As our weapons in the fight against mycotoxins become more sophisticated, so must the methods we use to test their efficacy. Simulating intestinal conditions and ‘stress gene’ technology are very helpful but sometimes no longer the best or fastest approach to help to develop more effective elimination strategies. A recent technique is the use of biomarkers.
Most mycotoxin adsorbents only bind within a certain pH-range and release the toxins as pH increases during gut propulsion. Nearly 2 decades ago, Impextraco introduced a gut simulation model enabling to select the most effective binders. Moreover, eliminating agents, such as enzymes and natural extracts were selected using a high throughput screening technique. The combined effect of this multiple strategy on the efficacy of eliminating mycotoxins in feed was extensively tested in vitro simulating the digestive tract. To do so Impextraco collaborated in a project to develop a gut simulator and still is intensively working on their in house gut simulation model.
Reference tool for in vitro research
In order to determine the real binding and inactivation of mycotoxins, simple measurements in the feed are not sufficient, since the animal itself may influence the effect and pH plays an underestimated role in the absorption and desorption of certain binders. A perfect in vitro model of the animal would eliminate individual variation and control all other factors. Of course, there is no such thing as a perfect model, but the digestive tract has been well studied and several factors are easy to simulate. The advantage of this method is that by relatively simple and constant means the effects of the complete range of factors affecting in vitro results of toxin-binding can be observed.
Developing anti-mycotoxin products requires in vivo research to confirm in vitro observations. Alongside partnerships with universities, Impextraco runs its own research facilities and has validated animal models. These accurate strategies led to a combination of effective ingredients being compiled in the product Elitox (hereafter named the product or mycotoxin binder), which has proven to be effective in eliminating a broad range of mycotoxins.
The use of biomarkers
A wide range of in vivo protocols have been developed recently to determine the effect of mycotoxins in an early stage, using biomarkers. Biomarkers are characteristics that are objectively measured and evaluated as indicators of biological processes, pathogenic or pharmacological responses to an intervention, such as mycotoxin ingestion. They need to be reliable, fast and easy to measure in order to find significant effects as early as possible. To find mycotoxin induced changes in the animals metabolism, different strategies need to be implemented at the same time: blood biochemistry, flow cytometry and histopathology are just a few amongst them.
All mycotoxins affect animal health status due to their toxic characteristics, but they all work in a different way. Each mycotoxin needs a specific blood parameter to investigate its effect on the animal. Aflatoxin for example is hepatotoxic and blood parameters indicating liver functionality can reveal information to further increase our knowledge on the best way to eliminate its toxicity. Ochratoxins on the other hand are nephrotoxic and need different biomarkers to investigate their effects on kidney impairment. Continuous improvement of knowledge is helping to understand the toxic effect of a wide range of mycotoxins and to develop fast methods to investigate the efficacy of their mycotoxin eliminator (Table 1).
Impextraco was one of the first companies investing in a spin-off company of the Catholic University of Parana to further develop the use of flow cytometry as a tool to gain information on the effect of mycotoxins on the animals immune reaction. Flow cytometry is a impedance-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in basic research, clinical practices and clinical trials. Impextraco uses it to quantify the number of circulating immune cells, namely lymphocytes such as helper T-cells, cytotoxic T-cells, macrophages and monocytes. This information (Table 2) enables determination of the effect of different mycotoxins on the immune response in the animal and the efficacy of the mycotoxin binder in supporting the animal immune reaction when encountering mycotoxicosis.
Fumonisin effect on poultry immune response
In one of the many trials performed in Impextraco’s own trial facility in Brazil, 96 male Cobb 500 day-old-chicks were contaminated with fumonisins. The contaminated diet was formulated by replacing control corn by a naturally contaminated corn with Fusarium mycotoxins. This resulted in a final contamination of 17 ppm fumonisins. This contaminated birds were compared with control birds, receiving no contamination and birds receiving the mycotoxin binder, mixed at 0.2% in the contaminated diet. Results (Figure 1) showed that fumonisin contamination had detrimental effects in broilers, resulting in decreased hematocrit and total white blood cells, indicating a health impairment. Birds supplemented with the mycotoxin binder had not such negative health effects due to the elimination of Fumonisins by the product. Fumonisin contamination resulted in an increased SA/SO ratio in the blood. Fumonisins have a structure which is very similar to the long chain base backbones of sphingolipids and hence interfere in the sphingolipid metabolism. It competes with the enzyme ceramide synthase, resulting in the production of more sphinganine (SA) and less sphingosine (SO) and hence altering the SA/SO ratio, what makes this parameter the perfect biomarker for fumonisin mycotoxicosis.
Figure 1 - Effect of Fumonisin mycotoxicosis in broilers on blood parameters which can be considered biomarkers allowing to detect toxicosis in early stage based on small variation.
The lower concentration of circulating cytotoxic and helper T-cells in their blood, indicates that birds are using these immune cells as a response to fumonisin mycotoxicosis (Figure 2). With the addition of the product, fumonisins were detoxified, resulting in lower immune response, saving T-cells to fight other invaders. The amount of monocytes and macrophages was even higher in the birds that received the product, compared to control birds, proving the immune enhancing effect of the carefully selected natural extracts that are added to the mycotoxin binder to support animals during mycotoxicosis.
Figure 2 - Effect of Fumonisin mycotoxicosis in broilers determined by the use of immune cell quantification.