Improving the diagnosis of deoxynivalenol ingestion in rainbow trout

In recent years, the industry has concentrated efforts on finding alternative sources of protein to substitute fishmeal in aquafeeds. Consequently, many new alternatives are available, e.g. insect meal, macroalgae meal or single-cell protein. However, high costs and limited availability are still challenges that must be overcome for these novel alternative protein sources. Plant-based meals seem to be one of the most promising and viable alternative solutions, but a common problem arising from the use of plant ingredients is the presence of mycotoxins. The trend to replace marine-derived ingredients with plant meals is expected to continue (Figure 1). Mycotoxin management is therefore an important step to avoid performance losses and disease vulnerability.

Fig. 1. Percentage of fishmeal inclusion in aquaculture species. Data obtained from
Tacon and Metian, 2008.

What is deoxynivalenol?

Mycotoxins are toxic secondary metabolites produced by moulds and fungi, which can be produced on agricultural commodities before and/or after harvest, during transportation or storage. Mycotoxins are a significant problem worldwide, causing adverse health outcomes when consumed by humans and animals, and are responsible for significant global economic losses due to condemned agricultural products. Deoxynivalenol (DON) is one of more than 400 mycotoxins that have been identified so far. DON is produced by Fusarium fungi, which are generally produced in the field rather than under storage conditions. This means that DON is present on the plant-based raw materials used to produce aquafeeds, as mycotoxins commonly occur in plant materials and are not destroyed during most processing operations.

Deoxynivalenol and the iceberg principle

The toxic effects of DON, a mycotoxin commonly known as “vomitoxin” as it causes vomiting in livestock, are well described including clinical symptoms for land-farmed animals. However, despite increasing knowledge of the effects of DON in aquaculture species, very little is known about the clinical signs of DON ingestion. Normally, when DON is ingested, no known distinct subclinical signs of DON toxicoses (i.e. no distinct lesions/pathologies) are shown, except accentuated anorexia and the evident decrease in feed intake (also a characteristic effect in livestock). In a very small number of cases, as reported by Gonçalves et al. 2018, a reduction in fish length in relation to width, and a protruding anal papilla was observed (Figure 2). Interestingly, protruding anal papilla or haemor-rhages are also typical clinical signs in swine fed DON. However, in the case of trout, this clinical manifes-tation was only observed in a small number of animals (Gonçalves et al., 2018).

Fig. 2. Fish presenting protruding anal papilla after being fed 2,745 ± 330 μg/kg DON. Results presented in Gonçalves et al., In Press.

One of the main constraints to detecting the impact of DON in aquaculture species is the lack of DON-induced clinical symptoms. Although it is true that several published reports describe some clinical signs for the most common mycotoxins (Anater et al., 2016), most of these clinical signs are very general and can be attributed to any other pathology or challenge faced by the animals, for example, anti-nutrition factors or lectins in the diet (Hart et al., 2010). Furthermore, the presence of some minor clinical signs, such as protruding anal papilla, are highly variable.

The iceberg principle (Figure 3) illustrates the problem of DON ingestion for aquaculture species very well. With the exception of some minor visible effects (e.g., protruding anal papilla) and histological damage (only detected by euthanizing the animal and analyzing tissues slides), many other effects are below the surface, making them harder to associate with DON ingestion. These sub-clinical effects normally lead to a decrease in performance and increased disease vulnerability, indirectly leading to profit losses without being noticed.

Fig. 3 . The iceberg principle, showing that visible clinical manifestations (above the surface) are minimal, with most of the symptoms being sub-clinical (under the surface) and therefore hard to detect.

Diagnosing DON ingestion without clinical symptoms

In order to understand the reasons behind the lack of clinical manifestation, an experiment was setup to evaluate and elucidate the impact of DON on rainbow trout, by exploring new tools and evaluating new diagnostic factors, which may be used later by the industry as a standard to better diagnose DON intake in fish.

In the experiment, quadruplicate groups of 50 rainbow trout (Oncorhynchus mykiss), with a mean ± standard deviation (s.d.) initial body mass (IBM) of 2.52 ± 0.03 g, were fed one of the three experimental diets for 60 days (Control, 4,714 ± 566 μg/kg and 11,412 ± 1,141 μg/kg). An accentuated reduction in growth performance was observed after trout were fed DON, which was expected and previously described in many other studies (Hooft and Bureau, 2017; Hooft et al., 2011; Matejova et al., 2014). Along with many other results, it was observed that despite the accentuated anorexia, especially at the higher exposure level of DON (Figure 4), no macroscopic lesions were found (e.g. internal or external haemor-rhages, dermal and oral lesions, abnormal pigmentation or damage to fins). This confirms that diagnosing DON ingestion is extremely difficult at farm level, which can lead to severe economic losses.

The experiment authors (Gonçalves et al., 2018) observed that DON contamination seemed to affect essential amino acid digestibility. In the study, it was observed that DON affected trypsin, which consequently influenced the levels of insulin, which ultimately influenced amino acid uptake. The influence of DON on this pathway may directly influence the decrease in growth performance.

The decrease in feed intake was also elucidated in the present work. Adenylate cyclase-activating polypeptide (PACAP), a neuroendo-crine satiety regulator, seemed to be influenced by the ingestion of DON. PACAP plays an important and direct role in the regulation of feed intake, and its upregulation in trout fed DON provides a possible link to the observed reduction in feed intake. Moreover, PACAP greatly decreases the frequency of gut motility waves, which might also have an impact on nutrient absorption.

The most curious aspect of DON intake is the lack of symptoms, especially when compared to livestock species also affected by DON ingestion. In the same study (Gonçalves et al., 2018), it was shown for the first time in rainbow trout that DON is metabolised into DON-3-sulfate, which is less toxic than DON. The formation of DON-3-sulfate can help to explain the absence of major clinical signs in trout fed DON, as the exposure to DON is minimised.

Fig. 4. Visual differences in growth between Rainbow trout given the three dietary treatments (Control, 4,714 ± 566 and 11,412 ± 1,141 μg/kg, from bottom to top). Fish shown are examples of the growth difference found in the different experimental groups. No visible clinical signs were observed, except the accentuated anorexia in DON 11 (top). Results presented in Gonçalves et al., 2018.

Going one step further to help the industry

The ingestion of DON significantly decreases the activity of trypsin, which seems to have a direct influence on the levels of insulin, which ultimately influences amino acid uptake. Suppression of appetite due to DON ingestion, and the observed increased gene expression of PACAP might be defence mechanisms in order to decrease exposure to DON, therefore reducing its potential negative impacts. Moreover, the biotransformation of DON into DON-3-sulfate minimises exposure of the gastrointestinal tract to the potential toxicological effects of DON, also helping to explain the lack of symptoms in animals fed DON.

The discovery of DON-3-sulfate as a novel trout metabolite makes it a potential biomarker of DON exposure, helping farmers to better diagnose the ingestion of DON by simply collecting and analysing a sample of faeces.

References

Anater, A., Manyes, L., Meca, G., Ferrer, E., Luciano, F. B., Pimpão, C. T. & Font, G. 2016. Mycotoxins and their consequences in aquaculture: A review. Aquaculture. 451. 1-10.

Gonçalves, R. A., Menanteau-Ledouble, S., Schöller, M., Eder, A., Schmidt-Posthaus, H., Mackenzie, S. & El-Matbouli, M. 2018. Effects of deoxynivalenol exposure time and contamination levels on rainbow trout. Journal of the World Aquaculture Society. 0.

Gonçalves, R. A., Navarro-Guillén, C., Gilannejad, N., Dias, J., Schatzmayr, D., Bichl, G., Czabany, T., Moyano, F. J., Rema, P., Yúfera, M., Mackenzie, S. & Martínez-Rodríguez, G. 2018. Impact of deoxynivalenol on rainbow trout: Growth performance, digestibility, key gene expression regulation and metabolism. Aquaculture. 490.362-372.

Hart, S. D., Bharadwaj, A. S. & Brown, P. B. 2010. Soybean lectins and trypsin inhibitors, but not oligosaccharides or the interactions of factors, impact weight gain of rainbow trout (Oncorhynchus mykiss). Aquaculture. 306. 310-314.

Hooft, J. M. & Bureau, D. P. 2017. Evaluation of the efficacy of a commercial feed additive against the adverse effects of feed-borne deoxynivalenol (DON) on the performance of rainbow trout (Oncorhynchus mykiss). Aquacul-ture. 473. 237-245.

Hooft, J. M., Elmor, A., Ibraheem, E. H., Encarnação, P. & Bureau, D. P. 2011. Rainbow trout (Oncorhynchus mykiss) is extremely sensitive to the feed-borne Fusarium mycotoxin deoxyniva-lenol (DON). Aquaculture. 311. 224-232

Matejova, I., Modra, H., Blahova, J., Franc, A., Fictum, P., Sevcikova, M. & Svobodova, Z. 2014. The effect of mycotoxin deoxynivalenol on haematological and biochemical indicators and histopathological changes in rainbow trout (Oncorhynchus mykiss). Biomed Res Int. 2014. 310680.

Tacon, A. G. J. & Metian, M. 2008. Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquaculture. 285.