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The Cumulative Effect of Multiple Mycotoxins

April 2, 2024
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Unlocking the Mysteries of Mycotoxin Levels in Feeds 

One of history’s enduring quotes originates from the philosopher Aristotle, who articulated, “The whole is greater than the sum of its parts.” Today, we apply this wisdom to agriculture, specifically in understanding the cumulative effect of mycotoxins on livestock health and productivity. 

We explore the effect of mycotoxin levels in livestock feeds and the significance of multiple mycotoxins in tandem, uncovering the holistic impact of these toxins on livestock well-being and performance. Also, we investigate historical research documenting threshold levels of concern for individual mycotoxins across various animal species. However, there is a notable gap in research exploring the combined effects of multiple different mycotoxins present in livestock diets.  

The Cumulative Effect of Stressors 

Producers want to understand the “safe” threshold of mycotoxin levels in livestock feed and consult various available guides. However, the speed at which mycotoxins become toxic is often contingent upon several factors, including environmental conditions, genetics, nutritional factors, and other stressors such as disease prevalence, heat, or overcrowding. Moreover, the presence of other mycotoxins in the diet can exacerbate these effects. These factors collectively influence the clinical expression of mycotoxicosis, adversely affecting weight gain, feed efficiency, production, and reproductive capabilities. 

Under most normal production conditions, more than one mycotoxin is present in an animal’s ration. Toxin-producing fungi often generate more than one mycotoxin. This process can contaminate a single feed ingredient, or several different toxins can be blended into a diet from several different ingredients that make up finished feed. Furthermore, animals can encounter toxins from other sources, causing additional stress. These would include endo and exotoxins from bacteria, viral toxins, or toxins from the environment, both synthetic and natural. 

Mycotoxin Interactions  

Most controlled research uses dietary mycotoxin concentrations much higher than those shown to affect field conditions. The reason for using these higher concentrations is to achieve a significant challenge response in research conditions. As a result, the combined effects of multiple dietary mycotoxins in research may sometimes appear less than cumulative. This discrepancy could arise because of the severity of the negative effects induced by each mycotoxin, preventing the typical response from the second mycotoxin from occurring simultaneously in the animal. 

The cumulative effects of multiple mycotoxins can manifest in various ways:  

  • Additive effects, where each mycotoxin independently decreases performance by the same amount as it would if present in the diet alone;  
  • Synergistic effects, where mycotoxins collaborate to exacerbate negative outcomes beyond their individual impacts; and rarely,  
  • Antagonistic effects, where one mycotoxin diminishes the activity of another, although this is not to be expected as supported by various research.  

In a 2003 mycotoxin report by the Council for Agricultural Science and Technology there was a review of 33 papers where researchers reported the effects of multiple mycotoxins fed concurrently throughout production. In this review, only one was believed to have had an antagonistic response.  

An interesting occurrence, often not discussed, occurs when multiple mycotoxins demonstrate potentiative effects. According to the CAST report, “Potentiative effects occur when one mycotoxin does not cause a toxic effect on a certain organ or system, but then fed with another mycotoxin it makes the latter much more toxic.”   

What requires consideration and management is the overall toxin exposure. Achieving control over the disease is not dependent on attaining 100% control of any given toxin. Instead, it can be achieved by reducing a portion of the toxin exposure so that the total exposure is controlled. 

Figure 1. Control of toxicosis is not dependent on 100% control of any given toxin but can be attained by reduction of part of the toxin exposure such that the total exposure is dropped below the point that production is economically impacted. 

Figure 2. Multiple sources of stress in an animal’s life will change the tolerance level it has for toxins at different times. If you can decrease the toxin concentration that is entering the body from the gastrointestinal tract you can minimize the amount of damage it can do. 

 The animal’s ability to tolerate total mycotoxins can fluctuate over time due to varying levels of additional stressors impacting the animal. 

These stressors encompass a wide range of factors including disease challenges, rapid growth, onset of egg production, weaning, pregnancy or nursing, exposure to other mycotoxins, or toxins in the diet, among others. In addition, the total concentration of mycotoxins in the diet will also change over time. Clinical disease becomes evident when the total mycotoxin level surpasses the animal’s tolerance threshold. To optimize performance, it is essential to both minimize animal stress and mitigate toxin exposure. 

 Calibrin®-Z:  The Broadest Spectrum for Biotoxin Control 

Calibrin®-Z, available in select international markets, is proven to adsorb a broad range of bacterial and fungal toxins that negatively impact livestock. It is supported by years of research at universities, research organizations, and on-farm use around the world.    

 

Calibrin-Z is not a traditional clay mineral. Amlan® International sources this unique, natural mineral in the U.S. and it is composed of calcium montmorillonite with high-capacity opal-lepispheres. It is a type of phyllosilicate constructed of nano-scale layers, providing an extensive surface area. Through proprietary thermal processing, Calibrin-Z is optimized to effectively bind the broadest range of biotoxins, including fungal and bacterial toxins. Selectively sourced for its exceptional binding capability, this mineral targets both polar toxins like aflatoxin and significant non-polar toxins such as zearalenone.  

Calibrin-Z: A Multiple Toxin Solution 

In the last year, numerous research projects have been undertaken to challenge and document the effectiveness of Calibrin-Z in mitigating the effects of multiple mycotoxins in the feed.   

These studies encompass a variety of approaches, ranging from a side-by-side comparison of high levels of a singular toxin, a combination of multiple toxins at lower levels, and a study that evaluated the effects of low levels of multiple toxins (all below a normal company rejection level for each toxin). 

In the first study, a comparison was made between broiler chickens fed a higher concentration of T-2 toxin (2 ppm) and broilers fed a combination of multiple mycotoxins consisting of T-2 (1 ppm), Aflatoxin (1.8 ppm), and Fumonisin (50 ppm). The treatment groups in the trial included an unchallenged control group, a challenged control group for both the singular toxin challenge and the multiple toxin challenge, and challenge groups with Calibrin-Z included in the diet for both the single toxin and the multiple toxin challenges. Due to the high concentrations of mycotoxins in this study, Calibrin-Z was added at an inclusion rate of 0.5%. In all traits measured (BW, FCR, Relative Liver Weight, and Villus Height) the negative effect on performance was greater for the multiple mycotoxin challenge as opposed to the higher single mycotoxin challenge. When Calibrin-Z was incorporated into the diet, the result was a statistically significant improvement in performance vs. the challenged control for body weight (63g), relative liver weight, and villus height, and a numerically significant (6 points) improvement in FCR under multiple mycotoxin challenge.. For the single high mycotoxin challenge, the birds fed Calibrin-Z improved in every KPI with BW and FCR performing even numerically better than the unchallenged control. 

  

   

While it is anticipated to observe performance improvements when employing a high-quality toxin binder like Calibrin-Z, in diets containing elevated levels of both single and multiple mycotoxins, the initial query raised in this article remains pertinent: When individual toxin levels are exceedingly low, can they still adversely impact performance? And if so, can a toxin binder improve performance with low levels of toxins enough to offset the cost of inclusion in the diet? 

To test this, a replicated pen study was run that did not spike the broiler diet with mycotoxins, but rather tested the ingredients to document the level of mycotoxins naturally present. The corn was found to contain naturally occurring Fumonisin, T-2, and DON at levels significantly below what is documented as “Levels of Concern” in poultry. 

 The trial contained 8 replicate pens of 23 male broilers for each treatment and compared a control group to a group fed the same diet with an inclusion of 1 KG/MT Calibrin-Z.  As demonstrated in the table below, even when individual mycotoxin levels in the feed are extremely low, there is a synergistic effect that adversely impacts economically significant KPIs. However, incorporating a standard inclusion of Calibrin-Z into the feed improved performance. 

While the numerical improvements of approximately two days of growth (60 g/Day), a 6-point adjusted FCR, and a 1.14% increase in livability are certainly notable, what is the economic impact on live cost? If we assume a standard feed cost of $350/ton, which is common with the current grain prices, and then include the cost of the standard 1 kg/MT dosage of Calibrin-Z, these differences in KPI performance translate to a live cost advantage of just over $280,000 annually for every 100,000 broilers processed per week. Therefore, for a 1 million broiler per week operation producing 2.75 kg (approximately 6 lb.) broilers, this would result in a live cost savings based on enhanced performance of approximately $2.8 million annually. 

 

As previously mentioned, much of the research, including the initial study cited, is conducted with mycotoxin levels significantly elevated. While this approach effectively demonstrates the negative impact of mycotoxins on performance and highlights the benefits of using a toxin binder like Calibrin-Z, it does not accurately reflect the levels typically encountered in real-world scenarios. Consequently, many producers opt to forego a toxin binder in their day-to-day feed formulations and only consider adding a binder during specific times of year when higher mycotoxin levels are anticipated. 

However, this second study clearly demonstrates the synergistic interaction between extremely low levels that are under the “acceptable” threshold of mycotoxins and the significant economic advantages of incorporating a proven toxin binder, like Calibrin-Z, into the diet to enhance a producer’s bottom line.  

To start a trial, visit Amlan.com 

© 2024 Amlan International. All Rights Reserved. Product availability may vary by country, associated claims do not constitute medical claims and may differ based on government requirements. 

Dr. Robin Jarquin Speaks with El sitio Avícola for an Interview on Sustainable Production Effeciency

March 1, 2024
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We recently conducted a highly informative Q&A session with El Sitio Avícola. Robin Jarquin, Amlan’s Director of Sales for Latin America, discussed pivotal strategies for optimizing poultry gut health with Calibrin-Z®, Varium®, and Phylox®. Want to learn more about sustainable solutions and productivity enhancement? Click to read more

Understand How Calibrin®-Z Controls T-2 Toxin in Broilers

January 19, 2024
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Poultry producers mostly know T-2 toxin by the horrific lesions seen on the beaks of poultry. The fast-acting T-2 toxin has a major impact on the growth and performance of poultry and livestock. Luckily it is not the most common trichothecenes toxin produced by Fusarium molds, deoxynivalenol would fill that spot, but T-2 is considered the most toxic of the trichothecenes. This poison can be inhaled or adsorbed through the skin or the gastro-intestinal tract and causes multiple problems in poultry and livestock. A short list of problems includes decreased gain and feed efficiency, decreased egg production and hatchability, decreased immune function, and increased mortality. It has been shown to have a synergistic negative effect with other mycotoxins in the diet or when administered in conjunction with lipopolysaccharide (aka LPS). This may be one reason why there is additional negative impact when there is a co-challenge with T-2 and gram-negative bacteria. Control of other dietary mycotoxins or LPS concurrently with T-2 may be important in any attempt to decrease its effects.   

 

T-2 toxin decreases the productivity of poultry and livestock by inhibiting protein synthesis at the cellular level and causing cell death. In eukaryotic cell’s DNA, RNA, and protein, synthesis is inhibited by T-2 toxin. It also induces apoptosis or programmed cell death.

A major concern in poultry is how T-2 affects the gastrointestinal tract starting with lesions of the beak and gizzard and going through the entire gut. These lesions will affect feed intake, gain, and feed efficiency. But T-2 can affect all aspects of production and reproduction, so egg production and hatchability also need to be considered. In early research looking at the effects of T-2 on hatchability, 2 ppm of T-2 toxin was fed to laying hens, egg production decreased by 3.8 percent, fertility of the eggs that were laid decreased by 1.7 percent, and hatchability of fertile eggs decreased by 5.6 percent. This is a substantial loss of hatched chicks because of the toxin in the feed.

T-2 is quickly adsorbed. And it can be adsorbed through the lungs, the skin, or through the gastrointestinal tract when ingested in the feed. Approximately 90% of T-2 is adsorbed into the body within 30 minutes of ingestion, but it does have a short half-life of less than 20 minutes. T-2 producing Fusarium molds can occur in feedstuffs either during a warm and moist growing season or during storage under high moisture, especially if stored grains have damage such as broken or cracked kernels. The best option for producers is to use feedstuffs free of all toxins, however, the reality is that this is not always possible. In those cases where feedstuffs are being fed that may contain T-2 it would be beneficial to have a fast-acting toxin binder in the diets.    

Because T-2 is so damaging and so rapidly absorbed, the toxin binder that is used needs to work and work fast. Calibrin®–Z, available in select international markets, adsorbed ~70% of T-2 toxin within 1 minute in research looking at speed-of-binding in vitro. This was approximately 24 times faster than the other products used in the trial. Additionally, Calibrin-Z had previously been shown to bind other mycotoxins and LPS in vitro and in vivo, which may be important during a T-2 challenge. A test to determine the binding ability of Calibrin-Z in vitro was conducted to look at seven common fungal biotoxins where the binder-to-toxin ratio was as if there was 1 kg of Calibrin-Z per metric ton of feed vs. observed concentrations of mycotoxins in feed. In vitro data showed that Calibrin-Z could bind LPS, but it has also been seen in vivo when Calibrin-Z was being fed to laying hens.

Calibrin-Z Mitigates the Effects of T-2 Toxin in Broiler Chicks

Recently, research was conducted at a large university in Brazil to determine the effects of Calibrin-Z on broiler chickens challenged with dietary T-2 Toxin. For this experiment, a total of 180 one-day-old male Cobb 500 broiler chicks were used. At the beginning of the trial the birds had an average body weight of 47 grams, with the average initial weight for each bird being equal. They were fed three different treatments 1) Unchallenged Control; 2) Challenged Control with 2 ppm T-2 Toxin; and 3) 2 ppm T-2 Toxin with 0.5% dietary Calibrin-Z. They were fed the treatment diets for 21 days. No aflatoxins, deoxynivalenol, diacetoxyscirpenol, fumonisins, ochratoxin A, T- 2 Toxin or zearalenone were detected in the feed ingredients that were tested before mixing the diets. The T-2 Toxin that was added to the feed for the challenged treatments was produced by Fusarium sporotrichioides fungi, and was 82% T-2 Toxin, 18% HT-2 Toxin.  There were 6 pens that were randomly assigned to each treatment and there were 10 chicks in each pen. Chicks had free access to a constant supply of food and water. The diet was corn-soybean meal-based and formulated according to requirements in the Cobb Broiler Management Guide.  

The intent of the study was to determine the effect of T-2 toxin on growth performance of broilers and how the addition of Calibrin-Z helped to mitigate any negative effects. Calibrin-Z is a unique calcium montmorillonite that has been shown to bind toxins, both fungal and bacterial, as well as lipopolysaccharides (LPS).

Feeding Calibrin-Z to the birds challenged with T-2 toxin increased body weight by 5% compared to the birds that were fed diets with T-2 toxin and no Calibrin-Z. This improvement returned body weight to that of the unchallenged control birds. In this experiment, there was no effect of feeding T-2 on feed intake with birds on all three treatments having equal feed intake.

Because there was no difference in feed intake the feed conversion ratio followed the same pattern seen in body weight. Feeding Calibrin-Z to birds challenged with T-2 toxin improved feed conversion by 8 points, with values of 1.50 for Calibrin-Z fed birds compared to 1.58 for birds that only received T-2 toxin in the feed.

While aflatoxin is the mycotoxin best known for its negative effects on the liver, T-2 toxin can also have bad effects. In this study, relative liver weight was higher in the challenged control, 3.33%, compared to the unchallenged control, 2.84%, but feeding Calibrin-Z again mitigated the negative effect of the T-2 toxin, with challenged birds fed Calibrin-Z having a relative liver weight of 2.96%.   

T-2 Toxin is a fast-acting mycotoxin that has a tremendous impact on animal performance. It acts in a synergistic way with challenges from other mycotoxins and lipopolysaccharide. To decrease its impact, you need a fast-acting toxin binder to control T-2 quickly while also controlling other potential problems. Calibrin-Z is a fast-acting multi-toxin binder that has proven results.   

 As the animal health business of Oil-Dri® Corporation of America, Amlan products are backed by Oil-Dri’s 80-plus years of mineral science expertise. Oil-Dri and Amlan are vertically integrated and own every step of the production process to consistently deliver safe, high-quality animal health products around the world. Calibrin-Z, a calcium montmorillonite clay, is sold as a broad-spectrum toxin binder. To understand how Calibrin-Z can work in your production system, contact your local Amlan representative.

 

References:  

Chi, M. S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates, and W. Shimoda. 1977. Effects of T-2 Toxin on Reproductive Performance and Health of Laying Hens. Poultry Sci. 56:628 – 637.  

Tai, J.-H. and J. J. Pestka. 1988. Synergistic interaction between the trichothecene T-2 toxin and Salmonella typhimurium lipopolysaccharide in C3H/HeN and C3H/HeJ mice. Toxicol Lett 44:191–200.  

Mycotoxins: Risks in Plant, Animal, and Human Systems. 2003. Task Force Report No. 139 Council for Agricultural Science and Technology. Ames, Iowa, USA.

Reduce Salmonella Prevalence and Load with NeutraPath®

December 21, 2023
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Salmonellosis is a foodborne pathogen that causes illness and death worldwide. A blended feed additive has been shown to have good effects in vitro on a wide range of Gram-negative and Gram-positive bacteria and in vivo to mitigate the effects of Clostridium perfringens in broilers and E. coli in swine. Thus, it was decided to investigate the ability of the proprietary blend of essential oils, medium-chain fatty acids, and an activated toxin-adsorbing mineral (NeutraPath® available in select international markets) to control Salmonella. In vitro and in vivo research with Salmonella typhimurium and Salmonella heidelberg showed the blend could reduce prevalence and bacterial load of Salmonella in broiler chickens. Based on these in vitro and in vivo data, feeding this blend could be a potential new method to help control Salmonella in broiler chickens and aid in control of pathogens at the farm level. 

 

A Foodborne Pathogen 

Salmonella is a very common pathogenic bacteria that can be passed from animals to humans. The typical symptoms of salmonellosis in humans are diarrhea, fever, and stomach cramps, with the occasional vomiting. Generally, this is mild and doesn’t require medical intervention, but it can be deadly, especially in young children. Worldwide, Salmonella is one of four main causes of diarrheal diseases, with diarrheal disease being the 2nd leading cause of death in children under five. In the United States approximately 1,350,000,000 people are infected annually, ~26,500 people are hospitalized, and ~420 die each year. The very young, the very old, pregnant women, and people with compromised immune systems are generally affected the most.  

According to the World Health Organization, Salmonella, a hardy bacteria, can survive in a dry environment for several weeks but several weeks turns into several months if it is in water.  

 

Sometimes salmonellosis can result from coming in direct contact with animals that carry the bacteria, typically reptiles or birds. There are two species of Salmonella, bongori and enterica. Salmonella bongori is normally associated with cold-blooded animals but can infect humans. Selling tiny turtles (those with shells less than 4 inches long) has been prohibited in the U.S. since 1975 because of their association with salmonellosis in children. Salmonella enterica has more serovars with approximately 80 that can infect humans and animals. With an increase in the popularity of raising your own chickens the CDC (Centers for Disease Control) has issued repeated reminders about the safe handling of chickens, including a reminder “don’t kiss your chickens” because of serious outbreaks of salmonellosis, especially among children, linked to raising chickens in the backyard.

But eating or mishandling raw or undercooked contaminated food is the source of most cases of salmonellosis in humans. Live poultry often don’t show signs of carrying Salmonella even if their intestines contain the pathogenic bacteria. There are multiple ways that birds can be exposed to Salmonella. Exposure can be through contaminated feed, from wild birds or rodents, or from a contaminated barn. It is even possible that poultry may be contaminated before the egg that they hatched from was laid. Hens can have bacteria in the ovary or oviduct and the egg can be contaminated before the shell forms around the egg, meaning that even clean, washed eggs could be contaminated. The Poultry Industry has been working diligently to control this problem and this source of contamination of broiler chickens has declined in recent years.  

 

Even though a majority of the foodborne illnesses due to Salmonella originate from non-poultry sources, twenty-three percent of the Salmonella outbreaks in the U.S. are linked to poultry consumption (16.8% from chicken, 6.6% from turkey) with another 6.3% coming from eggs. Salmonella in the intestine of poultry can lead to contamination of poultry meat during processing. Proper cooking will kill the bacteria, but improper handling may spread bacteria around the kitchen and raw vegetables, undercooked meat or uncooked foods containing eggs (i.e., cookie dough) may still be contaminated with live bacteria. Together the CDC, FDA (Food and Drug Administration), and USDA (United States Department of Agriculture) have a goal of reducing Salmonella illnesses by 25% by 2030. In order to do this, they need to decrease Salmonella infections from all products regulated by the Food Safety and Inspection Service division of the USDA by 25%. One way to help reach this goal is to help minimize the amount of Salmonella that comes into the processing plant making it less likely that contamination of poultry meat will occur.  

Poultry producers need help to accomplish this goal. Research has shown that a proprietary blend of essential oils, medium-chain fatty acids, and an activated toxin-adsorbing mineral (NeutraPath®, Amlan International, available in select international markets) may be of assistance. The blend has anti-virulence effects because of its ability to bind quorum sensing molecules, exotoxins, and endotoxins associated with bacteria. It also has direct bacteriostatic/ bactericidal effects against both Gram-positive and Gram-negative bacteria. Its efficacy has been proven over years of in vitro and in vivo trials at multiple research sites, against multiple bacteria, in multiple animal species.  

In vivo research in chickens showed that the blend decreased the effects of Clostridium perfringens. A summary of the studies showed that it improved mortality, gain, and feed conversion in challenged broilers. In weaning pigs, it was shown to decrease the impact of enterotoxigenic E-coli (F-18+). In the pigs challenged with E. coli the blend improved feed efficiency and decreased frequency of diarrhea. When the fecal microbiome was examined, there was a higher relative abundance of Lactobacillaceae and a lower relative abundance of Enterobacteriaceae. Enterobacteriaceae is a family of Gram-negative bacteria that includes both E. coli and Salmonella. These positive results led researchers to investigate its effects on Salmonella, with research being conducted both in vitro and in vivo.  

 

Salmonella Research 

 

In vitro tests were used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of NeutraPath against S. heidelberg. Three different concentrations (1, 3 and 5 mg/ml) of NeutraPath with a control of 0 mg/ml were added to samples of an S. heidelberg strain. The MIC of NeutraPath for S. heidelberg was found to be 5 mg/ml. To determine the MBC Salmonella cultures were incubated at 37ºC for 16 hours without agitation. After incubation, bacterial counts were measured by serial dilution. A 30 μl aliquot of each dilution was plated onto lysogeny broth and incubated overnight. The MBC of NeutraPath for S. heidelberg was determined to be 4 μg/ml. These assays demonstrated that the blend has strong in vitro inhibitory and bactericidal activities against this key pathogen.  

Because of the results seen in vitro researchers conducted an in vivo experiment. The researchers wanted to know how the blend would affect the Salmonella prevalence in birds previously challenged with S. heidelberg and fed for a short period of time before sampling.  

After feeding the treatments for seven days, pre-moistened boot-sock swab sampling showed that there was S. heidelberg contamination in 100% of the pens. Cecal digesta samples and cloacal swabs were also collected from 10 of the broilers that had been directly challenged at hatch to determine Salmonella prevalence. The prevalence of Salmonella decreased by 40% in the cecal digesta (83.3% vs. 50.0%) and by 55% (60.0% vs. 26.7%) in the cloacal swabs when the blend was added to the diet for 7 days prior to testing. 

 

Comparable studies were done at second location with another group of researchers to establish that the results from the initial studies could be repeated in other Salmonella serovars. This time studies looked at the effects of the blend on Salmonella typhimurium.  

 

First an in vitro study digestion was used to simulate the crop, proventriculus and intestinal section of the gastrointestinal tract. Each “section” had pH and enzymatic conditions that would correspond to that area of the gut. Adding the antimicrobial blend inhibited the growth of S. typhimurium, reducing the total colony forming units recovered in each section. This positive result meant that an in vivo study was warranted. This time the broilers were started at one day of age. Thirty male broiler chicks were placed on two treatments. The treatments were a challenged control with non-treated feed, or birds fed that diet + 0.25% of the blend. At nine days-of-age chickens were given an oral dose of 106 CFU (Colony Forming Units) of live S. typhimurium. Twenty-four hours after the challenge ceca and cecal tonsils were removed so that they could be evaluated for Salmonella recovery. Both the number of positive samples and the amount of Salmonella bacteria found in those positive samples decreased in the treated birds. Feeding the blend decreased the ceca that tested positive for S. typhimurium by 41.7%, 100% of the tested ceca were positive in birds fed the untreated control compared to only 58.3% of the ceca in the birds that were fed the blend. In the birds that tested positive the total S. typhimurium bacterial load recovered in the ceca also dropped by 1.84 log10 CFU/g compared to the untreated control.

Conclusion 

Around the world foodborne salmonellosis continues to be a problem. Regulators are targeting aggressive goals for salmonella reduction in poultry meat. A proprietary blend of essential oils, medium-chain fatty acids, and an activated toxin-adsorbing mineral has been shown to work against a variety of bacteria. Research has shown efficacy against C. perfringens, E. coli, and S. typhimurium and S. heidelberg. Based on the current in vitro and in vivo research adding the blended product has the potential to reduce Salmonella colonization in broiler chickens. This is a viable option for use in poultry health programs including those controlling Salmonella contamination. To learn more about NeutraPath or to contact a local representative, visit amlan.com.

 

NeutraPath® Featured in WATT Poultry International

December 13, 2023
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WATT Poultry® International has published an article on NeutraPath®, available in select international countries. NeutraPath is a non-pharmaceutical treatment specifically designed to improve feed efficiency and intestinal health in poultry and livestock. The natural ingredients of NeutraPath® target pathogens and their toxins through several modes of action, providing equivalent performance to industry-standard antibiotics. Follow the link to learn more.

 

Seeing is Believing! Calibrin®-Z Binding Effect

December 7, 2023
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(Figure 1) Platinum octaethylporphyrin molecules seen adsorbed by Calibrin-Z using Cryogenic Transmission Electron Microscopy. Platinum (pink) seen under microscopy indicates where the organic compound was bound in the interconnected pores of the unique clay mineral. 

Providing Economic Value for More Than a Decade  

For more than 16 years, Calibrin®-Z (available in select international markets) has helped poultry producers mitigate the damage that mycotoxins cause to their livestock, their sustainability, and their bottom line. Calibrin-Z’s ability to bind mycotoxins, fungal toxins that negatively affect the health and performance of livestock, has been shown both in experimental settings and on the farm. The implication from this research was that the unique clay mineral that comprises Calibrin-Z was blocking the negative effects of mycotoxins on animal performance by adsorbing the toxins in the pores of the clay.  The way the binding occurred was known but had not been seen directly. 

Until Now!  

Scientists from Oil-Dri worked with university scientists to enable us to see organic molecules binding to Calibrin-Z (Figure 1). To do this they used Cryogenic Transmission Electron Microscopy.  This type of microscopy is used to look at biological and materials structures at an almost atomic level. The material of interest is flash-frozen to keep from damaging the structure of the organic material that is being observed. 

Octaethylporphyrin, is an organic molecule that was chosen to represent the mycotoxins that Calibrin-Z normally adsorbs. It has a general size and planar orientation similar to that of mycotoxins. Platinum is not an element that is typically found in the clay mineral that makes up Calibrin-Z and can be seen using cryogenic transmission electron microscopy. This combination of factors makes it an excellent marker to use to visualize Calibrin-Z’s binding sites. When the organic portion of the platinum octaethylporphyrin molecule is adsorbed onto the Calibrin-Z binding sites you can see the platinum with a cryogenic transmission electron microscope. The platinum in the picture taken under the microscope was interspersed between the layers and on the outer surface of the Calibrin-Z particles. This shows that the organic compound was bound in the interconnected pores as was anticipated.   

Selection and Quality 

Calibrin-Z’s natural ability to adsorb biotoxins is based on the clay mineral used in its manufacture. The source of the clay mineral was chosen after years of testing and comparisons of a multitude of different potential sites. This source was selected based off its innate ability to bind toxins, the ability to improve that binding with processing, and its benign chemical profile. With vertical integration, mine to market traceability, and decades of reserves, this unique clay mineral is the foundation of Amlan International’s animal health products.   

Calibrin-Z is composed mainly of calcium montmorillonite with opal lepispheres. The opal lepispheres are intimately interwoven within the nano-scale layers of montmorillonite. They help Calibrin-Z maintain its structure during a proprietary processing step that expands the number of biotoxins Calibrin-Z adsorbs. The unique structure of Calibrin-Z is vital to its toxin binding capacity.

 

Absorption and Adsorption 

A kilogram of Calibrin -Z has approximately the same surface area as 60 soccer fields.  This is because over 99% of Calibrin-Z’s total surface area is inside the particle. Calibrin-Z’s internal network of interconnected channels and pores is ~50% of its total volume. When Calibrin-Z is fed to livestock or poultry, fluid in the intestine rapidly absorbs into the mineral’s pores through capillary action. Biotoxins in the fluid move inside via the networks of capillary channels. From a molecular perspective it is as if they are traveling on a superhighway. Biotoxins adsorb once they reach the binding sites on the pores’ surfaces.  

The biotoxin molecules are attracted onto the pore surfaces via adsorption, this is both chemisorption and physisorption.  Biotoxins will structurally coordinate themselves onto charged surfaces and bind via ion-dipole and electrostatic interactions. While mycotoxins tend to be smaller and can enter the pores of Calibrin-Z and bind there, bacterial toxins tend to be larger but may also bind. Theoretically, there are special physical properties that allow the molecular conformation of the bacterial toxin to become distorted, which allows them to adsorb onto macro-surfaces within the pore spaces. Someday we may be able to use microscopy to see that, too. 

Because of its structure, the clay mineral that Calibrin-Z is made from is naturally hydrophilic and will bind to polar molecules. But Calibrin-Z undergoes a proprietary processing method  that causes dehydroxylation of the clay mineral’s crystal structure. During this process the opal lepispheres spread between the layers maintain its channels and binding sites. Thus, Calibrin-Z continues to bind polar molecules, such as the mycotoxin aflatoxin, but processing also allows it to have the ability to bind non-polar mycotoxins such as zearalenone. The ability to mitigate the effects of multiple mycotoxins has been shown using both in vitro and in vivo research.  

Because of Calibrin-Z’s proprietary heat treatment the toxins that it adorbs include a broad-spectrum of polar and non-polar toxins. Therefore, Calibrin-Z has shown high adsorption properties for mycotoxins, enterotoxins, and endotoxins.  

 

Examples of Biotoxins Bound by Calibrin-Z 

 Natural and Reliable to Use 

Calibrin-Z is shown to be a reliable and effective biotoxin binder. When added to animal feed at up to 5X the recommended dose it showed no negative effects. In fact, there was often a numerical improvement in gain, feed intake, or feed conversion when Calibrin-Z was added to an unchallenged diet. This indicates that there was no significant negative effect of Calibrin products on nutrient utilization.  

The unique surface chemistry and structural properties of the calcium montmorillonite in Calibrin-Z, added to its proprietary thermal-processing method, are what provide its optimal toxin binding capacity. This is what sets Calibrin-Z apart from other clay-based products. We have long known this because of its structure, how it works in vitro and how for more than a decade it has improved the performance of livestock and poultry. And now we, and you, are able to see it with our own eyes.  

To learn more about broad spectrum biotoxin binder Calibrin-Z, and how you can add it to your poultry and livestock feed, visit amlan.com 

Mineral-based solution for dairy cow gut health

October 26, 2023
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During the 2023 World Dairy Expo held in Madison, Wisconsin, Ann Hess from Feedstuffs 365 spoke with Amlan, the Animal Health business of Oil-Dri, about mineral technology as a solution to support gut health and improve diary performance. Listen to Dr. Marc Herpfer, VP of New Technologies, Regan Culbertson, VP of Strategic Marketing, and Jay Hughes, Director of Technical Services, Americas discuss how the unique, thermally processed calcium montmorillonite with opal lepispheres are driving economic, bottomline, performance in the dairy industry.

Watch the interview here.

Industry Report Shows Damaging Mycotoxins Present in Silage

September 14, 2023
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Mycotoxins are a worldwide challenge for livestock producers. The presence of mycotoxins in feed materials represents a threat of disease and death in animals. Perhaps more of a problem is the subclinical losses to production, reproduction, and thus, efficiency that they can cause. Mycotoxins are formed as secondary metabolites by molds. Molds require nutrients and water to grow. Feedstuffs such as corn and silage can provide both, thus allowing for mycotoxin contamination to easily occur.  

Recently Dairyland Laboratories, one of the leading agricultural testing labs in the US, released a report summarizing the amount of mycotoxin contamination they are finding in samples of the 2022 silage crop submitted by their customers. Only 2.5% of the tested samples were positive for aflatoxin. However, two of the six most important mycotoxins were found in more than 50% of the samples submitted. 

The toxins coming in at over 50% were two mycotoxins produced by Fusarium molds: zearalenone and vomitoxin (aka DON, deoxynivalenol). Due to its chemical structure, zearalenone mimics estrogen (Figure 1) and once ingested and absorbed into the body, causes significant repercussions on all aspects of fertility in most livestock species. Zearalenone has also been shown to decrease immune responses, feed digestibility, and can result in inefficient animal production and reproduction. Vomitoxin is a mycotoxin that mainly affects swine. It depresses feed intake and gain, and in high enough concentrations, will cause pigs to vomit. Vomitoxin has also been used as a marker to indicate that the feedstuff may be contaminated with other mycotoxins.

Figure 1. Chemical structure of (a) zearalenone and (b) 17b-estradiol a form of estrogen

From the Dairyland Laboratories report, both zearalenone and vomitoxin increased in the 2022 corn silage crop. First, they increased in the percentage of samples that tested positive compared to the previous two years. Zearalenone was found in 88% of the current samples versus 76% in earlier years. Vomitoxin was in 92% of the new samples compared to 85% in the previous two years. Secondly, they increased in the median amount of mycotoxin that was present in the samples. The median number for zearalenone increased slightly from 0.079 ppm vs. 0.075 ppm in earlier samples. The same was true for vomitoxin, which increased from 1.1 ppm from October 2021 – September 2022 to 1.6 ppm in the new crop samples that were analyzed.

Table 1. Percent of submitted samples that tested positive for zearalenone, vomitoxin, or fumonisin.

Table adapted from Mycotoxin Trends – Corn Silage produced by Dairyland Laboratories, Inc. Arcadia, WI

 

Table 2. Concentration of zearalenone or vomitoxin present in submitted samples 

Table adapted from Mycotoxin Trends – Corn Silage produced by Dairyland Laboratories, Inc. Arcadia, WI

Dairyland Laboratories did report an “uptick” in Fumonisin another Fusarium mycotoxin. Of the samples tested, 47% of the 2022 crop samples tested positive compared to 43% in the previous year. Of all species, horses and pigs have the worst reactions to dietary fumonisin. Horses can be affected at relatively low levels of fumonisin and develop leukoencephalomalacia, also called moldy corn syndrome or blind staggers. In this disease, there is damage to the horse’s brain. Fumonisin can also cause severe problems in pigs: pulmonary edema, and damage to the liver along with decreased growth performance. The other livestock species are thought to tolerate higher concentrations of fumonisin than horses or swine. However, numerous studies have shown that low levels of fumonisin or combinations of mycotoxins can have an impact on intestinal integrity.  

Often a ration may be contaminated with more than one mycotoxin. This can be from one feed ingredient or several. This can result in additive or even synergistic negative effects, especially in non-research settings where other challenges such as heat stress, crowding, and disease pressure from viruses and bacteria are common.  

Clostridium is ubiquitous bacteria. Increased concentrations of it in silage has been shown to decrease productivity. Research has shown that combinations of mycotoxins can exacerbate the effects of Clostridium on gut health. Low levels of fumonisin, for instance, make it easier for bacteria such as Clostridium and E. coli to attach to the gut, allowing them to proliferate. Clostridium bacteria, such as Clostridium perfringens release toxins that injure the intestine. This allows for a condition known as leaky gut; allowing unwanted substances into the body while decreasing the gut’s ability to absorb nutrients. Research has shown that combinations of mycotoxins can exacerbate the effects of Clostridium on gut health.  

Mycotoxins in animal feed threatens their health. Measures must be taken by producers to manage and protect their livestock against mycotoxins to preserve the economic viability of their production. Sourcing effective technologies will be the key to moving producers to greater production, improved efficiencies, and increased profitability. At Amlan, we offer advanced feed additive solutions, including Calibrin®-Z (available in select international countries), that can mitigate mycotoxin challenges and optimize the intestinal environment to keep livestock healthy for improved performance. To learn more, click here.

Calibrin®-Z: A Solution for Performance-Degrading Challenges Caused by Lethal Toxins

August 8, 2023
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Normally when we think of problems caused by bacteria, we think of the diseases and the damage that they do when they are alive. We believe if we kill the bacteria, we will stop the disease that they cause to ourselves or our livestock. Unfortunately, the death or destruction of some bacteria does not end the damage that they can cause.

Gram negative bacteria such as Escherichia coli have a component of the outer membrane of the cell wall called lipopolysaccharides (LPS). Lipopolysaccharides, also referred to as endotoxins, are highly toxic and are released when the cell dies, is damaged, or destroyed. Lipopolysaccharides range in size from 10 thousand to 1 million daltons. Because of their small size they can easily enter the bloodstream through a damaged or “leaky” intestine. When this happens endotoxins can be very toxic to humans and animals. How toxic depends first on the species of animal and the amount of endotoxins that enter the bloodstream. Humans have a much more severe reaction than do mice.

One of the main ways that endotoxins enter the animal’s bloodstream is when intestinal integrity is compromised. There can be several reasons for this. The gut can be compromised as the result of diseases such as necrotic enteritis or coccidiosis in chickens or ileitis in pigs. Other factors such as heat stress can also increase the permeability of the intestine and allow increased amounts of endotoxin to enter the bloodstream. The toxicity seen from endotoxins is associated with responses of the animal’s immune system to the LPS in the animal’s bloodstream, once the endotoxins enter the bloodstream they can cause fever, inflammation, and decreased growth. With high levels of endotoxins entering the bloodstream, shock, organ failure, and death can occur.  However, if the endotoxin could be bound and contained in the intestine, it would not have the ability to cause these reactions. 

Calibrin®-Z (available in select international markets), a proprietary thermally processed calcium montmorillonite, has been shown to effectively bind a broad range of bacterial (e.g., alpha toxin and NetB from Clostridium perfringens, E. coli Shiga–like toxins, etc.) and fungal (e.g., zearalenone, fumonisin, aflatoxin, ochratoxin, T-2 toxin, ergovaline, and ergotamine) toxins. Because it binds these toxins, it prevents them from damaging the intestine or from being absorbed through the intestine and entering the body to cause damage to internal organs (e.g., liver, kidneys) hence reducing animal performance. Because of this ability of Calibrin-Z, it was thought that it might also bind lipopolysaccharides. Therefore, a laboratory test was conducted to determine if Calibrin-Z could bind the lipopolysaccharide from E. coli. 

This was done using an in vitro test called the Limulus Amebocyte Lysate assay, which uses the fact that the blood cells (amebocytes) of the horseshoe crab (Limulus) secrete an enzyme (lysate) that clumps when mixed with endotoxins. In preparation for this test LPS solutions with concentrations of 5, 10, 50, or 500 milligrams of LPS per kilogram were made. When these solutions were then treated with Calibrin-Z it resulted in different ratios of Calibrin-Z to LPS, ranging from 20:1 to 10,000:1. The Calibrin-Z was removed using a 0.2 micrometer filter and the remaining solutions were tested using the Limulus Amebocyte Lysate assay to determine the amount of lipopolysaccharide that remained in the solution (Figure 1).

 

These results show that the processed calcium montmorillonite bound the E. coli LPS. The percentage of toxin that was bound increased as the amount of Calibrin-Z to LPS toxin increased. When the binder to toxin ratio was 10,000: 1 Calibrin-Z removed 95% of the LPS from the solution. Calibrin-Z effectively bound lipopolysaccharides, the deadly toxins released when gram-negative bacteria die. Based on this information, feeding Calibrin-Z could help protect poultry and livestock performance from toxic LPS.  To learn more about Calibrin-Z or details about this and other studies, visit amlan.com or contact your local Amlan sales representative.

The Role of Quorum Sensing in Necrotic Enteritis Development

July 11, 2023
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The fast onset of necrotic enteritis and the devastating production losses it inflicts make it one of the most challenging diseases for the poultry industry, particularly for antibiotic-free producers. Clostridium perfringens, the cause of necrotic enteritis, possesses a number of virulence factors that allow it to mount a fast, efficient attack on the host including potent toxins and cell-to-cell communication (quorum sensing). However, natural mineral-based products that can disrupt quorum sensing and reduce the virulence of C. perfringens are available.

The Pathogenesis of Necrotic Enteritis

C. perfringens is an anerobic, spore-forming pathogen found in the normal microbiota of poultry, as well as the poultry house. Necrotic enteritis occurs when predisposing conditions, such as a change in diet, immune status or intestinal pathophysiology, promote an overgrowth of C. perfringens. Coccidiosis can also increase the incidence of necrotic enteritis, as the damage that Eimeria spp. cause to intestinal epithelial cells promotes the invasion of C. perfringens (as well as other pathogens).

C. perfringens’ Rapid Infection Rate

C. perfringens is one of the fastest growing bacterial pathogens. Under optimal conditions it can replicate every 8 to 10 minutes — outgrowing other resident bacteria to achieve intestinal colonization.1 As well as a rapid growth rate, C. perfringens infection involves multiple steps, which likely occur simultaneously, including colonization, replication, nutrient procurement, evasion of host immune defenses, host tissue damage and transmission.2

Exotoxin Roles in Necrotic Enteritis Development

Multiple exotoxins can be produced by C. perfringens, including alpha-toxin and necrotic enteritis toxin B-like toxin (NetB). Alpha-toxin is cytotoxic to endothelial cells, red blood cells, white blood cells and platelets, while NetB toxin forms pores in cell membranes that allow electrolytes to rupture cells, causing cell death and necrotic lesions in the small intestinal mucosa.3 These two toxins are known to have a role in necrotic enteritis development.

C. perfringens Growth Depends on Host Nutrients

C. perfringens relies on nutrients from the host to live and multiply — a process which results in the destruction of host tissues (formation of necrotic lesions). C. perfringens lacks enzymes needed for amino acid biosynthesis and subsequent protein synthesis, so enzymes and toxins are released to degrade structural proteins from the host.4 The host amino acids and/or peptides are then taken in by C. perfringens for use in its own protein synthesis. 4

To produce energy, C. perfringens degrades large sugar compounds from the host and ferments them, producing gas that enhances the anerobic environment.4 C. perfringens also produces hyaluronidases that increase connective tissue permeability and help C. perfringens spread into deeper tissues.4

Quorum Sensing Controls Exotoxin and Enzyme Production

C. perfringens uses quorum sensing (cell-to-cell communication) to coordinate exotoxin and enzyme production to occur when its population reaches a density that supports the most efficient use of its metabolic resources.5 For example, to determine the optimum time to start producing NetB, the accessory gene regulator-like (Agr-like) quorum-sensing system sends out signals that are recognized by the VirR/VirS two-component regulatory system.5 Once the VirR/VirS system detects that the C. perfringens population has reached the threshold density, it switches on the expression of NetB and other virulence and related metabolism genes.2

Quorum Quenching Reduces Pathogen Virulence

Quorum quenching is an approach that can disrupt the quorum-sensing system of pathogenic bacteria, preventing cell-to-cell communication and the expression of quorum-sensing-controlled genes that produce toxins and other virulence factors. Additionally, quorum-quenching products should reduce the chance of antibiotic resistance, since they are modifying bacteria behavior rather than killing them.

Natural Quorum-Sensing Control

One product that has displayed quorum-quenching properties is the mineral-based feed additive Calibrin®-Z (available in select international markets). This all-natural single-ingredient product binds bacterial pathogens and the toxins they produce, as well as multiple mycotoxins, to help protect the intestinal barrier against enteric disease. Other natural mineral-based products can also help manage necrotic enteritis; read this article to learn more.

An in vitro study found that Calibrin-Z separated out quorum-sensing molecules by adsorption or catalytically broke them down into small fragments. By reducing the concentration of quorum-sensing biochemicals, Calibrin-Z can potentially disrupt the ability of pathogenic bacteria (including C. perfringens) to produce toxins, since quorum sensing controls this function. Calibrin-Z was also shown to effectively bind alpha-toxin and NetB toxin, further reducing the virulence of C. perfringens.

 

The global reduction in the use of in-feed antibiotics has compelled producers to rely on other management methods to maintain a healthy intestinal environment in poultry and reduce the risk of necrotic enteritis. The use of best-practice management strategies and inclusion of mineral-based feed additives that reduce the virulence of C. perfringens can assist in promoting intestinal health and maximizing production efficiency. To learn more about necrotic enteritis and natural mineral-based methods to control it, contact your local Amlan representative.

 

References

  1. Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens. Emerg Microbes Infect. 2018;7:141.
  2. Prescott JF, Parreira VR, Mehdizadeh Gohari I, Lepp D, Gong J. The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review. Avian Pathol. 2016;45:288–94.
  3. Chi, F. A Viable Adjunct or Alternative to Antibiotics: Meta-Analysis of Broiler Research Shows Natural Growth Promoter Delivers Feed Efficiency Equal to Antibiotics. Amlan International.
  4. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A. 2002;99:996–1001.
  5. Yu Q, Lepp D, Mehdizadeh Gohari I, Wu T, Zhou H, Yin X, Yu H, Prescott JF, Nie SP, Xie MY, Gong J. The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry. Infect Immun. 2017;85:e00975-16.

 

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