading to higher permeability of the intestinal wall, with bacteria, respectively endotoxins being transferred into the bloodstream. Another “source” of endotoxins can be the udder, as the prevalence of gram-negative bacteria in the mammary glands is remarkable (Morkoc et al., 1983).

The endotoxins can lead to an endocrine dysfunction: ↑ Cortisol, ↓ PGF2α, ↓Prolactin, ↓ Oxytocin. MMA stands for:

– Mastitis, a bacterial infection of the udder.

Mastitis can be provoked from two sides: on the one hand, endotoxemia leads to an elevation of cytokines (IL1, 6, TNFα). Lower Ca- and K-levels cause teat sphincter to be less functional, facilitating the entry of environmental pathogens into the udder and resulting in mastitis. On the other hand,  due to farrowing stress, Cortisol concentrations get higher. The resulting immunosuppression enables E. coli to proliferate in the udder.

– Metritis, an infection of the uterus with vulvar discharges:

It leads to reduced contractions and, therefore, to prolonged and/or complicated farrowing or dead piglets. Metritis can be promoted by stress causing a decrease in oxytocin and prostaglandin F2α secretion. Morkoc and co-workers (1983) didn’t find a relation between metritis and endotoxins.

– Agalactia, a reduction or total loss of milk production:

In many cases, agalactia is not detected until the nursing litter shows signs of hunger and/or weight loss. At worst, the mortality rate in piglets increases. Probably, milk deficiency is caused by lower levels of the hormones involved in lactation. Prolactin levels e.g., may be dramatically reduced by small volumes of endotoxin (Smith and Wagner, 1984). The levels of oxytocin are often half those in normal sows (Pig Progress, 2020).

Endotoxin shock

A septic shock can be the response to the release of a high amount of endotoxins.

In the case of an infection with gram-negative bacteria, the animals are treated with (often bactericidal) antibiotics. Also, the immune system is eliminating the bacteria. Due to bacterial death, endotoxins are massively released. When not bound, they activate the immune system including macrophages, monocytes, and endothelial cells. Consequently, high amounts of cellular mediators like TNFα, Interleukin 1 (IL-1), IL-6, and leukotrienes are released. High levels of pro-inflammatory cytokines activate the complement and coagulation cascade. In some animals, then the production of prostaglandins and leukotrienes is stimulated, implicating high fever, decreased blood pressure, generation of thrombi in the blood, collapse, damaging several organs, and lethal (endotoxic) shock.

Endotoxic shock only occurs to a few susceptible animals, although the whole herd may have been immune-stimulated. A more severe problem is the decrease in the normally strong piglets’ performance, deviating resources from production to the immune system because of the endotoxemia.

Amplified diarrhea

Lipopolysaccharides lead to an augmented release of prostaglandins, which influence gastrointestinal functions. Together with leukotrienes and pro-inflammatory mediators within the mucosa, they reduce intestinal absorption (Munck et al., 1988; Chiossone et al., 1990) but also initiate a pro-secretory state in the intestine. Liang and co-workers (2005) observed a dose-dependent accumulation of abundant fluid in the small intestine resulting in increased diarrheagenic activity and a decreased gastrointestinal motility in rats.

Conclusion

Acting against Gram- bacteria can result in an even more severe issue – endotoxemia. Endotoxins, besides having a direct negative impact on the organism, also contribute to some diseases. Supporting gut health by different approaches, including the binding of toxins, helps to keep animals healthy.

References

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Andersen, P.H., N. Jarløv, M. Hesselholt, and L. Bæk. “Studies on in vivo Endotoxin Plasma Disappearance Times in Cattle.” Zentralblatt für Veterinärmedizin. Reihe A 43 no. 2(1996): 93-101. DOI: 10.1111/j.1439-0442.1996.tb00432.x

Baker, B., S.L. Gaffin, M. Wells, B.C. Wessels and J.G. Brock-Utne. “Endotoxaemia in racehorses following exertion.” Journal of the South African Veterinary Association June (1988): 63-66. https://journals.co.za/docserver/fulltext/savet/59/2/1341.pdf?expires=1598542211&id=id&accname=guest&checksum=E50C766D318776E09CA41DA912F14CAD

Beutler, B. and T. Rietschel. “Innate immune sensing and its roots: The story of endotoxin.” Nature Reviews / Immunology 3(2003): 169-176. DOI: 10.1038/nri1004

Brandenburg, K. “Kleines Molekül – große Hoffnung – Neue Behandlungsmöglichkeit gegen Blutvergiftung in Sicht.“ Newsletter 70 (Okt.); Bundesministerium für Bildung und Forschung (2014). https://www.gesundheitsforschung-bmbf.de/de/kleines-molekul-grosse-hoffnung-neue-behandlungsmoglichkeit-gegen-blutvergiftung-in-sicht-2716.php

Braun-Fahrländer, C., J. Riedler, U. Herz, W. Eder, M. Waser, L. Grize, S. Maisch, D. Carr, F. Gerlach, A. Bufe, R.P. Lauener, R. Schierl, H. Renz, D. Nowak and E. von Mutius. „Environmental exposure to endotoxin and its relation to asthma in school-age children. ”The New England Journal of Medicine 347 (2002): 869-877. DOI: 10.1056/NEJMoa020057.

Brock-Utne, J.G., S.L. Gaffin, M.T. Wells, P. Gathiram, E. Sohar, M.F. James, D.F. Morrel, and. R.J. Norman. “Endotoxemia in exhausted runners after a long-distance race.” South Afr. Med. J. 73 (1988): 533-536. https://www.researchgate.net/publication/19780279_Endotoxaemia_in_exhausted_runners_after_a_long-distance_race

Chiossone, D. C., P.L. Simon, P.L. Smith. “Interleukin-1: effects on rabbit ileal mucosal ion transport in vitro.” European Journal of Pharmacology 180 no. 2-3 (1990): 217–228. DOI: 10.1016/0014-2999(90)90305-P.

Deopurkar R., H. Ghanim, J. Friedman, et al. “Differential effects of cream, glucose, and orange juice on inflammation, endotoxin, and the expression of Toll-like receptor-4 and suppressor of cytokine signaling-3.” Diabetes care 33 no. 5 (2010):991–997.

Erridge, C., E. Bennett-Guerrero, and I.R. Poxton. “Structure and function of lipopolysaccharides.” Microbes and Infection 4 no. 8 (2002): 837-851. DOI: 10.1016/s1286-4579(02)01604-0

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Hanslin, K., J. Sjölin, P. Skorup, F. Wilske, R. Frithiof, A. Larsson, M. Castegren, E. Tano, and M. Lipcsey. “The impact of the systemic inflammatory response on hepatic bacterial elimination in experimental abdominal sepsis.” Intensive Care Medicine Experimental 7 (2019): art. 52. https://doi.org/10.1186/s40635-019-0266-x

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Most grains used as feed raw materials are susceptible to mycotoxin contamination. These toxic secondary metabolites are produced by fungi before or after harvest and cause severe economic losses all along agricultural value chains. For livestock, negative consequences include acute effects such as impaired liver and kidney function, vomiting, or anorexia, as well as chronic effects such as immunosuppression, growth retardation, and reproductive problems. Mycotoxin management is, therefore, of the utmost priority for animal producers worldwide.

But how is it that mycotoxins cause such damage in the first place? This article delves into the complex processes that take place when mycotoxins come into contact with the gastrointestinal tract (GIT). The intestinal epithelium is the first tissue to be exposed to mycotoxins, and often at higher concentrations than other tissues. A deeper understanding of how mycotoxins affect the GIT allows us to appreciate the cascading effects on animal health and performance, why such damage already occurs at contamination levels well below official safety thresholds – and what we can do about it.

The intestinal epithelium: the busy triage site for nutrients and harmful substances

When mycotoxins are ingested, they encounter the GIT’s intestinal epithelium (Figure 1). This single layer of cells lining the intestinal lumen serves two conflicting functions: firstly, it must be permeable enough to allow the absorption of nutrients. On the other hand, it constitutes the primary physiological barrier against harmful agents such as viruses, microorganisms, and toxins.

Within the intestinal epithelium, several types of highly specialized cells are involved in epithelial regeneration, nutrient absorption, innate defense, transport of immunoglobulins, and immune surveillance. The selective barrier function is maintained due to the formation of complex networks of proteins that link adjacent cells and seal the intercellular space. Besides, the intestinal epithelium is covered with mucus produced by goblet cells, which isolates its surface, preventing the adhesion of pathogens to the enterocytes (intestinal absorptive cells).

Due to its dual involvement in digestive and immune processes, the intestinal epithelium plays a pivotal role in the animal’s overall health. Importantly, the epithelium is directly exposed to the entire load of ingested mycotoxins. Hence their effects can be problematic even at low concentrations.

Figure 1: The intestinal epithelium

Problematic effects of mycotoxins on the intestinal epithelium

Most mycotoxins are absorbed in the proximal part of the gastrointestinal tract (Table 1). This absorption can be high, as in the case of aflatoxins (~90%), but also very limited, as in the case of fumonisins (<1%); moreover, it depends on the species. Importantly, a significant portion of unabsorbed toxins remains within the lumen of the gastrointestinal tract.

Some of the mycotoxins that enter the intestinal lumen can be bio-transformed into less toxic compounds by the action of certain bacteria. This action, however, predominantly happens in the large intestine – therefore, no detoxification takes place before absorption in the upper parts of the GIT. Part of the absorbed mycotoxins can also re-enter the intestine, reaching the cells from the basolateral side via the bloodstream. Furthermore, they re-enter through enterohepatic circulation (the circulation of substances between the liver and small intestine). Both actions increase the gastrointestinal tract’s overall exposure to the toxins.

Table 1: Rate and absorption sites of different mycotoxins

Adapted from: Biehl et al., 1993; Bouhet & Oswald, 2007; Devreese et al., 2015; Ringot et al., 2006

The damaging impact of mycotoxins on the intestinal epithelium initially occurs through:

• A decrease in protein synthesis, which reduces barrier and immune function (Van de Walle et al., 2010)
• Increased oxidative stress at the cellular level, which leads to lipid peroxidation, affecting cell membranes (Da Silva et al., 2018)
• Changes in gene expression and the production of chemical messengers (cytokines), with effects on the immune system and cellular growth and differentiation (Ghareeb et al., 2015)
• The induction of programmed cell death (apoptosis), affecting the reposition of immune and absorptive cells (Obremski & Poniatowska-Broniek, 2015)

Importantly, studies based on realistic mycotoxin challenges (e.g., Burel et al., 2013) show that the mycotoxin levels necessary to trigger these processes are lower than the levels reported as safe by EFSA, the Food Safety Agency of the European Union. The ultimate consequences range from diminished nutrient absorption to inflammatory responses and pathogenic disorders in the animal (Figure 2).

Figure 2: Mycotoxins’ impact on the GIT and consequences for monogastric animals

1. Alteration of the intestinal barrier‘s morphology and functionality

The mycotoxins DON, fumonisin, and T2 induce a reduction in the rate of epithelial cell proliferation and differentiation. This causes a decrease in the height and the surface of the intestinal villi, which in turn leads to a reduction in nutrient absorption. Additionally, some nutrient transporters are inhibited by the action of mycotoxins such as DON and T2, for example, negatively affecting the transport of glucose.

Several studies indicate that mycotoxins such as aflatoxin B1, DON, fumonisin B1, ochratoxin A, and T2, can increase the permeability of the intestinal epithelium of poultry and swine (e.g. Pinton & Oswald, 2014). This is mostly a consequence of the inhibition of protein synthesis. As a result, there is an increase in the passage of antigens into the bloodstream (e.g., bacteria, viruses, and toxins). This increases the animal’s susceptibility to infectious enteric diseases. Moreover, the damage that mycotoxins cause to the intestinal barrier entails that they are also being absorbed at a higher rate.

2. Impaired immune function in the intestine

The intestine is a very active immune site, where several immuno-regulatory mechanisms simultaneously defend the body from harmful agents. Immune cells are affected by mycotoxins through the initiation of apoptosis, the inhibition or stimulation of cytokines, and the induction of oxidative stress. Studies demonstrate that aflatoxin, DON, fumonisin, T2, and zearalenone interact with the intestinal immune system in such a manner that the animal’s susceptibility to viral and bacterial infections increases (e.g., Burel et al., 2013). Moreover, by increasing their fecal elimination, the horizontal transmission of pathogens is extended.

For poultry production, one of the most severe enteric problems of bacterial origin is necrotic enteritis, which is caused by Clostridium perfringens toxins. Any agent capable of disrupting the gastrointestinal epithelium – e.g. mycotoxins such as DON, T2, and ochratoxin – promotes the development of necrotic enteritis. The inhibition of the intestinal immune system caused by mycotoxins such as aflatoxin, DON, and T2 also collaborates with the development of this disease.

3. Alteration of the intestinal microflora

The gastrointestinal tract is home to a diverse community of bacteria, fungi, protozoa, and viruses, which lines the walls of the distal part of the intestine. This microbiota prevents the growth of pathogenic bacteria through competitive exclusion and the secretion of natural antimicrobial compounds, volatile fatty acids, and organic acids.

Recent studies on the effect of various mycotoxins on the intestinal microbiota show that DON and other trichothecenes favor the colonization of coliform bacteria in pigs. DON and ochratoxin A also induce a greater invasion of Salmonella and their translocation to the bloodstream and vital organs in birds and pigs – even at non-cytotoxic concentrations. It is known that fumonisin B1 may induce changes in the balance of sphingolipids at the cellular level, including for gastrointestinal cells. This facilitates the adhesion of pathogenic bacteria, increases in their populations, and prolongs infections, as has been shown for the case of E. coli.

From the perspective of human health, the colonization of the intestine of food-producing animals by pathogenic strains of E. coli and Salmonella is of particular concern. Mycotoxin exposure may well increase the transmission of these pathogens, posing a risk for human health.

4. Interaction with bacterial toxins

When mycotoxins induce changes in the intestinal microbiota, this can lead to an increase in the endotoxin concentration in the intestinal lumen. Endotoxins or lipopolysaccharides (LPS) are fragments of Gram-negative bacteria’s cell walls. They are released during bacterial cell death, growth, and division. Hence endotoxins are always present in the intestine, even in healthy animals. Endotoxins promote the release of several cytokines that induce an enhanced immune response, causing inflammation, thus reducing feed consumption and animal performance, damage to vital organs, sepsis, and death of the animals in some cases.

The synergy between mycotoxins and endotoxins can result in an overstimulation of the immune system. The interaction between endotoxins and estrogenic agents such as zearalenone, for example, generates chronic inflammation and autoimmune disorders because immune cells have estrogen receptors, which are stimulated by the mycotoxin. The combination of DON at low concentrations and endotoxins in the intestine, on the other hand, has been shown to engender a decrease in transepithelial resistance and to alter the balance of the microbiota.

What to do? Proactive toxin risk management

To prevent the detrimental consequences of mycotoxins on animal health and performance, proactive solutions are needed that support the intestinal epithelium’s digestive and immune functionality and help maintain a balanced microbiome in the GIT. Moreover, it is crucial for any anti-mycotoxin product to feature both anti-mycotoxin and anti-bacterial toxin properties and that it supports the organs most targeted by mycotoxins, e.g., the liver. EW Nutrition’s Mastersorb® Gold premix is based on the synergistic combination of natural clay minerals, yeast cell walls, and phytomolecules. Its efficacy has been extensively tested, including as a means for dealing with E. coli endotoxins.

Mastersorb® Gold: anti-mycotoxin activity stabilizes performance and strengthens liver health

A field trial conducted in Germany on male Ross 308 broilers showed that for broilers receiving a diet contaminated with DON and zearalenone, adding 1kg of Mastersorb® Gold per ton of feed to their diet led to significant performance enhancements. Not only did they recuperate the mycotoxin-induced weight loss (6% increase relative to the group receiving only the challenge), but they gained weight relative to the control group (which received neither the challenge nor Mastersorb® Gold). Feed conversion also improved by 3% relative to the group challenged with mycotoxins.

A scientific study of crossbred female pigs showed that Mastersorb® Gold significantly reduced the deleterious effects of fumonisin contamination in the feed. The decrease in weight gain and the decline of feed conversion could be mitigated by 6.7% and 13 FCR points, respectively (Figure 3). Also, the sphinganine/sphingosine (Sa/So) ratio, a biomarker for fumonisin presence in the blood serum, could be decreased by 22.5%.

Figure 3: Mastersorb® Gold boosts performance for pigs fed a fumonisin-contaminated diet

Another study of crossbred female piglets, carried out at a German university, investigated whether Mastersorb® Gold could support performance as well as liver health under a naturally occurring challenge of ZEA (~ 370ppb) and DON (~ 5000ppb).  Mastersorb® Gold significantly improved weight gain and feed conversion in piglets receiving the mycotoxin-contaminated diet: daily body weight gain was 75g higher than that of a group receiving only the challenge, and the FCR improved by 24% (1.7 vs. 2.25 for the group without Mastersorb® Gold). Moreover, Mastersorb® Gold significantly improved the liver weight (total and relative) and the piglets’ AST levels (aspartate aminotransferase, an enzyme indicating liver damage). A tendency to improve spleen weight and GGT levels (gamma-glutamyl transferase, another enzyme indicative of liver issues) was also evident, all of which indicate that Mastersorb® Gold effectively counteracts the harmful impact of mycotoxin contamination on liver functionality.

In-vitro studies demonstrate Mastersorb® Gold’s effectiveness against mycotoxins as well as bacterial toxins

Animal feed is often contaminated with two or more mycotoxins, making it important for an anti-mycotoxin agent to be effective against a wide range of different mycotoxins. Besides, to prevent mycotoxins damaging the GIT, an effective product should ideally adsorb most mycotoxins in the first part of the animal’s intestine (under acidic conditions). In-vitro experiments at an independent research facility in Brazil showed that an application of 0.2% Mastersorb® Gold binds all tested mycotoxins at rates from 95 to 97% at a pH level of 3, using realistic challenges of 1000ppb (Aflatoxin B1 and ZEA) and 2500ppb (Fumonisin B1 and DON). The binding results achieved for Fumonisin and DON, which are often considered outright “nonbinding,” under challenging close to neutral conditions (pH 6), are particularly encouraging.

Figure 4: Mastersorb® Gold binding capacity against different mycotoxins (%)

Concerning its efficacy against endotoxins, an in vitro study conducted at Utrecht University, among other studies, has shown Mastersorb® Gold to be a strong tool against the LPS released by E. coli. For the test, four premium mycotoxin binders were suspended in a phosphate buffer solution to concentrations of 0.25% and 1%. E. coli LPS were suspended to a final concentration in each sample of 50ng/ml. Against this particularly high challenge, Mastersorb® Gold achieved a binding rate of 75% at an inclusion rate of 1%: clearly outperforming competing products, which at best showed a binding rate of 10%.

Conclusion

A healthy gastrointestinal tract is crucial to animals’ overall health: it ensures that nutrients are optimally absorbed, it provides effective protection against pathogens through its immune function, and it is key to maintaining a well-balanced microflora. Even at levels considered safe by the European Union, mycotoxins can compromise different intestinal functions such as absorption, permeability, immunity, and microbiota balance, resulting in lower productivity and susceptibility to disease.

To safeguard animal performance, it is important to continually strive for low levels of contamination in feed raw materials –  and to stop the unavoidable mycotoxin loads from damaging the intestinal epithelium through the use of an effective anti-mycotoxin agent, which also supports animals against endotoxins and boosts liver function. Research shows that Mastersorb Gold is a powerful tool for proactive producers seeking stronger animal health, welfare, and productivity.

By Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition

In animal feed, multi-mycotoxin contamination is found quite frequently and seems to be the rule rather than the exception in practical diets. Here is a quick overview of the known interactions.

What are the most common mycotoxins in feed?

Mycotoxins represent an exceptional challenge for feed and animal producers: they are produced by common molds, occur in a great variety and number, are sporadic or heterogeneous in their distribution, and their effects on farm animals are seldom recognized as mycotoxicosis. Among hundreds of known mycotoxins, aflatoxins, mainly produced by Aspergillus species, ochratoxin A, produced by Aspergillus and Penicillium species, as well as fumonisins, trichothecenes (especially DON and T-2 toxin) and zearalenone, primarily produced by many Fusarium species stand out as the most common contaminants.

Consequences of mycotoxin contamination

Ingestion of these mycotoxins may cause an acute toxicity or chronic disorders, depending on the concentration and duration of exposure. In farm animals, this might manifest as decreased performance, feed refusal, poor feed conversion, reduced body weight gain, immune suppression, reproductive disorders, and residues in animal food products.

Due to their frequent occurrence and their severe toxic properties, many countries appointed legal regulations or guidance for the major mycotoxins to protect animals and human consumers. The current regulations are typically very specific in terms of animal species and even for the production stage considering that mycotoxins affect for example poultry in a different way than cattle and broilers in a different way than breeders or laying hens. The threshold and/or guidance values for each species, however, were determined based on toxicological data from studies investigating a monoexposure leaving out the possibility of any combined effects of mycotoxins.

Multi-contamination: the rule, not the exception

If we were able to ensure that the animals were exposed to only one mycotoxin at a time, following the regulatory guidelines would allow us to protect our animals in most of the cases. Several worldwide surveys show, however, that mycotoxin multicontamination of animal feed is found very frequently* and seems to be the rule rather than the exception in practical diets. The concurrent appearance of mycotoxins in feed can be explained as follows: each mold species has the capacity to produce a number of mycotoxins simultaneously. Each species, in turn, may infest several raw materials leaving behind one or more toxic residue. In the end, a complete diet is made up of various raw materials with individual mycotoxin loads resulting in a multitude of toxic challenges for the animals.

Several researchers showed that the effects observed during multiple mycotoxin exposure can differ greatly from the effects observed in animals exposed to a single mycotoxin, indicating that the simultaneous presence of mycotoxins may be more toxic than predicted from the mycotoxins alone. This is because mycotoxins interact with each other. The interactions can be classified into three main different categories: antagonistic, additive, and synergistic.

Types of mycotoxin interactions

Additivity occurs when the effect of the combination equals the expected sum of the individual effects of the two toxins (Figure 1a).
Synergistic interactions of two mycotoxins lead to a greater effect of the mycotoxin combination than would be expected from the sum of their individual effects (Figure 1b). A special form of synergy, sometimes called potentiation, occurs when one or both of the mycotoxins do not induce effects whereas the combination induces a significant effect.
When the effect of the mycotoxin combination is lower than expected from the sum of their individual effects, antagonism can be observed (Figure 1c). In general, most of the mycotoxin mixtures lead to additive or synergistic effects, highlighting a significant threat to animal health and being the major reason that impedes risk assessment. Synergistic actions may occur when the single mycotoxins of a mixture act at different stages of the same mechanism, e.g. T-2 increases ROS production while AFB1 decreases its clearance when the presence of one mycotoxin increases the absorption of another or decreases its metabolic degradation.

Be aware of contaminations

Given their complex interactions, the toxicity of combinations of mycotoxins cannot merely be predicted based upon their individual toxicities. Knowing that even low levels of mycotoxin combinations can harm animal productivity, health, and welfare, it is useful for feed and animal producers to be aware of present contaminations, to be able to link them to the risk they pose for the animal and consequently take actions before the problems appear in the field.

*References are available on request.

By Marisabel Caballero, Global Technical Manager, Poultry
Published on ALL ABOUT FEED | Reprint 2018.