THE EFFECTS OF FEED-BORNE FUSARIUM MYCOTOXINS ON THE PERFORMANCE
AND HEALTH OF RAINBOW TROUT (ONCORHYNCHUS MYKISS)
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
JAMIE MARIE HOOFT
In partial fulfilment of requirements for the degree of
Master of Science
May, 2010
© Jamie M. Hooft, 2010 Library and Archives Bibliothèque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada
Your file Votre référence ISBN: 978-0-494-67487-1 Our file Notre référence ISBN: 978-0-494-67487-1
NOTICE: AVIS:
The author has granted a non- L'auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis.
¦+¦ Canada ABSTRACT
THE EFFECTS OF FEED-BORNE FUSARIUM MYCOTOXBVS ON THE PERFORMANCE AND HEALTH OF RAINBOW TROUT (ONCORHYNCHUSMTKISS)
Jamie Marie Hooft Advisor: University of Guelph, 2010 Professor D.P. Bureau
This thesis is an examination ofthe effects of low levels of naturally occurring
Fusarium mycotoxins, predominately deoxynivalenol, on the performance and health of rainbow trout. Fish performance parameters including weight gain, growth rate, feed
efficiency and nutrient utilization efficiency responded linearly or quadratically to low
levels of deoxynivalenol ranging from 0.3 (control) to 2.6 ppm. Additionally, significant differences in growth rate, feed efficiency and carcass crude protein content between fish
fed the diet containing 2.6 ppm and fish pair-fed the control diet were observed, suggesting that reduction of feed intake is not the sole mechanism of action of deoxynivalenol in rainbow trout. No significant differences in the apparent digestibility of crude protein and gross energy offish fed diets containing 0.3 (control) to 2.0 ppm deoxynivalenol were determined. However, histological examination indicated morphological changes of the livers of some fish fed diets containing 2.6 ppm DON. ACKNOWLEDGEMENTS
This thesis is the result of generous contributions from many people and organizations. I want to extend my sincere thanks to my advisor, Dr. Dominique Bureau, whose passion for research and dedication to his students are unquestionable. Dom, thank you for your guidance through every step of this process and for the opportunities you have given me. The expertise and guidance of my advisory committee members, Dr. Jim Atkinson and Dr. Trevor Smith, throughout this process have also been critical to the
successful completion of my research and thesis. I would like to thank Hakeem Elmor of Zagazig University, Egypt for his contribution to the histopathological examination of tissues during his time in the Fish Nutrition Research Laboratory and Dr. Pedro Encarnaçao for his advice and assistance over the course of the project. Thank you to Dr. Margaret Quinton, whose statistical . advice and help was critical in the interpretation of the results.
The funding of this project by Biomin was instrumental to the research presented in this thesis and is greatly appreciated. Additionally, the co-operation of research
scientists and staffai Biomin in Austria and Singapore is gratefully acknowledged. My appreciation and thanks to the staff and students ofthe Fish Nutrition Research Laboratory for their support, friendship, encouragement and help with my experiments. It has been my privilege and good fortune to work with all of you.
Thank you to my family and friends for your love and support during this process and always. To my parents, Jim and Pat Hooft, there are not enough words to thank you for everything you have given me and done for me. Thank you for teaching me by example that with hard work, perseverance and determination anything is achievable; for
i your constant reassurance; for supporting my decisions; for your wisdom and advice; for listening to countless stories about the trials and tribulations of graduate school; and
above all, for your unconditional love and support.
11 TABLE OF CONTENTS
1 - GENERAL INTRODUCTION 1 1.1 - Objectives 4 2 - LITERATURE REVIEW 6 2.1 - Introduction 6 2.2 - Mycotoxins .9 2.3 - Fungal species and mycotoxin production 10 2.4 - Factors affecting fungal growth and mycotoxin production 11 2.5 - Occurrence and global distribution of mycotoxins 14 2.6 - Fusarium mycotoxins 19 2.6.1 - Trichothecenes 19 2.6.2 - Zearalenone 34 2.6.3 - Fumonisins 37 2.7 - Conclusion 42 3 - THE EFFECTS OF FEED-BORNE FUSARIUM MYCOTOXINS ON THE PERFORMANCE AND HEALTH OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) 44 3.1 -Abstract 44 3.2 - Introduction 45 3.3 - Materials and Methods 48 3.3.1 - Fish and experimental conditions 48 3.3.2 - Experimental diets and feeding protocol 49 3.3.3 - Mycotoxin analysis 51 3.3.4 - Digestibility trial 53 3.3.5 - Chemical analysis 54 3.3.6 - Histopathological examination 54 3.3.7 - Calculations 56 3.3.8 - Statistical Analysis 57 3.4 -Results 58 3.4.1 - Growth performance 58 3.4.2 - Apparent digestibility 59 3.4.3 - Histological examination 60
iii 3.4.4 - Carcass composition 60 3.5 - Discussion 70 3.6 - Conclusion 78 4 - GENERAL DISCUSSION 80 5 - REFERENCES 88
IV LIST OF TABLES
Table 2.1 - Cost comparison offish meal and plant ingredients for inclusion in salmonid aquaculture feeds 7 Table 2.2 - Minimum, optimal and maximum temperatures and water activities required for the growth of common mycotoxin-producing fungal genera...... „ 14 Table 2.3 - The influence of temperature on the relative quantities of DON and ZON produced by F. graminearum 14 Table 2.4 - Occurrence and levels of mycotoxins in agricultural commodities according to geographical region 17 Table 2.5 - Occurrence and levels of mycotoxins according to commodity 18 Table 3.1 - Analyzed mycotoxin content of the three corn sources used to formulate the experimental diets 51 Table 3.2 - Comparison of experimental diets and dietary mycotoxin concentrations.... 52 Table 3.3 - Growth, feed intake, feed efficiency ratio and mortality of rainbow trout (initial average weight = 24.3 g/fish) fed the experimental diets for 56 days 61 Table 3.4 - Retained nitrogen, recovered energy, nitrogen retention efficiency and energy retention efficiency of rainbow trout (initial average weight - 24.3 g/fish) fed the experimental diets for 56 days 62 Table 3.5 - Apparent digestibility coefficients (ADC) for Diets 1, 3, 4 and 6 fed to rainbow trout (initial average weight = 8.5 g/fish) calculated over two collection periods (N=4 for each diet) 65 Table 3.6 - Chemical body composition of the whole carcass of rainbow trout fed the experimental diets for 56 days 69 Table 4.1 - Concentration of DON (ppm) in samples of corn gluten meal used as a feed ingredient of experimental diets for rainbow trout determined using ELISA 86 Table 4.2 - Comparison of the concentration of DON (ppm) in four samples determined using an ELISA kit and HPLC. 86
? LIST OF FIGURES
Figure 2.1 - Chemical structures of DON and T-2 toxin 20 Figure 2.2 - Weight gains of rainbow trout fed diets containing graded levels of DON for 4 weeks 31
Figure 2.3 - Chemical structures of ZON and 17ß-estradiol 34
Figure 2.4 - Chemical structures of FBi, sphinganine and sphingosine 38 Figure 3.1 - Growth curves of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from a naturally contaminated source of corn 63
Figure 3.2 - Growth curves of rainbow trout (initial average weight = 24.3 g/fish) fed a diet containing 2.6 ppm DON and a pair-feeding treatment of the control diet 63 Figure 3.3 - Weight gain of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from naturally contaminated corn 64
Figure 3.4 - Feed efficiency of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from naturally contaminated corn 64
Figure 3.5 - Liver of a rainbow trout fed Diet 1 (control, 0.3 ppm DON) showing normal hepatic and sinusoidal architecture 66 Figure 3.6 - Liver of a rainbow trout fed Diet 3 (1.4 ppm DON) showing congestion and subcapsular edema with a fibrinous network 66 Figure 3.7 - Liver of a rainbow trout fed Diet 5 (2.6 ppm DON) showing multifocal areas of fatty infiltration 67 Figure 3.8 - Liver of a rainbow trout fed Diet 5 (2.6 ppm DON) showing phenotypically altered hepatocytes; pyknosis and karyolysis 67 Figure 3.9 - Pancreas of a rainbow trout fed Diet 5 (2.6 ppm DON) showing hydropic degeneration in the islet of Langerhan 68 Figure 3.10 - Comparison of the sensitivity of rainbow trout from two different studies and starter pigs to DON 71
vi 1 - GENERAL INTRODUCTION
Feed represents the single largest production cost of an intensive aquaculture operation. Consequently, the development of more cost-effective feeds is a key objective
in fish nutrition (Adelizi et al., 1998). The high cost of these feeds is largely attributable to their high content of expensive feed ingredients, namely fish meal and fish oil.
Historically, fish meal has been used as the main protein source in salmonid feeds due to its excellent amino acid profile. However, in recent years, the rising cost offish meal,
mainly as a result of its increased demand, has created a significant need for more economical protein sources (FAO, 2008). Increased criticism surrounding the reliance on
wild pelagic fish stocks for the production of fish meal is also a growing concern to the aquaculture industry (Hardy, 1996; Naylor et al., 2009; Tacón and Metían, 2009).
Therefore, the evaluation of alternative protein sources to fish meal is highly important for the development of economically and environmentally sustainable salmonid
aquaculture production. Considerable progress in the replacement offish meal in salmonid feeds with more economical plant protein sources, including soybean meal and corn gluten meal, has been achieved in recent years (e.g. Cho et al., 1974; Alexis et al., 1985; Moyano et al.,
1992; Gomes et al., 1995; Kaushik et al., 1995; Carter and Hauler, 2000). The use of certain plant ingredients in salmonid feeds must, however, generally be limited for various reasons including high starch and fibre contents, specific nutrient deficiencies and/or the presence of antinutritional factors which may affect diet palatability and fish performance (Ketola, 1983; Hardy, 1996; Naylor et al., 2009). As such, many studies have focused on the characterization of these problems, the development of potential
1 solutions to these issues and the optimization of levels and/or combinations of plant ingredients which support good health and growth performance of salmonid fish (Francis et al., 2001). One additional issue arising from the increased inclusion of plant ingredients in salmonid feeds which has been largely underestimated in aquaculture is the potential presence and effect of mycotoxins; naturally occurring, potentially toxic secondary metabolites produced by filamentous fungi (D'Mello and Macdonald, 1997;
Whitlow and Hagler, 2002; Sudakin, 2003). Mycotoxins are found in many agricultural commodities commonly used as animal feedstuffs. An additional challenge in animal
nutrition is the accumulation or increased concentration of mycotoxins in processed plant ingredients (e.g. corn gluten meal, corn steepwater, DDGS) relative to the raw materials from which they are derived. Futhermore, mycotoxins are, in general, chemically and thermally stable, rendering them unsusceptible to typical animal feed manufacturing processes such as extrusion (CAST, 2003; Leung et al., 2006). Mycotoxins have
attracted worldwide attention because of the significant economic losses associated with their impact on human health, animal productivity and feed and agricultural industries. It
is estimated that 25 % of the world's crop production may be contaminated with mycotoxins and losses due to mycotoxins in animal industries have been estimated to be
as much as several hundred million dollars per annum (CAST, 2003). The major mycotoxin-producing fungal genera, both in terms of relevance to agricultural industries and research efforts, are Aspergillus, Pénicillium and Fusarium. Although there are several hundred known mycotoxins produced by these fungal species, those of most importance to animal health and production based on their occurrence and potential toxicity are anatoxin B], ochratoxin A, the trichothecenes (e.g. deoxynivalenol,
2 T-2 toxin), zearalenone and fumonisin Bi (Hussein and Brasel, 2001). Environmental conditions, primarily temperature and moisture, are critical determinants of which fungal species and subsequent mycotoxin(s) are produced (Whitlow and Hagler, 2002). Variable climatic conditions can lead to a high prevalence of certain mycotoxins in
different years and different geographical regions. For example, warm temperatures and frequent rainfall at the time of anthesis (flowering) were cited as major contributing
factors to a widespread DON epidemic in Ontario grains in 1996 (Schaafsma et al., 2001). On a global basis, the Fusarium mycotoxins are the most common and widely
distributed (Morgavi and Riley, 2007). A recent survey of a large number of samples of cereal grains and plant by-products from different geographical regions indicated a high percentage of samples containing detectable levels of the Fusarium mycotoxins, zearalenone (29 %), fumonisin Bi (52 %) and deoxynivalenol (57 %) (Rodrigues and
Griessler, 2009). The high prevalence and widespread occurrence of deoxynivalenol (DON) in many different commodities and regions has made it a particular concern in
animal production. Average concentrations of DON in samples of corn, corn gluten meal, soybean meal and DDGS were determined to be 0.8, 0.7, 0.6 and 1 .3 ppm respectively, while the corresponding maximum concentrations of DON found in these
commodities were 6.1, 3.1, 1.5 and 13.9 ppm (Rodrigues, 2008). Trichothecene mycotoxins, including DON, exert their toxicity in animals largely through the inhibition of protein synthesis at the ribosomal level. Generally, the most common symptoms resulting from consumption of DON-contaminated feeds in farm animals are reduced feed intake, feed refusal and/or vomiting, hence its colloquial name of vomitoxin. Other toxic effects of DON include diarrhea, intestinal inflammation,
3 gastrointestinal hemorrhage and either immunosuppression or immunostimulation depending on dose and duration of exposure (Ueno, 1984; Pestka and Smolinski, 2005; Pestka, 2007). Significant variations exist among terrestrial species with regard to sensitivity and response to DON-contaminated feeds. While ruminants and poultry are capable oftolerating high concentrations of DON without corresponding decreases in production, concentrations as low as 1 to 2 ppm have been shown to result in significant decreases in feed intake in swine (Forsyth et al., 1977). The impacts of DON on fish, especially salmonids, are not well researched or understood. To date, only one previous study has examined the effects of DON on the performance of rainbow trout. Significant decreases in growth, feed intake and feed efficiency were associated with increasing concentrations of dietary DON ranging from 0 to 13 ppm (Woodward et al., 1983). Although reductions in these parameters were noted at levels as low as 1 ppm DON, the response offish at these low concentrations was quite highly variable. Therefore, there is a critical need for more applicable and comprehensive studies examining the effects of low levels of DON that could potentially be present in aquaculture feeds on the health and performance of salmonid species.
1.1 - Objectives
The general objective of this study was to provide an accurate representation of the potential impact of DON on farmed salmonid fish. More specifically, this study was aimed at determining the effects of commercially realistic levels of DON from corn naturally contaminated with Fusarium mycotoxins on performance and health parameters of rainbow trout, namely growth, feed intake, feed efficiency, nutrient digestibility and
4 utilization and pathological changes of the liver, pancreas and intestine, as well as to differentiate between adverse effects of DON related to associated decreases in feed
intake or deleterious metabolic effects through the use of a pair-feeding treatment. It is hoped that the results obtained will provide a frame of reference for nutritionists, feed manufacturers and producers with regard to the acceptable level of DON contamination of salmonid aquaculture feeds.
5 2 - LITERATURE REVIEW
2.1 - Introduction
Aquaculture is currently the fastest growing food producing sector in the world. In 2006, the United Nations' Food and Agriculture Organization estimated the total production of aquatic animals and plants from aquaculture to be 52 million tonnes valued at US $ 79 billion, with freshwater finfish accounting for more than half of this output. Aquaculture continues to play an increasingly important role in satisfying the demand for human consumption offish and fishery products. The contribution of aquaculture to per capita fish available for human consumption increased from 14 percent in 1986 to 47 percent in 2006 and is expected to continue to rise (FAO, 2008). Like many animal production systems, feed quality is one of the most important issues in the aquaculture industry. Adequate nutrition and proper feeding are essential aspects in intensive fish culture. Animal growth is dependent on the quality and nutritional adequacy of the feed. Improper feeding, nutritional deficiencies, inefficient utilization of nutrients or poor quality feeds will greatly impair fish performance and health. The formulation and manufacturing of commercial feeds also requires many practical considerations, including ingredient availability and price, crucial aspects in maintaining profitability and economic sustainability (NRC, 1993). Significant increases in the cost offish meal, traditionally a major ingredient of salmonid feeds, have resulted in considerable efforts focused on the development of cost-effective aquaculture feeds through the use of more economical feedstuffs (FAO, 2008). Plant ingredients, several of which appear to be of good nutritional quality (i.e. are high in digestible protein) for
6 salmonid species, are considerably cheaper than fish meal on a cost per tonne of crude protein and cost per tonne of digestible protein basis (Table 2.1). Given that feed constitutes the largest expense in intensive fish farming, replacement offish meal with
more economical plant based alternatives could have a substantial impact on the
production costs of these operations (Adelizi et al., 1998).
Table 2.1 - Cost comparison offish meal and plant ingredients for inclusion in salmonid aquaculture feeds. _ . CP ADCCP Cost Cost ingredient ^ (%) (USD/tonne) (USD/tonne DP) Fish meal (herring) 68a 92* 1,350* 2,158 Corn gluten meal 60b 96a 537b 932 Soybean meal 48a 89a 379b 887 Wheat middlings 17c 92a 75b 480 DDGS 29^ 90^ 116^ 444 a Cho and Kaushik, 1990. ADC for rainbow trout. b USDA Market News for October 6, 2009. Average price for available states. c Cho et al., 1991. d Lim and Yildirim-Aksoy, 2008. e Cheng and Hardy, 2004. ADC for rainbow trout. f FAO Globefish, fish meal market report September 2009.
Successful incorporation of plant ingredients in salmonid aquaculture feeds which support optimal growth, feed efficiency and health requires careful consideration of the
quality ofthese ingredients and accurate assessment of their nutritive value. Generally, plant ingredients have characteristics which make them inferior to fish meal as components of salmonid feeds, the most routinely studied of which include high starch and fibre levels, limiting essential amino acids and antinutritional factors, such as protease inhibitors, lectins, phytic acid and saponins (Ketola, 1983; Encarnaçao et al.,
2006; Hardy, 1996; Francis et al., 2001). Additionally, of increasing concern, is the
7 recognition that changes in the formulation of salmonid feeds to include higher levels of plant ingredients have significantly increased the likelihood of exposing farmed fish to mycotoxins; naturally occurring, potentially toxic fungal metabolites commonly found in
many cereal grain and oilseed commodities (Sudakin, 2003; Spring and Fegan, 2005).
It is estimated that approximately 25 % of the world's crops are contaminated with mycotoxins, resulting in average yearly economic losses of $ 932 million in the United States alone. Annual mitigation costs and livestock losses resulting from contaminated feeds have been predicted to be as high as $ 466 million and $ 6 million,
respectively (CAST, 2003). Consumption of mycotoxin-contaminated feeds by animals results in a variety of symptoms ranging from decreased production efficiency to mortality. General, unspecific symptoms associated with mycotoxin exposure make diagnosis difficult. Further complications in the diagnosis of mycotoxicoses in farm
animals can be caused by synergistic effects resulting from the presence of multiple mycotoxins in feeds and by secondary symptoms resulting from opportunistic disease related to the suppression of the immune system. Additionally, sensitivity to mycotoxins varies greatly between species and is dependent on several factors which can modify the expression of toxicity including age, gender, nutritional and health status prior to exposure and environmental conditions (Whitlow and Hagler, 2002). Mycotoxins which are widely found in agricultural commodities and have the potential to adversely affect animal health and performance are produced mainly by Aspergillus, Pénicillium and Fusarium fungal species. Numerous fungal metabolites can be found in commodities destined for use as animal feedstuffs, however, those which occur with the greatest frequency and are associated with particularly undesirable
8 consequences in animal production are anatoxin Bi (AFBi), ochratoxin A (OTA), zearalenone (ZON), fumonisin Bi (FBi), and the trichothecenes, most notably deoxynivalenol (DON) and T-2 toxin. Surveys have indicated that worldwide the Fusarium mycotoxins are the most prevalent and widespread. On a global basis, the percentage of samples of cereal grains, oilseed meals and processed plant by-products containing detectable levels of DON, FBi and ZON were recently determined to be 57, 52 and 29 %, respectively (Rodrigues and Griessler, 2009). Based on the ubiquitous presence of the Fusarium mycotoxins in a variety of feedstuffs and geographical regions
and the increased risk of exposing salmonid fish to these mycotoxins due to increased inclusion of plant material in aquaculture feeds, the main focus of this review is to examine the effects of the trichothecenes, ZON and FBi on the performance and health of intensively cultured finfish species.
2.2 - Mycotoxins
Mycotoxins are naturally occurring, potentially highly toxic secondary metabolities produced primarily by the mycelial structure of filamentous fungi (Hussein and Brasel, 2001). They are a structurally diverse group of low molecular weight compounds which vary greatly in their chemistry, potential toxicity and mechanism of toxicity (Sudakin, 2003). Some fungi are capable of producing more than one mycotoxin and some mycotoxins may be produced by more than one fungal species (Hussein and Brasel, 2001). Fungal infection and mycotoxin contamination of agricultural crops and commodities is largely dependent on environmental factors and can occur at various
9 stages of production including pre-harvest, harvest or post-harvest during storage, transport, processing and feeding (Whitlow and Hagler, 2002). Relative to the number of mycotoxins known, few are considered ofprime importance to animal health and productivity. These include aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins (CAST, 2003). Consumption or exposure of
humans and animals to different mycotoxins causes a diverse variety of adverse symptoms referred to as mycotoxicoses (D'Mello and Macdonald, 1997). In general, mycotoxins exert their adverse effects on animals through four primary mechanisms: (1) reduction of feed intake or feed refusal, (2) alterations in the nutrient content of feed and/or in nutrient absorption and metabolism, (3) endocrinological effects and (4)
immunological effects (Whitlow and Hagler, 2002).
2.3 - Fungal species and mycotoxin production
The main species of mycotoxin producing fungi in commodities destined for the production of animal feeds are Aspergillus, Pénicillium and Fusarium (Sweeney and Dobson, 1998). The important mycotoxins produced by Aspergillus species include the
aflatoxins, ochrâtoxin A, sterigmatocystin and cyclopiazonic acid (Santin, 2005). The aflatoxins are a group of mycotoxins comprised primarily of aflatoxin Bi, B2, Gi and G2, with Bi usually being the aflatoxin found at the highest concentration in food and feed (Sweeney and Dobson, 1 998). Aflatoxin Bi and B2 are produced mainly by A. flavus, while A. parasiticus often produces all four of the primary aflatoxins (D'Mello and Macdonald, 1997). Pénicillium species produce a number of mycotoxins including patulin, citrinin and ochratoxin. Ochratoxins are a group of structurally similar
10 metabolites of which Ochratoxin A (OTA) is the major mycotoxin of interest. Ochratoxins are produced by both Pénicillium and Aspergillus species of fungi, namely
P. verrucosum in temperate climates and A. ochraceus in warm climates (Pitt and Leistner, 1991).
The Fusarium mycotoxins constitute one of the largest and most economically significant species of mycotoxin producing fungi worldwide (Placinta et al., 1999; Santin,
2005). The major Fusarium mycotoxins include trichothecenes, zearalenone, fumonisins and moniliformin (D'Mello and Macdonald, 1997). The trichothecenes are a diverse group of compounds, the most commonly studied of which are diacetoxyscirpenol (DAS), T-2 toxin and deoxynivalenol (DON). There are several species of toxigenic
Fusarium species capable of producing trichothecenes. F. sporotrichioides and F. poae produce mainly T-2 toxin and DAS, while four species, F. crookwellense, F. culmorum, F. sambucinum and F. graminearum, produce both DAS and DON (Desjardins et al.,
1993). Zearalenone is commonly produced by many of these same species including F. culmorum, F. sporotrichioides and F. graminearum and thus is often found to co-exist in agricultural commodities with the trichothecene mycotoxins, most notably DON. The fumonisins and moniliformin are produced primarily by F. verticillioides (synonymous with F. moniliforme) (D'Mello and Macdonald, 1 997; Placinta et al., 1 999).
2.4 - Factors affecting fungal growth and mycotoxin production
The types of fungal species and mycotoxins produced in agricultural crops and commodities are dependent on a wide variety of environmental, physical and biological factors (D'Mello and Macdonald, 1997). Fungal infection and subsequent mycotoxin
11 production can occur during various stages ofproduction including pre-harvest, harvest and post-harvest (Abramson, 1998; CAST, 2003). Thus, mycotoxin contamination of
animal feed ingredients is often considered to be an additive process, beginning in the field and continuing or increasing throughout the course of production (CAST, 2003).
In the field, variations in climatic conditions, most importantly temperature and moisture, interact to produce differential growth of fungal species. The timing of these
environmental factors in relation to plant development also appears to be critical to the growth of some fungal species and subsequent mycotoxin production. For example, the
incidence of Fusarium diseases is most affected by precipitation at the time of anthesis (Abramson, 1998). Damage to plants as a result of insects or birds can break the outer
seed coat of cereals and further facilitate infestation and colonization by mycotoxin- producing fungi. Additionally, field management practices including crop rotation, cultivation, irrigation and fertilization can greatly influence the pre-harvest mycotoxin contamination of crops (Coulombe, 1993; Munkvold, 2003). Most toxigenic fungi, such as F. graminearum, the principal fungal species responsible for Fusarium head blight of wheat, Gibberella ear rot of corn and the production of DON, survive in crop residue. Significant reductions in the DON content of wheat have been achieved by plowing crop residues under the soil surface (Dill-Macky and Jones, 2000). Conversely, average DON concentrations in wheat crops grown in fields previously containing corn were determined to be over two-fold higher compared to the levels of DON in wheat grown in rotation with soybeans (Schaafsma et al., 2001). Similarly, other practices including irrigation to alleviate drought stress and optimal levels of nitrogen fertilization have been associated with the réduction of anatoxin levels in corn (Munkvold, 2003).
12 Environmental conditions and management decisions are also critical variables in determining the potential for mycotoxin contamination of feedstuffs at, during and after harvest. Timing of harvest can affect the ultimate level of mycotoxin contamination. In general, earlier harvest minimizes moisture accumulation and damage from insects, resulting in lower concentrations of mycotoxins (Munkvold, 2003). During storage and transport, temperature, moisture content, humidity, physical damage to the grain or seed either as a result of mechanical injury during harvest or insects and rodents, oxygen
availability, pH and degree of inoculation determine the probability, extent and type of mycotoxin contamination (Marquardt, 1996; Moss, 1991). Heat and moisture content
during storage can be further increased by insect activity and by the metabolism of the fungi themselves. Ideally, grain temperature should be maintained between 1 to 4°C for the duration of storage in order to minimize fungal metabolism (Munkvold, 2003). Moisture that initiates the spoilage process may also come from rain seepage, the melting of snow during winter and condensation (Abramson, 1 998). Some generalizations with regard to the occurrence ofAspergillus, Pénicillium and Fusarium fungal species can be made based on the aforementioned factors, most notably temperature and moisture (Table 2.2). Mycotoxins produced by Aspergillus species are most often associated with high temperatures, while Pénicillium and
Fusarium mycotoxins are of particular importance in temperate regions of the world (Marquardt, 1996; Whitlow and Hagler, 2002). Likewise, Aspergillus and Pénicillium species are able to grow at lower moisture levels than their Fusarium counterparts. Consequentially, Aspergillus and Pénicillium species are typically classified as storage fungi, while Fusarium species, in contrast, are most commonly referred to as field fungi
13 (CAST, 2003). However, the optimum conditions for fungal growth may not necessarily coincide with those required for mycotoxin production (Moss, 1991). Similarly, the production of mycotoxins by the same fungal species may not occur optimally under identical conditions (Table 2.3).
Table 2.2 - Minimum, optimal and maximum temperatures and water activities required for the growth of common mycotoxin-producing fungal genera. ______Temperature (0C)* Water activity (aw)a P Min. Optimal Max. Min. Optimal Max. Aspergillus 15 35 44 0.72 0.90-0.95 Pénicillium 0 24-28 31 0.78-0.84 0.95-0.97 Fusarium - 25-28 - 0.90 0.98-0.99 a Adapted from Moss, 1 99 1 .
Table 2.3 - The influence of temperature on the relative quantities of DON and ZON produced by F. graminearum*. Temperature^) Mycotoxin concentration (p^prn) 19.5 6 58 25 149 120 ______28 367 98 a Adapted from Moss, 1 99 1 .
2.5 - Occurrence and global distribution of mycotoxins
While contamination is closely related to the factors affecting fungal growth and mycotoxin production, the occurrence and concentrations of specific mycotoxins in certain crops and geographic regions can be highly variable for a variety of reasons including annual variations in weather conditions and global trade of agricultural commodities (Coulombe, 1993; CAST, 2003). Despite the ubiquitous nature of many mycotoxins, surveys focused on the occurrence and concentrations ofAspergillus,
14 Pénicillium and Fusarium species of mycotoxins in different commodities from various geographic regions provide important information for mycotoxin risk assessment strategies of ingredients used in animal feeds. Recent summaries of mycotoxin
contamination according to geographical region and commodity are presented in Tables 2.5 and 2.6, respectively.
Anatoxins are generally found in areas with tropical and semi-tropical climates.
Commodities commonly contaminated with anatoxins include groundnuts, tree nuts, cottonseed, rice and corn (Yoshizawa, 1991; CAST, 2003). Widespread occurrence of anatoxins in cereal grains, ground-nut (peanut) meal and cottonseed meal has been found
in areas of Uganda, Brazil, Nigeria and India (Shotwell, 1991; Strange, 1991; Yoshizawa,
1991). Binder et al. (2007) reported high incidences of anatoxin Bi in samples from South and South East Asia, especially in peanut meal.
The highest reported incidences and levels of ochratoxin A have been in cereal grains such as barley, oats, wheat and corn produced in Northern European (e.g. United Kingdom, Denmark and Sweden) or Balkan (e.g. Yugoslavia) countries and in India (Shotwell, 1991; CAST, 2003). Surveys of U.S. barley, oats, wheat, corn and sorghum
revealed some ochratoxin A in all samples except sorghum, but at low incidences (0.5 %
for corn, 1 % for wheat, 2 % for oats and 14 % for barley) and low concentrations (< 0.2
ppm for all samples) (CAST, 2003). Despite the high prevalence of anatoxins and ochratoxins in many feedstuffs, the
Fusarium mycotoxins appear to pose a greater risk as contaminants of animal and fish feeds based on incidences and contamination levels. In particular, DON, ZON and FBi
are found in a variety of cereal grains and regions worldwide (Placinta et al., 1999).
15 Globally, the incidences of DON, ZON and FB] in samples analyzed between 2007 and
2008 were 54, 46 and 54 % respectively, compared to only 3 1 % for AFBi and 19 % for OTA (Rodrigues, 2008). Many Fusarium mycotoxins are frequently found in Canadian,
U.S. and European wheat and corn crops, especially during cool, wet growing and harvest seasons (CAST, 2003). Scott (1997) determined the incidence of DON in Ontario
corn samples between 1980 and 1995 to be 86 %, with contamination levels ranging from 0.02 to 4.1 ppm. Contamination of cereals and feeds with DON in tropical countries,
including South Africa and Egypt, has also been reported (Scott, 1989). The co-occurrence of DON with low levels of other trichothecenes mainly
nivalenol (NIV), 3-acetyl DON (3-ADON), 15-acetyl DON (15-ADON), T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS) and with ZON and FBi is a predominant concern
worldwide (Placinta et al., 1999; Schollenberger et al., 2006). Binder et al. (2007) and Rodrigues (2008) reported a high prevalence of DON, ZON and FBi in a variety of
commodities, particularly, corn, corn gluten meal and DDGS from several different regions (Table 2.5). In North Asia, the average levels of DON, ZON and FBi were 0.9, 0.4 and 1 .9 ppm respectively, while the corresponding maximum concentrations were
27.9, 4.2 and 21.5 ppm (Table 2.4). Similarly, in other regions including South and South East Asia, Oceania and Europe the average and maximum concentrations of DON,
ZON and FBi were high relative to the levels of AFBj and OTA (Binder et al., 2007;
Rodrigues, 2008; Rodrigues and Griesslef, 2009).
16 Table 2.4 - Occurrence and levels of mycotoxins in agricultural commodities according to geographical region. AFB OTA FB1 ZON DON North and South America8 Number of samples analyzed 48 9 48 48 48 Incidence (% positive) 6 11 63 33 50 Mean level (ppm) 0.2 0.002 1.1 1.1 0.9 Maximum level (ppm) 0.7 0.002 7.9 2.9 2.7 Europe Number of samples analyzed 73 53 19 238 297 Incidence (% positive) 10 36 84 15 66 Mean level (ppm) NDh 0.003 2.8 0.2 0.7 Maximum level ¿pm) 0.006 0.007 7.7 1.0 4.7 Africa0 Number of samples analyzed 140 7 135 125 135 Incidence (% positive) 10 36 84 15 84 Mean level (ppm) 0.1 0.01 1.2 0.07 1.2 Maximum level (ppm) 0.4 0.01 4.4 0.3 4.4 North Asia Number of samples analyzed 553 87 397 571 566 Incidence (% positive) 17 37 66 40 80 Mean level (ppm) 0.02 0.3 1.9 0.4 0.9 Maximum level (ppm) 0.3 1.6 21.5 4.2 27.9 South East Asia0 Number of samples analyzed 269 24 269 269 269 Incidence (% positive) 46 33 54 33 15 Mean level (ppm) 0.06 0.004 1.0 0.2 0.3 Maximum level (ppm) 1.0 0.01 10.8 4.0 4.0 South Asia Number of samples analyzed 23 7 20 23 23 Incidence (% positive) 74 100 45 22 22 Mean level (ppm) 0.1 0.02 0.4 0.05 0.2 Maximum level (ppm) 0.9 0.09 1.5 0.08 0.3 Australia and New Zealand8 Number of samples analyzed 50 N/A 49 49 49 Incidence (% positive) 2 N/A 10 29 18 Mean level (ppm) 0.005 N/A 1.5 2.9 0.4 Maximum level (ppm) 0.005 N/A 2.8 26.7 1.3 a'd"g Adapted from Rodrigues, 2008. Values based on samples collected between October 2006 and September 2007. b' c Adapted from Rodrigues and Griessler, 2009. Values based on samples collected between January and June 2009. hND = not detectable. Limits of detection (ppm): AFBi = 0.004; OTA = 0.002; FBi 0.1; ZON = 0.032; DON - 0.05.
17 Table 2.5 - Occurrence and levels of mycotoxins according to commodity". ______AFBi OTA FBi ZON DON Corn Number of samples analyzed 192 39 185 196 196 Incidence (% positive) 32 18 78 35 65 Mean level (ppm) 0.1 0.02 1.5 0.2 0.9 Maximum level (ppm) 1.0 0.09 13.0 1.7 27.9 Corn gluten meal Number of samples analyzed 16 2 17 16 17 Incidence (% positive) 63 50 94 81 94 Mean level (ppm) 0.1 0.008 5.7 1.9 1.2 Maximum level (ppm) 0.9 0.008 21.1 4.2 5.0 Soybean meal Number of samples analyzed 65 6 64 65 65 Incidence (% positive) 0 33 8 20 18 Mean level (ppm) NDb 0.007 2.6 0.07 0.4 Maximum level (ppm) ND 0.009 7.9 0.1 2.7 DDGSC Number of samples analyzed 44 1 43 44 44 Incidence (% positive) 14 0 88 77 80 Mean level (ppm) 0.02 0 1.0 0.4 2.7 Maximum level (ppm) 0.07 ND 4.1 2.5 24.2 Wheat/bran Number of samples analyzed 14 3 14 15 15 Incidence (% positive) 14 33 29 13 87 Mean level (ppm) 0.006 0.02 0.5 0.05 0.3 Maximum level (ppm) 0.006 0.02 0.6 0.05 0.5 Rice/bran Number of samples analyzed 32 4 32 32 32 Incidence (% positive) 19 0 34 34 6 Mean level (ppm) 0.01 ND 0.2 0.08 0.1 Maximum level (ppm) 0.04 ND 0.4 0.3 0.2 a Adapted from Rodrigues, 2008. Includes samples from North and South America, North Asia, South East Asia, South Asia and Australia and New Zealand collected between October 2006 and September 2007. bND = not detectable. Limits of detection (ppm): AFBi = 0.004; OTA = 0.002; FB1 = 0. 1 ; ZON = 0.032; DON = 0.05. c Dried distillers grains with solubles.
18 2.6 - Fusarium mycotoxins
2.6.1 - Trichothecenes
The trichothecenes are a large group of structurally related sesquiterpenoid metabolites produced by several species of toxigenic fungi, the most important being F. graminearum. All of the trichothecenes share a common tricyclic ring system, a double bond at C-9,10 and an epoxide at the C- 12, 13 position, which is responsible for their toxicological activity (Figure 1; Sudakin, 2003; Pestka and Smolinski, 2005). Trichothecenes are divided into four groups (type A, B, C and D) according to their chemical properties. Type B trichothecenes (e.g. deoxynivalenol and nivalenol) are distinguished by the presence of a carbonyl group at the C-8 position, while type A trichothecenes (e.g. T-2 toxin, HT-2 toxin and diacetoxyscirpenol) are either not oxidized, hydroxylated or esterified at that position (Ueno, 1984; Sudakin, 2003). Type
C trichothecenes which include crotocin and baccharin have an additional epoxide group at the C-7,8 or C-9,10 position. Type D trichothecenes (e.g. satratoxin and roridin) contain a macrocyclic ring between the C-4,15 positions (Sudakin, 2003). Ofthese compounds, deoxynivalenol (DON), also known as vomitoxin, is among the most commonly encountered in cereal grains throughout the world (Ueno, 1984).
Acute exposure of experimental animals to high doses oftrichothecenes has been shown to result in a variety of symptoms such as diarrhea, vomiting, intestinal inflammation and gastrointestinal hemorrhage (Ueno, 1984). At very high doses, these effects can be accompanied by circulatory shock, reduced cardiac output and death. Chronic exposure to trichothecenes produces anorexia, reduced weight gain, altered
19 nutritional efficiency, neuroendocrine changes and immune modulation (Rotter et al., 1996).
H3C
= CH3 6h,CH3 Ô OH CH2 J C-CH3 C—CH3
Figure 2.1 - Chemical structures of DON and T-2 toxin (Hussein and Brasel, 2001).
2.6.1.1 - Cellular and molecular effects of trichothecenes
The toxicity of trichothecenes is partially explained by their ability to disrupt eukaryotic protein synthesis at both the transcriptional and translational levels, particularly in highly proliferating cells and tissues (Kiessling, 1986). Inhibitory activity requires the presence of an intact C-9,10 double bond and the C- 12, 13 epoxide group (Wei and McLaughlin, 1974). Trichothecenes bind to the 60S subunit of ribosomes and interfere with peptidyl transferase activity, thereby inhibiting the initiation, elongation or termination step of protein synthesis (Ueno, 1984; Feinberg and McLaughlin, 1989). Structure-activity studies have revealed that trichothecenes with substituents at both C-3 and C-4 primarily inhibit polypeptide chain initiation (e.g. T-2 toxin), whereas those lacking one substituent at either site inhibit the elongation or termination step of protein synthesis (e.g. DON). A common feature of the later type of inhibitor is replacement of one of the substituents at C-3 or C-4 by a hydrogen (Ehrlich and Daigle, 1987; Rotter et al., 1996). The effect of trichothecenes on tissue protein synthesis has also been assessed
20 in vivo. Hepatic protein synthesis was significantly reduced in laying hens fed a diet containing 1 1.9 mg/kg DON and 1.1 mg/kg 15 -^acetyl DON (Chowdhury and Smith, 2004). Likewise, Dänicke et al. (2006) found that protein synthesis significantly decreased in the kidneys, spleen and ileum of pigs fed chronically or acutely with diets containing 5.7 mg/kg DON from naturally contaminated wheat. More recent studies have suggested that alterations in cell signaling, particularly at the level of mitogen-activated protein kinases (MAPKs), are critical to the toxicity of trichothecenes (Pestka and Smolinski, 2005). MAPKs are responsible for the modulation of physiological processes including cell growth, differentiation and apoptosis and are critical for signal transduction in the immune response. Trichothecenes and other translational inhibitors which bind to ribosomes can rapidly active MAPKs and induce apoptosis in a process known as the ribotoxic stress response (Iordanov et al., 1997; Laskin, 2002). Inhibitor and gene silencing studies have revealed that hematopoietic cell kinase (Hck) and double-stranded RNA-(dsRNA)-activated protein kinase (PKR) are important upstream mediators of DON-induced MAPK phosphorylation. The effect of subsequent activation of MAPKs on downstream signaling cascades is dependent on trichothecene dose and exposure frequency. Low dose trichothecene exposure upregulates the expression of cytokines, chemokines and proinflammatory genes resulting in immune stimulation. In contrast, high dose exposure promotes apoptosis of cells including leukocytes leading to immunosuppression (Pestka et al., 2004; Pestka,
2007). Trichothecenes have also been shown to inhibit nucleic acid synthesis (Ueno, 1991). However, inhibition of DNA and RNA synthesis has only been observed at levels
21 similar to or higher than the concentrations required for eliciting the inhibition of protein synthesis. Although the exact mechanism for the inhibition of DNA and RNA synthesis is not fully understood, it appears to be a secondary effect of the inhibition of protein synthesis or of the apoptotic effect of trichothecenes (Eriksen and Pettersson, 2004). In addition to these ribosomal-mediated effects, trichothecenes can act on cell membranes. Bunner and Morris (1988) found that at very low levels T-2 toxin alters various myoblast cell membrane functions such as calcium efflux, rubidium uptake or residual cellular lactate dehydrogenase activity. Liver lipid peroxidation was increased in rats fed a single oral dose of T-2 toxin (2 ppm) up to 24 hours post treatment, suggesting that exposure to T-2 toxin and other trichothecenes may result in the production of free radicals (Suneja et al., 1989). Further evidence of this was provided by the findings of Rizzo et al. (1994) who observed that dietary selenium, alpha-tocopherol and ascorbic acid had a protective effect against free radical-mediated lipid peroxidation induced by exposure to DON or T-2 toxin in rats.
2.6.1.2 - Effects of DON on brain neurochemistry Interest in the neurotoxicological effects of DON has been generated by observations that consumption of feeds contaminated with DON can alter feeding behaviour in rodents and pigs (Fitzpatrick, 1988; Prelusky and Trenholm, 1993). At low dietary concentrations DON reduces feed intake (anorexia), while at higher acute doses it is associated with vomiting (emesis). Although DON is considered to be one of the least potent trichothecenes, its anorectic and emetic effects are equal to or greater than those reported for more acutely toxic trichothecenes (e.g. T-2 toxin) (Rotter et al., 1996).
22 Several studies have demonstrated a possible correlation between DON-induced emesis and the serotoninergic system. Prelusky et al. (1992) found that acute intravenous (i.v.) administration of 0.25 mg/kg DON to barrows resulted in an initial increase in hypothalamic serotonin (5-hydroxytryptamine, 5-HT) one hour post-dosing. Similarly, feeding of diets naturally contaminated with DON, 15-ADON, ZON and fusaric acid (FA) to pigs elevated the hypothalamic 5-HIAA: 5-HT ratio (Swamy et al., 2002). In rats, oral dosing with 2.5 mg DON per kg body weight resulted in increased levels of 5- HT in several regions of the brain. Furthermore, Prelusky and Trenholm (1993) found that certain 5-HT3 receptor antagonists efficaciously prevented vomiting in pigs dosed intravenously (i.v.) or orally with DON, supporting the involvement of 5-HT in DON- induced emesis. The mechanism by which DON alters brain neurochemistry, primarily the concentration of 5-HT and subsequently produces an anorectic or emetic effect is not entirely understood. It is proposed that inhibition of protein synthesis in the liver and possibly in other tissues causes increased concentrations of free amino acids in the blood (hyperaminoacidemia). Tryptophan, the precursor of serotonin, appears to be of critical importance to the effects of DON on feeding behaviour. Elevated levels of tryptophan in the blood result in increased amounts of tryptophan in the brain. Consequently, serotonin synthesis is increased (Smith, 1992). Serotonin is known to be an important mediator of a variety of behaviours including sleep patterns, muscle coordination and feed intake (Leathwood, 1987). Stimulation of the chemoreceptor trigger zone (CTZ) in the medulla appears to mediate the emetic response associated with increased levels of serotonin (Hesketh and Gándara, 1991). DON was also found to inhibit small-intestinal motility in
23 rodents, mediated through 5-HT3 receptors in the gastrointestinal tract. Interestingly, gastric relaxation and/or delayed gastric emptying are important components of emesis and feed intake. Thus, in addition to its effects on the emetic regions of the brain, elevated concentrations of serotonin as a result of DON exposure may also act peripherally on the gastrointestinal tract to cause reduced feed intake and/or vomiting
(Fioramonti et al., 1993).
2.6.1.3 - Potential role of fusaric acid in trichothecene mycotoxicoses Fusaric (5-butylpicolinic) acid, a metabolite produced by F. verticillioides, has received particular attention due to its common occurrence in feedstuffs and its potential synergistic interaction with trichothecene mycotoxins, especially DON (Smith and MacDonald, 1991; Smith and Sousadias, 1993). Like DON, fusaric acid has been observed to alter brain neurochemistry and feeding behaviour in experimental animals
(Smith and MacDonald, 1991). It is a potent inhibitor of dopamine ß-hydroxylase, a key enzyme in the regulation of the synthesis of norepinephrine (Nagatsu et al., 1970). Additionally, fusaric acid can affect serotonin synthesis. However, in contrast to DON, fusaric acid does not alter tryptophan concentration, but rather acts as a tryptophan analog. Chemical similarity to tryptophan enables fusaric acid to compete with tryptophan for binding sites on blood albumin, thereby raising the levels of free tryptophan in the blood. Consequently, tryptophan levels and serotonin synthesis in the brain are increased (Chaouloff et al., 1986). Smith and MacDonald (1991) found behavioural changes including vomiting and lethargy associated with a trend toward increased concentrations of tryptophan, serotonin and 5 -HIAA in the hypothalamus of
24 pigs orally dosed with a toxic level (200 mg/kg of body weight) of fusaric acid. A linear depression in growth rate was also observed in starter pigs fed diets containing relatively constant amounts of DON (0.5, 2.2, 2.5 and 2.4 ppm) and increasing levels of fusaric acid ranging from 2.9 to 15.9 ppm (Smith et al., 1997).
2.6.1.4 - Effects of DON on terrestrial animals
Significant variations exist among terrestrial species with regard to sensitivity and response to DON-contaminated feeds. While swine can display symptoms of toxicity at
concentrations as low as 1 to 2 ppm DON, other species such as ruminants and poultry have been shown to tolerate much higher concentrations of DON (Pestka, 2007). Generally, the order of decreasing sensitivity of experimental animals to DON is considered to be swine > mice > rats > poultry ~ ruminants (Rotter et al., 1996).
The most common effects ofprolonged dietary exposure of animals to DON are decreased weight gain, feed intake and altered nutritional efficiency (Pestka and Smolinski, 2005). Studies with swine showed that reduced feed consumption and lower weight gain are the principal clinical effects following ingestion of DON in naturally contaminated feedstuffs. In growing pigs, reductions in feed intake have been observed at 1 to 2 ppm DON, whereas levels of 12 and 20 ppm led to complete feed refusal and vomiting respectively (Forsyth et al., 1977; Young et al., 1983; Abbas et al., 1986). Diets containing 2 and 4 ppm DON from naturally contaminated oats resulted in a dose-related decrease in weight gain of 25 kg pigs, while feed intake and feed efficiency were adversely affected by 4 ppm DON (Bergsjo et al., 1992). Conversely, DON at concentrations up to 8 ppm does not appear to affect the productivity ofpoultry
25 (Hamilton et al., 1985). Similarly, DON fed to dairy cows at 66 ppm for 5 days (Côté et al., 1986) or at 6 ppm for six weeks (Trenholm et al., 1985) did not result in reduced performance. In addition to the aforementioned effects on performance, exposure to dietary DON from naturally contaminated feedstuffs has also been associated with lesions of the gastrointestinal tract in swine (Smith et al., 2005) and hepatotoxicity in both rats and pigs (Sahu et al., 2008; Tiemann et al., 2006). Liver histopathology following a single intraperitoneal (i.p.) administration of 10 mg DON per kg body weight to adult male rates was consistent with early mild hepatotoxicity. Drochner et al. (2006) reported an increase in plasma aspartate aminotransferase activity suggesting liver damage in female piglets fed 0.3 to 1 .2 ppm DON for 8 weeks. Likewise, hepatotoxicity was also reported in pigs fed naturally contaminated feeds containing 0.2 to 9.6 ppm DON and low levels of ZON. Although there was no gross liver pathology in any group and limited changes in enzyme activities, histological and ultrastructural examination did provide evidence of liver dysfunction including loss of glycogen, increased hemosiderin, increased thickness of interlobular connective tissue, loss of ribosomes and increased fatty and autophagic vacuoles (Tiemann et al., 2006).
2.6.1.5 - Metabolism of DON in terrestrial animals
An understanding of the metabolism and toxicokinetics of DON is critical in explaining its effects on a particular species and in evaluating variations in sensitivity among different species. The transformation of DON via intestinal or ruminai microbial activity to de-epoxy DON (DOM-I), the principal product of DON detoxification in
26 animals, has been well-established by both in vitro and in vivo studies (Pestka, 2007). The de-epoxidation of DON to yield DOM-I results from the reduction of the C- 12, 13 epoxide group (which is responsible for the toxicity of DON) and consequential formation of a double bond at this position. The transformation of DON to DOM-I has been correlated with a loss of cytotoxicity, demonstrated using a cell culture assay (Kollarczik et al., 1994). DOM-I was first isolated from the urine and feces of rats orally dosed with DON (Yoshizawa et al., 1983). Transformation of DON to DOM-I has also been demonstrated to occur within the gastrointestinal tract ofpoultry, cattle and swine (Côté et al., 1986; He et al., 1992; Dänicke et al., 2004). He et al. (1992) found complete transformation of DON to DOM-I after incubation of DON with contents of the large intestines of chickens. The transformation rate was high in media containing 143 and 1,419 ppm DON relative to that containing 14 ppm DON, suggesting that elevated substrate concentrations stimulated de-epoxidation of DON by inducing greater microbial activity. Similarly, nearly complete transformation of DON was achieved after 24 hours of incubation of up to 10 ppm DON with rumen fluid from a cow (King et al., 1984). In pigs, DOM-I appeared in increasing proportions from the distal small intestine to the rectum relative to the sum of DON and DOM-I concentrations (Dänicke et al., 2004). Further evidence of this was provided by Kollarczik et al. (1994) who determined that DOM-I was only present when DON was incubated with suspensions of intestinal contents originating from the caecum, colon and rectum ofpigs and not with contents of the duodenum or jejunum. In contrast to the metabolism of many other xenobiotics, phase I reactions (oxidation, reduction and hydrolysis) of the liver do not appear to contribute to the
27 metabolism of DON in animals (Côté et al., 1987; Swanson and Corley, 1989). However, phase II hepatic conjugation of DON and DOM-I with glucuronic acid, which increases the solubility of these compounds and thus facilitates their excretion, has been noted in ruminants. The proportion of glucuronide conjugated DON to free DON was notably higher following oral administration of DON to sheep compared to i.v. dosing, suggesting conjugation of DON by the liver following absorption from the gastrointestinal tract (first-pass metabolism) (Prelusky et al., 1986). Furthermore, in dairy cattle fed a DON-contaminated diet, urinary recovery of glucuronide conjugated DOM-I indicated absorption and hepatic metabolism of DOM-I following transformation of DON by the rumen microflora (Côté et al., 1986). The sensitivity of swine to DON relative to poultry and ruminants can be explained, in part, by the rapid and efficient absorption, extensive systemic distribution and poor metabolism of DON in pigs. In pigs, plasma radioactivity levels following intragastic administration of DON reached peak concentrations within 15 to 30 minutes after dosing, indicating rapid absorption of the toxin from the gastrointestinal tract (Prelusky et al., 1988). Considering that the transformation of DON to DOM-I occurs mainly in the distal segments of the gastrointestinal tract, this rapid absorption (primarily in the stomach and proximal small intestine), would severely limit the detoxification of DON by the gut microflora (Dänicke et al., 2004). The elimination half-life of DON following intragastric and oral dosing was determined to be approximately 7.1 hours in pigs compared to only 2.1 hours in sheep (Prelusky et al., 1985; Prelusky et al., 1988). The half-life of a xenobiotic is a function of the intrinsic ability of the body to eliminate that compound as well as the extent of binding of the compound in blood and
28 extracellular (tissue) space. Binding of DON to plasma proteins or red blood cells is similar in pigs and sheep, thus greater tissue uptake ofthe toxin by pigs is the most probable reason for differences in elimination half-life of DON between these species. Interestingly, the calculated volume of distribution of DON in swine is about eight times higher than that of sheep (Prelusky et al., 1988). Furthermore, in sheep, metabolism appears to be the major process of elimination of DON, whereas, in pigs, 90 to 95 % of the excreted toxin was the unchanged parent compound, suggesting that very little of the
clearance was metabolic (Friend et al., 1986; Prelusky et al., 1988).
2.6.1.6 - Effects of DON on fish Research efforts examining the effects of DON on fish are very limited relative to other farmed species. To date, studies examining the effects of DON on farmed aquaculture species have been focused on rainbow trout (Salmo gairdneri) and channel catfish (Ictalurus punctatus). Additionally, these studies have been limited in their scope due to the use of high dietary concentrations of DON and/or purified DON which do not accurately represent what occurs in practice. Thus, it is difficult to make inferences from existing studies with regard to the potential effects of DON on farmed fish in a commercial setting. There is a strong need for studies examining the effects of commercially relevant concentrations of dietary DON in practical feeds formulated using naturally contaminated feedstuffs. In juvenile rainbow trout, Woodward et al. (1983) reported that diets containing graded levels of DON ranging from 1.0 to 12.9 ppm from artificially infected corn (also noted to contain 4 ppm ZON and a trace of 7-deoxyvomitoxin) caused progressively
29 greater reductions in live weight gains. Although the magnitude of the effects on weight gain, feed intake and feed efficiency at very low concentrations of DON (i.e. < 2 ppm) were difficult to determine, the authors reported that performance was significantly affected by as little as 1 ppm DON. The depression in weight gains ranged from 12 to 92
% of the control. Both feed intake and feed efficiency were adversely affected by the increasing levels of DON in the experimental diets. Complete feed refusal was observed in fish fed diets containing 20 ppm DON or greater. Fish that had previously been fed diets containing high levels of DON for 4 weeks resumed feeding when offered a non- contaminated diet. Conversely, fish fed the control diet during the first 4 weeks of the study showed signs of feed refusal when offered the diet containing 20 ppm DON. These results suggest that fish exposed to high concentrations of DON can recover when offered a non-contaminated diet and that reduced feed intake of rainbow trout associated with exposure to DON is transitory. Channel catfish appear to be much more resistant to dietary DON than rainbow trout. Manning (2005) observed no adverse effects on feed consumption, growth, hematocrit values or liver weights in juvenile channel catfish (initial weight of 6.8 g/fish) fed diets containing up to 10 ppm purified DON. In an additional trial, catfish of a similar size fed diets containing 15 and 17.5 ppm DON from naturally-contaminated wheat experienced reductions in growth and poorer feed efficiency compared to the control group. No adverse effects on performance were noted in fish fed dietary concentrations of DON up to 10 ppm and there was no effect on hematocrit values as a result of feeding any of the diets containing naturally contaminated wheat.
30 The variability in sensitivities to DON among several species offish has recently been attributed to species differences in the abilities of the intestinal microbes to transform DON to DOM-I . Digesta from nine species including the brown bullhead catfish (Ameiurus nebulosus), pink salmon {Oncorhynchus gorbuscha) and brown trout {Salmo trutta) were screened for their ability to metabolize DON. Only digesta from the brown bullhead catfish exhibited the ability to detoxify DON. In fact, complete transformation of DON to DOM-I by the microbial contents of this species occurred when it was incubated in full medium with DON at 1 5 0C for 96 hours (Guan et al.,
2009).
R' = 0.99
SP 15 H
0 4 6 8 10 12 14 Dietaiy DON Level (ppm)
Figure 2.2 - Weight gains of rainbow trout fed diets containing graded levels of DON for 4 weeks (adapted from Woodward et al., 1983).
2.6.1.7 - Effects of DAS and T-2 Toxin on fish
Negative effects of type A trichothecenes have also been noted in fish. Koski (1985) observed slight gastritis in 60 g rainbow trout fed a diet containing 0.1 ppm DAS
31 and 0.02 ppm DON. In contrast, intramuscular injection with a DAS concentration of 1 mg/kg body weight resulted in death of all experimental animals. Pathological examination revealed haematopoetic necrosis and necrosis of the epithelium, gastric glands and primary lamellae of the gills. Several studies have examined the response of different species offish to T-2 toxin. Diets containing greater than 2.5 ppm of chemically pure T-2 toxin significantly reduced weight gain and feed efficiency of rainbow trout fry. T-2 toxin also depressed hematocrit and hemoglobin concentrations and increased the storage of hepatic vitamin A in a dose-dependent manner at dietary levels of 5 ppm or higher. However, levels of T-2 toxin up to 15 ppm did not alter the function of the intestinal digestive proteases (trypsin and chymotrypsin) in young trout, nor did it impact the metabolizable energy or nitrogen digestibility ofthe diets in rainbow trout with an average weight of 300 g (Poston et al., 1982). In an earlier study, Marasas et al. (1969) determined that long term chronic exposure of rainbow trout to low doses ofpurified T-2 toxin did not improve their tolerance to subsequent acute exposure to higher doses of the toxin, suggesting that the metabolism of T-2 toxin by rainbow trout is unaffected by previous exposure or adaptation to the toxin. Despite a high tolerance to DON, channel catfish appear to be quite highly sensitive to T-2 toxin. Significant reductions in growth were observed in juvenile channel catfish fed diets containing 0.625, 1.25, 2.5 and 5 ppm purified T-2 toxin. Significantly poorer feed efficiency occurred only in fish fed the highest level of T-2 toxin, while hematocrit values were adversely affected by the inclusion of T-2 toxin at levels equal to or greater than 1 .25 ppm. In a concurrent study, three pair-fed treatments
32 offish received the control diet (0 ppm T-2 toxin) at the same daily intake levels as the fish fed the diets containing 1.25, 2.5 and 5.0 ppm T-2 toxin. The survival offish fed diets containing the two highest levels of T-2 toxin was significantly lower than that of the control group and their pair-fed counterparts. Additionally, pair-fed catfish had a significantly greater increase in body weight and significantly better feed efficiency than fish fed the diet containing 5 ppm T-2 toxin. Furthermore, diagnostic assessment revealed pathological changes in the head kidneys, stomach and intestines offish exposed to the three highest levels of toxin (Manning et al., 2003). In addition to its effects on performance and organ histology, exposure to T-2 toxin has-been shown to adversely affect the immune response in warm water species, more specifically channel catfish and Nile tilapia. Feeding of diets contaminated with low levels of T-2 toxin significantly increased the mortality of channel catfish challenged with Edwardsiella ictaluri compared to fish fed a non-contaminated diet (Manning et al., 2005). Previously, histological examination of the anterior kidneys of catfish fed diets containing T-2 toxin revealed a reduction in cellularity of areas of the anterior kidney normally associated with hematopoiesis (Manning et al., 2003). In addition to the production of erythrocytes, these cells are associated with the production of cells involved in the cell-mediated immune response such as macrophages and granulocytes. Thus, reductions in the number and activity of these cells related to consumption of T-2 toxin may reduce the disease resistance of catfish (Manning et al., 2005). In agreement with these findings, Gogal Jr. et al. (2000) reported increased leukocyte apoptosis in the head kidney ofNile tilapia exposed to T-2 toxin by intraperitoneal (i.p.) injection.
33 2.6.2 - Zearalenone Zearalenone (ZON) is a resorcyclic acid lactone, chemically described as 6-[10- hydroxy-6-oxo-trans-l-undecyl]-ß-resorcyclic acid, which is produced by several Fusarium species including F. graminearum and F. culmorum. Although it can be present in a variety of agricultural commodities including wheat, barley, oats and sorghum, it is typically found with a high prevalence and concentration in corn. ZON is unique among the Fusarium mycotoxins due to its ability to adversely affect reproductive processes in farm animals (Fink-Gremmels, 2008).
OH HO
HO HO
ZON 17ß-Estradiol
Figure 2.3 - Chemical structures of ZON and 17ß-estradiol (Arukwe et al., 1999).
2.6.2.1 - Mechanism of action of ZON
ZON and its metabolites are non-steroidal, estrogenic mycotoxins capable of competitively binding to estrogen receptors. The lactone ring of ZON is structurally similar to the aromatic ring of estradiol, allowing it to fit into the binding pocket of mammalian estrogen receptors (Figure 2.3). ZON passively crosses the cell membrane and binds to the cytoplasmic estrogen receptor. Once bound, the ZON-receptor complex is rapidly transferred to the nucleus, where it binds to estrogen-responsive elements, thereby activating gene transcription (Fink-Gremmels, 2008).
34 Kuiper et al. (1998) demonstrated that ZON interacts with both types of estrogen receptors, ERa and ERß. The binding affinities of ZON to ERa and ERß were found to be 7 and 15 % respectively, relative to the binding affinity of 17ß-estradiol. Previously, Kiang et al. (1978) demonstrated that ZON and some of its derivatives competed with 17ß-estradiol at estrogen cytosol receptor sites. They also noted that ZON derivatives had a longer duration of nuclear retention than 17ß-estradiol. Consequently, they proposed that ZON may have a longer biological half-life than 17ß-estradiol and may therefore stimulate a second wave of nuclear translation following replenishment of the cytosolic receptors.
2.6.2.2 - Effects of ZON on terrestrial animals
The effects of ZON on farm animals have been widely studied. In pigs, symptoms of hyperestrogenism generally appear when contamination of ZON in corn exceeds 1 ppm, but can occur at concentrations as low as 0. 1 ppm. Long-term exposure of sows during three reproductive cycles to feed concentrations as low as 0.18 ppm ZON resulted in a prolonged return to estrus and abortions. Other common adverse effects in sexually mature females related to the consumption of ZON-contaminated feed include decreased fertility, ovarian atrophy, reddening of the vulva and prolapsed uterus and rectum (Fink-Gremmels, 2008). Poultry and ruminants are less sensitive to ZON than swine. Chi et al. (1980) administered high levels of crystalline ZON, either orally or intramuscularly, at doses of 50, 200, 400 and 800 mg ZON per kg body weight per day to growing female White Leghorn chickens. Although the oviduct weight increased with increasing levels of ZON
35 in both groups, the estrogenic potency of ZON administered intramuscularly was determined to be only 1.4 % that of estradiol dipropionate. Similarly, in dairy heifers, a decrease in conception rate over three heat cycles occurred when 250 mg crystalline ZON was fed per day (Weaver et al., 1986). However, Coppock et al. (1990) observed frequent episodes of behavioural estrus which were not synchronized with the ovarian cycle following the accidental feeding of corn containing 1.5 ppm ZON and 1.0 ppm DON to dairy cows.
2.6.2.3 - Effects of ZON on fish
Little information is known about the effects of ZON on fish. Using an in vitro model, Arukwe et al. (1999) determined the ER binding affinities for a-zearalenol and ZON in rainbow trout to be 1/150 and 1/300 that of estradiol respectively. In a comparative study, Matthews et al. (2000) found a particularly high affinity for ZON and its metabolites towards the ER receptors of rainbow trout compared to receptors from humans, mice, chickens and frogs. Rainbow trout and Atlantic salmon receptors have been reported to have similar affinity for estrogenic compounds (Tollefsen et al., 2002). The in vivo effects of ZON on vitellogenesis and zonagenesis, two ER-mediated responses that are integral parts of fish oogenesis, have also been studied. Juvenile salmon were exposed to a single intraperitoneal (i.p.) injection of ZON, a-zearalenol and ß-zearalenol (each at 1 and 10 ppm) and compared to fish injected with 17ß-estradiol. A dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr- proteins) were observed 7 days after exposure to ZON and a-zearalenol. The estrogenic potencies of ZON and a-zearalenol were determined to be approximately 50 % compared
36 to that of estradiol. These findings indicate that in vivo, the estrogenicity of ZON may be a result of its metabolic product, a-zearalenol (Arukwe et al., 1999).
2.6.3 - Fumonisins
Fumonisins are a large group of Fusarium mycotoxins mainly produced by F. verticillioides and F. proliferatum and are found predominantly in corn and corn-based feeds (Ross et al., 1992). At least 28 different fumonisins belonging to three primary groups (A, B and C) have been characterized based on structural similarities. With regard to toxicity, the B series of fumonisins (fumonisin Bi, B2, B3 and B4) pose the greatest risk to animal health and productivity. Of these, fumonisin Bi (FBi) is the most widely studied and most prevalent, accounting for approximately 70 % of the total fumonisin content of feedstuffs (Krska et al., 2007).
2.6.3.1 - Mechanism of action of FBi One of the primary effects of FB] in biological systems is the disruption of sphingolipid metabolism. Although sphingolipids are minor components of cell membranes compared to phospholipids, they constitute one of the most chemically and functionally diverse classes of biomolecules and play critical roles in the regulation of cell survival and differentiation (Ghosh et al., 1997; Merrill et al., 2001). In experimental animals, most studies to date have demonstrated a correlation between FBi exposure and perturbations in the levels of sphinganine and sphingosine, sphingoid bases used in the formation of ceramide. Significant elevations in the ratio of free sphinganine to free sphingosine (Sa:So) after consumption of diets containing FBi have been noted in several
37 species including fish (Goel et al., 1994; Carlson et al., 2001), rats (Riley et al., 1994) and ponies (Wang et al., 1992). Alterations in the concentrations of sphinganine and sphingosine also result in the accumulation and/or depletion of other sphingolipid intermediates, the most physiologically active of which with regard to cell regulation are ceramide, a precursor of complex sphingolipids and sphingosine- 1 -phosphate, a phosphorylated product of sphingosine. Much of the evidence to date indicates that ceramide is an important mediator of apoptosis. Conversely, sphingosine- 1 -phosphate stimulates cell growth and differentiation and is an important mediator of calcium homeostasis (Ohanian and Ohanian, 2001). The mechanism by which FBi disrupts the de novo synthesis and turnover of complex sphingolipids is based on its structural similarity to sphinganine and sphingosine (Figure 2.4). Using radioactively labeled [14C] serine, Wang et al. (1991) determined that FBi disrupts the formation of ceramide from the sphingoid bases by inhibiting sphinganine (sphingosine) N-acyltransferase (ceramide synthase), a critical enzyme in the
CH-, FBi CH3 OR CH3 OH NH-, R = CCObCHiCOOH)CHjCOOH
CH2OH H3C Sphinganine NH-,
CH2OH HiC Sphingosine
Figure 2.4 - Chemical structures of FBi, sphinganine and sphingosine (Griessler and Encarnacäo, 2009).
38 metabolism of these lipids. Ceramide synthase recognizes the amino group of the fatty CoA domain of FBi which allows it to bind to the catalytic site of the enzyme (Riley et al., 2001). The absence of a hydroxy1 group at the C-I position of FBi may alter its orientation in the active site of ceramide synthase and preclude acylation or, if acylated, result in an inhibitory ceramide that cannot be removed by the addition of a sphingolipid head group (Merrill et al., 2001). As a result, the sphingoid bases cannot be converted to ceramide resulting in changes in the concentrations of sphinganine, sphingosine and other sphingolipid intermediates in the serum or tissues. This FBi -induced disruption of sphingolipid metabolism is associated with a diversity of animal diseases.
2.6.3.2 - Effects of FBx on terrestrial animals The effects of FBi have been extensively studied in laboratory animals, most notably rodents. Riley et al. (1994) observed increasingly severe pathological changes in the mitochondria from livers and kidneys of Sprague-Dawley rats exposed to 15, 50 and 150 ppm dietary FBi. Ultrastructural renal and hepatic lesions were also noted in male rats fed 15 ppm FBi. As well, chronic dietary exposure to FBi (> 50 ppm) was determined to be nephrocarcinogenic in male rats and hepatocarcinogenic in male rats and female mice (Gelderblom et al., 1991; Howard et al., 2001). Similarly, reproductive and developmental studies in rodents have revealed that FBi is teratogenic in mice, resulting in neural tube defects when administered intraperitoneally (i.p.) to pregnant
dams (Gelineau-van Waes et al., 2005; Voss et al., 2006). Among terrestrial domestic and livestock species, horses and pigs appear to be the most sensitive to FBi toxicity based on the manifestation of specific clinical conditions
39 following exposure. These diseases, equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE), refer to critical species-specific target organs; the brain in horses and the lungs in pigs. ELEM is generally characterized by liquefactive necrosis of the white matter of the brain, primarily in the cerebrum and is often associated with exposure to relatively low levels of FBi. The clinical course of ELEM is generally short with an acute onset of symptoms followed by death within hours or days. Decreased feed intake, depression, ataxia, blindness and hysteria have been reported. Hepatotoxicity has also been noted in horses at higher levels of FBi (Voss et al., 2007). Like equine species, swine are also sensitive to FBi toxicity. In pigs, the onset of PPE is related to the cardiovascular effects of FBi. Some studies have shown that the concentrations of the sphingoid bases affect the function of the L-type calcium channels in myocardial cells, thereby influencing cardiac contractility (Voss et al., 2007). More recently, sphingosine 1 -phosphate has also been implicated in the pathogenesis of cardiovascular alterations in vitro (Hsiao et al., 2005). In accordance with these findings, Smith et al. (1996) observed
decreases in the maximal rate of change of left ventricular pressure, heart rate, cardiac output and mean aortic pressure in pigs fed diets containing 20 ppm FBi. Cardiac distress or failure typically leads to severe pulmonary edema with death occurring within hours of respiratory distress. Non-lethal pulmonary edema has also been reported following longer term, lower dose exposures to FBi (Voss et al., 2007).
2.6.33 - Effects of FBi on fish Corn is a common ingredient in catfish and carp diets, thus many of the studies on FBi related to aquaculture have focused on these species. However, as with research on
40 other types of mycotoxins in fish, these efforts have not been extensive and in some cases conflicting results have been reported. Like other species, the susceptibility of channel catfish to FBi toxicity is dependent on age (Spring and Burel, 2008). A significant reduction in weight gain and feed intake and a significant increase in FCR (feed:gain) was observed in young channel catfish with an average initial weight of 1.5 g fed diets containing 20 ppm FB1. As expected, dose-dependent increases in the Sa:So ratio were observed with increasing levels of FBj, while hematocrit was significantly reduced in fish fed a diet containing 40 ppm FBi (Yildirim et al., 2000). The sensitivity of fingerling Nile tilapia towards FBi seems comparable to channel catfish (Tuan et al., 2003). Conversely, two-year old channel catfish weighing 3 1 g were able to tolerate up to 80 ppm dietary FBi over a 14-week period without experiencing a reduction in weight gain relative to the control group. In the same study, FBi was also found to impair the immune response offish. Antibody production by fish fed 20 or 80 ppm FBi and inoculated with killed Edwardsiella ictaluri cells was significantly lower after 14 days than antibody production by inoculated fish fed the control diet (Lumlertdacha et al., 1995; Lumlertdacha and Loveil, 1995). Additionally, microscopic hepatic lesions and significant reductions in hematocrit were observed in fish fed diets contaminated with 20 and 320 ppm FB] respectively (Lumlertdacha et al., 1995). In contrast to these findings, Brown et al. (1994) reported no histological evidence of toxicity in adult channel catfish fed a diet containing 313 ppm FBi for periods of up to 5 weeks. Channel catfish appear to be more tolerant to FBi than carp. Exposure of one- year old carp to feed contaminated with 0.5 and 5.0 mg FBi per kg body weight resulted in a loss of body weight and alterations of physiological parameters in target organs,
41 including increased activities of liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In another study with carp of a similar age, signs of toxicity were observed at dietary levels of 10 ppm FBi. Scattered lesions in the exocrine
and endocrine pancreas and inter-renal tissue, likely due to ischemia and/or increased endothelial permeability, were reported (Petrinec et al., 2004).
Given the evidence of carcinogenic effects in rodents, Carlson et al. (200 1 ) explored the potential of FBi as a carcinogen in rainbow trout, a species known to have a very low spontaneous tumor incidence. Although FBi was not a complete carcinogen in trout, it did promote AFBi initiated liver tumors, suggesting that the co-occurrence of these mycotoxins in aquaculture feeds is a potential concern with regard to the health and consequently the performance of cultured salmonid species.
2.7 - Conclusion
The steadily increasing price offish meal, once considered to be a staple ingredient of salmonid feeds, has resulted in considerable efforts related to the development of nutritionally adequate diets containing alternative feed ingredients, including a wide variety of ingredients of plant origin (e.g. Cho et al., 1974; Alexis et al., 1985; Moyano et al., 1992; Gomes et al., 1995; Kaushik et al., 1995; Carter and Hauler, 2000). Increased reliance on plant ingredients as components of aquaculture feeds and the high probability of mycotoxin contamination ofthese feedstuffs has significantly
increased the likelihood of feeding intensively cultured fish diets containing mycotoxins. Based on their high global prevalence and ubiquitous distribution, the Fusarium mycotoxins, DON, ZON and FBi, appear to pose the greatest risk to animal production
42 industries (Rodrigues, 2009). Exposure to the Fusarium mycotoxins can potentially result in a wide range of adverse effects on the performance and health of terrestrial livestock and fish and ultimately affect the profitability of these industries. Annual economic losses in plant and animal agriculture due to mycotoxins have been estimated to be as high as several hundred million dollars each year, highlighting the potential importance of this issue to aquaculture production (CAST, 2003). Properly assessing the risk mycotoxins pose to the aquaculture industry is a difficult task. Although many studies have focused on the effects oíFusarium mycotoxins on warm water species, comparatively few studies have addressed the effects of DON, ZON and FBi on cold water species such as rainbow trout and Atlanic salmon. In addition, studies using unrealistic concentrations of mycotoxins and artificial sources or a combination of both do not provide information that is applicable to a production setting. Thus, there is a critical need for research investigating the impact of feedstuffs naturally contaminated with commercially realistic levels of relevant Fusarium mycotoxins on fish, especially salmonid species.
43 3 - THE EFFECTS OF FEED-BORNE FUSARIUM MYCOTOXINS ON THE
PERFORMANCE AND HEALTH OF RAINBOW TROUT (ONCORHYNCHUS
MYKISS)
3.1 - Abstract
Deoxynivalenol (DON), a Fusarium mycotoxin, is a major contaminant of cereal grains worldwide which has previously been shown to adversely affect many livestock species. Increased use of plant protein sources in aquaculture feeds due to the high cost offish meal may result in an increased risk of exposing aquaculture species to DON. The effects of graded levels of DON from naturally contaminated sources of corn on the performance, health and apparent nutrient digestibility of rainbow trout were investigated. Feeding diets with increasing dietary levels of DON ranging from 0.3 to 2.6 ppm for eight weeks to rainbow trout (initial weight = 24 g/fish) resulted in significant linear or quadratic decreases in feed intake, weight gain, growth rate (expressed as thermal-unit growth coefficient, TGC), feed efficiency (gainrfeed), retained nitrogen (RN), recovered energy (RE), energy retention efficiency (ERE), and nitrogen retention efficiency (NRE). Fish pair-fed the control diet (0.3 ppm DON) had significantly higher TGC (PO.0 1 ), FE (PO.000 1 ) and CP carcass content (P<0.0 1 ) compared to their counterparts fed the diet containing 2.6 ppm DON. No significant differences (P>0.05) were observed in the apparent digestibility coefficients (ADC) of crude protein (CP) and gross energy (GE) offish fed diets containing 0.3 (control) to 2.0 ppm DON. In addition, some morphological changes of the liver were noted in samples obtained from fish fed the diet containing 2.6 ppm DON. These results suggest that, relative to other species,
44 rainbow trout are extremely sensitive to DON from plant ingredients naturally contaminated with Fusarium mycotoxins. More research is required to identify the specific mechanism(s) of toxicity of DON in rainbow trout. Keywords: Deoxynivalenol (DON), mycotoxin, feeding, fish
3.2 - Introduction
Mycotoxins are structurally diverse, potentially highly toxic secondary metabolites produced by filamentous fungi which frequently contaminate agricultural commodities used as animal feedstuffs (Hussein and Brasel, 2001; Sudakin, 2003). It is estimated that approximately 25 % of the world's crop production is contaminated to some extent with mycotoxins, resulting in substantial, widespread financial losses to agricultural and animal industries. Economic impacts associated with mycotoxins are the result of a number of contributing factors, including the condemnation of highly contaminated crops for human or animal consumption, costly mitigation efforts and mycotoxin screening programs and reduced production efficiency, impaired health or mortality of livestock exposed to contaminated feeds (CAST, 2003). Research characterizing the adverse effects of mycotoxins on the performance and health of animals has, in large part, been focused on terrestrial species (Rotter et al., 1996; D'Mello and Macdonald, 1997; Pestka, 2007). However, the high cost offish meal and consequential increase in the use of more economical plant protein and energy sources in commercial salmonid feeds has significantly increased the potential of exposing intensively cultured trout and salmon to low, but noñ-negligible, concentrations of mycotoxins (Hardy, 1996; Carter and Hauler, 2000; Naylor et al., 2009). Further
45 concerns regarding mycotoxins in aquaculture feeds stem from their inherent chemical and thermal stability, which render them unsusceptible to commonly used feed manufacturing techniques (e.g. extrusion) and from the typically elevated mycotoxin concentrations in processed plant ingredients (e.g. corn gluten meal), considered to be among the most nutritionally and economically suitable plant-based alternatives to fish meal (Hardy, 1996; CAST, 2003; Leung et al., 2006) The most relevant mycotoxins to animal production, based on their occurrence and toxicity, are primarily produced by three genera of fungi, namely Aspergillus, Pénicillium and Fusarium. Deoxynivalenol (DON), a Fusarium mycotoxin belonging to the trichothecene family, is one of the most frequently found mycotoxins in cereal grains worldwide. In recent surveys, nearly 60 % of samples analyzed were determined to contain detectable levels of DON, with average concentrations ranging from 0.2 to 2.7 ppm depending on commodity type and geographical region of origin (Rodrigues, 2008; Rodrigues and Griessler, 2009). The ubiquitous presence of DON, both in terms of the diversity of commodities and geographical regions affected, makes it a significant concern as a potential contaminant of animal feeds. Consumption of feedstuffs contaminated with DON has been shown to cause adverse effects in several species. Common clinical symptoms of DON toxicity include reduced growth and feed intake, vomiting, diarrhea, gastrointestinal hemorrhaging, inflammation and alteration of the immune response (Pestka and Smolinski, 2005; Rotter, 1996). At the cellular level, trichothecenes inhibit protein synthesis via binding to the 60S ribosomal subunit (Ueno, 1984). Additionally, DON acts on the serotoninergic system, resulting in its ability to mediate feeding behaviour and cause an emetic
46 response, hence its colloquial name of vomitoxin (Smith, 1992; Prelusky and Trenholm, 1993). The effects of DON on animals vary greatly depending on a variety of factors including nutritional and health status prior to exposure, dose and duration of exposure, age and species. Generally, the order of decreasing species sensitivity to DON is considered to be swine > mice > rats > poultry ~ ruminants (Rotter et al., 1996). Reduced feed intake of growing pigs has been attributed to dietary DON concentrations as low as 1 to 2 ppm, whereas, conversely, the performance of poultry and ruminants was not significantly affected by 8 and 6 ppm DON, respectively (Hamilton et al., 1985; Trenholm et al., 1985). In fish, there appear to be considerable differences in sensitivity to DON- contaminated feeds among species. Feeding of diets containing up to 10 ppm DON from either a purified source or naturally contaminated wheat had no effect on the feed consumption, growth, hematocrit values or liver weights ofjuvenile channel catfish (Manning, 2005). Conversely, Woodward et al. (1983) found significant reductions in the weight gains, feed intakes and feed efficiencies ofjuvenile rainbow trout fed diets containing graded levels of DON ranging from 1 to 12.9 ppm from an artificially infected source of corn. Fish appeared to be affected by low levels (< 3 ppm) of DON, however, the response of trout at these concentrations was quite highly variable and difficult to accurately determine. Considerably better growth rates of commercially produced rainbow trout are achieved nowadays and thus, feeds containing low levels of DON may have a more pronounced effect on fish performance parameters, specifically weight gain and feed efficiency. Given the apparent sensitivity of rainbow trout to DON and the relatively common occurrence of low levels of DON in many plant feedstuffs, there is a
47 need for more research regarding the effect of DON on salmonid species, notably the impact of realistic (i.e. practically relevant), commonly observed levels of DON. To our knowledge, the impact of DON originating from ingredients naturally contaminated with Fusarium mycotoxins on the performance and health of salmonids, including rainbow trout, has not been previously examined. The objectives of this study were, therefore, to investigate the effects of diets containing DON from naturally contaminated grains in a realistic range (i.e. concentrations that could be encountered in practice) on the growth, feed intake, feed efficiency, carcass composition and apparent digestibility of rainbow trout and to determine if any potentially adverse effects of DON on the abovementioned parameters were strictly the result of a commonly observed
DON-induced reduction in feed intake. Clinical and histological examinations of selected tissues, namely the liver, pancreas and intestine, were also studied in order to determine if consumption of feeds contaminated with DON could affect fish health.
3.3 - Materials and Methods
3.3.1 - Fish and experimental conditions Rainbow trout {Oncorhynchus mykiss) were obtained from the Alma Aquaculture Research Station (Elora, ON, Canada). Groups of twelve fish with an initial average weight of 24.3 g/fish were randomly distributed into 21 tanks, with three tanks per diet. Tank was considered the experimental unit. The fish were maintained in a flow-through system consisting of 60 L fiberglass tanks, individually aerated and supplied with well water at a rate of approximately 3 L/min. Water temperature was maintained at 1 1 .9 ± 0.7 ° C. Photoperiod was maintained at 12 h light: 12 h dark in a windowless laboratory.
48 The animals were kept in accordance with the guidelines of the Canadian Council on Animal Care (CCAC, 1 984) and the University of Guelph Animal Care Committee.
Fish were acclimated to the experimental conditions for two weeks prior to the start of the experiment. During this period, they were fed a commercial trout feed
(Martin Mills Inc., Elmira, Ontario, Canada) once daily. Throughout the eight-week
experimental period, feed intake was recorded on a weekly basis and fish were weighed every 28 days.
At the beginning of the experiment, a pooled sample of five fish was taken for determination of initial carcass composition. At the end of the experiment, five and three
fish per tank were randomly sampled for carcass composition analysis and histopathological examination, respectively. Prior to sampling, an external examination was conducted according to Noga (1996) in which the fish were observed in the tanks for any significant change in general behaviour, abnormal colouration and/or respiratory distress. Fish were killed by a lethal dose of tricaine methane sulfonate (200 mg/L water). Fish to be analyzed for carcass composition were cooked in an autoclave, ground into homogeneous slurry using a food processor, freeze-dried, reground and stored at — 200C until analysis.
3.3.2 - Experimental diets and feeding protocol Three batches of whole corn were obtained from the Arkell Feed Mill (University of Guelph, Guelph, ON, Canada) and ground to a fine, homogeneous consistency using a plant grinder. Corn samples were analyzed by mass spectrometry (MS) and high performance liquid chromatography (HPLC) (Veterinary Diagnostic Laboratory, North
49 Dakota State University, Fargo, ND, USA) and were found to contain 0.6, 10.5 and 20.9 ppm DON, respectively (Table 3.1). Therefore, the three corn sources were subsequently referred to as "clean", "moderately contaminated" and "highly contaminated", respectively. Traces of 1 5-acetyl DON (clean = 0.5 ppm; moderately contaminated = 1 .2 ppm; highly contaminated = 1 .5 ppm) and zearalenone (moderately contaminated = 3.0 ppm; highly contaminated = 2.1 ppm) were also determined to be present in these grains (Table 3.1). Eight isonitrogenous and isoenergetic experimental diets were formulated to contain low, graded levels of DON by replacement of clean corn with moderately contaminated or highly contaminated corn (Table 3.2). Diets 1, 2, 3, 4 and 5 were found to contain 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON respectively from the highly contaminated source of corn. Diet 6 was formulated to be similar to Diet 4 with regard to mycotoxin contamination and contained 1 .9 ppm DON from the moderately contaminated corn. Diet 7 was identical to Diet 1 (control, 0.3 ppm DON) and represented a pair-feeding treatment with Diet 5. Yttrium oxide (Sigma-Aldrich Inc., St. Louis, MO, USA) was incorporated into all diets at 100 ppm to serve as a digestibility indicator.
Diets were mixed using a Hobart mixer (Hobart Ltd., Don Mills, ON, Canada), steam pelleted using a laboratory pellet mill (California Pellet Mill Co., San Francisco, CA, USA), dried under forced-air at room temperature for 24 hours, sieved and stored at
4 ° C until used.
Fish receiving Diets 1 through 6 were hand-fed to satiety three times daily on weekdays and once daily on weekends throughout the duration of the experiment. Fish in
50 the pair-feeding treatment (Diet 7) were hand-fed to satiety during the first week of the trial. In subsequent weeks, these fish were fed Diet 7 (0.3 ppm, control) at the same amount as the tank with the lowest feed intake in the previous week. In most cases, this was a tank receiving the highest level of DON (Diet 5, 2.6 ppm DON).
3.3.3 - Mycotoxin analysis Samples of the three sources of corn and the eight experimental diets were analyzed for DON, 3-acetyl DON, 15-acetyl DON, nivalenol, T-2 toxin, iso T-2 toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpentriol, 15-acetoxyscirpentriol, neosolaniol, zearalenone, zearalenol, anatoxin Bi and fumonisin Bi by mass spectrometry (MS) and high performance liquid chromatography (HPLC) (Veterinary Diagnostic Laboratory, North Dakota State University, Fargo, ND, USA). The analyzed mycotoxin content (detected mycotoxins) of the corn sources and experimental diets are presented in Tables 3.1 and 3.2, respectively.
Table 3.1 - Analyzed mycotoxin content of the three corn sources used to formulate the experimental diets. ______Mycotoxin concentration (ppm) Corn source Dexoynivalenol(DON) 15-AcetylDON(15-ADON) Zearalenone(ZON)
Clean 0.6 ^ 0.5 NDa Moderately contaminated 10.5 1.2 3.0 Highly contaminated 20.9 L5 2.1 a Not detectable.
51 Table 3.2 - Comparison of experimental diets and dietary mycotoxin concentrations. Ingredient (g/100 g diet) Diet
Fish meal, herring 25.0 25.0 25.0 25.0 25.0 25.0 25.0 Poultry by-products meal 9.0 9.0 9.0 9.0 9.0 9.0 9.0 Feather meal 6.5 6.5 6.5 6.5 6.5 6.5 6.5 Blood meal, porcine, spray-dried 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Corn, clean3 25.0 21.1 17.2 13.2 9.3 2.8 25.0 Corn, moderately contaminatedb - - - - - 22.2 Corn, highly contaminated0 - 3.9 7.8 11.8 15.7 Soy protein concentrate 300 7.2 7.2 7.2 7.2 7.2 7.2 7.2 Biolys®, 52 % lysine 1.0 1.0 1.0 1.0 1.0 1.0 1.0 DL-methionine 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Vitamin premixd 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Mineral premix6 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Fish oil, herring 12.0 12.0 12.0 12.0 12.0 12.0 12.0 Soybean oil 6.0 6.0 6.0 6.0 6.0 6.0 6.0 Binder (lignosol) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Total 100 100 100 100 100 100 100 Analyzed composition (dry matter basis) Dry matter (%) 95.9 94.4 93.9 96.1 96.1 94.5 95.6 Crude protein (%) 43.0 43.3 43.0 42.8 42.6 44.0 44.0 Lipid (%) 25.0 24.7 24.7 24.1 24.7 24.3 24.7 Ash(%) 6.4 6.4 6.1 6.2 6.2 6.3 6.3 Gross energy (KJ/g) 24.8 24.7 24.8 24.5 24.9 24.7 24.7 Analyzed mycotoxin concentrations (ppm) Deoxynivalenol (DON) 0.3 0.8 1.4 2.0 2.6 1.9 0.3 15-acetylDON(15-ADON) 0.2 0.2 0.3 0.3 0.4 0.4 NDf Zearalenone (ZON) ND 0.2 0.3 0.4 0.4 0.6 ND a Contains: 0.6 ppm deoxynivalenol (DON); 0.5 ppm 15-acetyl DON(15-ADON). b Contains: 1 0.5 ppm DON; 1 .2 ppm 1 5-ADON; 3.0 ppm zearalenone (ZON). c Contains: 20.9 ppm DON; 1.5 ppm 1 5-ADON; 2.1 ppm ZON. d Provides per kg of diet: retinyle acetate (Vitamin A), 3750 IU; cholecalciferol (vitamin D), 3000 IU; dl-a-tocopherol-acetate (vitamin E), 75 IU; menadione Na-bisulfate (vitamin K), 1.5mg; cyanocobalamine (vitamin B 12), 0.03mg; ascorbic acid monophosphate, 75mg; D-biotin, 0.2 lmg; choline chloride, 1500mg; folic acid, 1.5mg; niacin, 15mg; D-calcium pantothenate, 30mg; pyridoxine-HCl (vitamin B6), 7.5mg; Riboflavin (vitamin B2), 9mg; thiamine-HCl, 1.5mg. e Provides per kg of diet: sodium chloride (NaCl, 39% Na, 61% Cl), 1200mg; ferrous sulphate (FeSO4-7H20, 20% Fe), 13mg; manganese sulphate (MnS04, 36% Mn), 32mg; zinc sulphate (ZnSO4-7H20, 40% Zn), 60mg; copper sulphate (CuSO4-5H20, 25% Cu), 7mg; potassium iodide (KI, 24% K, 76% I), 8mg; selenium (Na2Se03 , 45.66% Se), 0.3mg. fND = not detectable.
52 3.3.4 - Digestibility trial
In order to determine if DON had any effect on the apparent digestibility
coefficient (ADC) ofthe nutrients in the experimental diets, Diets 1, 3 and 4, containing 0.3 (control), 1 .4 and 2.0 ppm from the highly contaminated source of corn and Diet 6, containing 1 .9 ppm DON from the moderately contaminated corn, were used. Groups of
25 rainbow trout, obtained from the same source as fish used in the growth trial, with an initial average weight of 8.5 g/fish were randomly distributed into an aquatic system equipped with feces settling columns (Guelph system) as described by Cho et al. (1982). Water temperature was maintained atl3.4±0.7°C and the velocity of the water flow
was adjusted to minimize settling of the feces in the drainpipe and maximize recovery of the feces in the settling columns. As in the first trial, fish were maintained under artificial
light, with a photoperiod regime of 12 h light: 12 h dark. The experimental diets were each randomly allocated to two collections units.
Fish were acclimated to both the tanks and the experimental diets for four days prior to the first fecal collection. The fish were hand-fed to satiety three times daily. After the last daily meal, the drainpipe and settling column were brushed out to remove feed residues and feces from the system. One-third ofthe water in the tanks was drained to ensure that the cleaning procedure was complete. Before feeding of the first daily meal the following morning, the settled feces and surrounding water were gently withdrawn from the base of the settling column into a large centrifuge bottle. These feces were free of uneaten particles and considered to be a representative sample of the feces produced throughout the 24 h period. The feces were centrifuged at 4000 ? g for 20 min and the supernatant discarded. The resulting samples were stored at - 20 ° C until the end of the
53 trial. A total of four samples per diet (two samples per settling column) were collected over an eight-week period. Following the end of the experiment, the fecal samples were
freeze-dried, ground and stored at - 20 ° C until analysis.
3.3.5 - Chemical analysis Diets, ingredients and carcass samples were analyzed for dry matter (DM) and ash according to AOAC (1995), crude protein (CP, %N ? 6.25) by LECO (LECO Corp., St. Joseph, MI, USA), lipids with an Ankom XT20 fat analyzer (Ankom Technology, Macedón, NY, USA) using petroleum ether and gross energy (GE) content using a Parr 1271 automated bomb calorimeter (Parr Instruments, Moline, IL, USA). Fecal samples were analyzed for CP and GE by the same methods described above. Additionally, fecal • samples and diets used in the digestibility trial were analyzed for yttrium content by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) by Laboratory Services Division of the University of Guelph (Guelph, ON, Canada).
3.3.6 - Histopathological examination At the end of the growth trial, clinical examinations were performed. A postmortem internal examination of three fish per tank was done according to Lucky (1977) and Noga (1996). Any abnormalities of the intestine, liver, pancreas, spleen, kidney and musculature were noted. The fish were then dissected and the intestine was freed of visceral fat. Samples from the distal intestine just before the anus (1.5 to 2.0 cm in length) were carefully removed. Liver and pancreas samples were also taken for histological examination. Immediately following removal, the liver, intestine and
54 pancreas samples were fixed in buffered formaldehyde (10 %). The samples were later dehydrated, processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H+E) by the Animal Health Laboratory of the Ontario Veterinary College (University of Guelph, Guelph, ON, Canada). Following preparation, the samples were
scanned for histopathological changes.
55 3.3.7 - Calculations
Growth rate, expressed as thermal-unit growth coefficient (TGC), was calculated for each tank as: TGC = 100 ? [(FBW173 - IBW1/3) ? (sum T ? D)-1] Where: FBW=final body weight (g/fish); IBW=initial body weight (g/fish); sum TxD = sum degrees Celsius ? days. Feed efficiency (FE, gain:feed) was calculated for each tank as: FE = live body weight gain/dry feed intake Where: feed intake = total dry feed/number offish; live body weight gain = (FBW/final number offish) - (IBW/initial number offish); FBW = final body weight (g); IBW = initial body weight (g). Retained nitrogen (RN, g/fish) and recovered energy (RE, KJ/fish) were calculated for each tank as:
RN = (FBW ? N contentfínai) - (IBW ? N contentini,iai)
RE = (FBW ? GE contentful) - (IBW ? GE contentfinai) Where: FBW = final body weight (g/fish); IBW = initial body weight (g/fish); N contentfmai = nitrogen content (%) of the final carcass sample; N contentjnitiai = nitrogen content (%) of the initial carcass sample; GEfinai = gross energy (KJ/g) content of the final carcass sample; GEinitjai = gross energy (KJ/g) content ofthe initial carcass sample. Nitrogen retention efficiency (NRE) and energy retention efficiency (ERE) were calculated for each tank as a percentage of ingested nitrogen (IN): NRE (% IN) = [[(FBW ? N contentful) - (IBW ? N contentini,iai)]/rN] ? 100 ERE (% IE) = [[(FBW ? GE content^,) - (IBW ? GE contentini,iai)]/IE] ? 100
56 Where: FBW = final body weight (g/fish); IBW = initial body weight (g/fish); N contentfinai = nitrogen content (%) of the final carcass sample; N contentjnitiai = nitrogen content (%) of the initial carcass sample; GEfinai = gross energy (KJ/g) content of the final carcass sample; GEinjtiai = gross energy (KJ/g) content of the initial carcass sample; IN = ingested nitrogen (g/fish); IE = ingested energy (KJ/fish). The apparent digestibility coefficients (ADC) for crude protein (CP) and gross energy (GE) of the experimental diets were calculated as follows (Cho et al., 1982):
ADC = 1 - (F/D ? Di/Fi) Where: D = % nutrient (or KJ/g gross energy) of diet; F = % nutrient (or KJ/g gross energy) of feces; Di = % digestion indicator (yttrium) of diet; Fi = % digestion indicator (yttrium) of feces.
3.3.8 - Statistical Analysis
All data were analyzed as a completely randomized design using the GLM procedure of SAS (SAS version 9.1.3, SAS Institute Inc., Cary, NC, USA). Pre-planned contrasts were used to determine the nature of the responses exhibited by different parameters to graded levels of DON and to compare specific treatment means. Data on weight gain, thermal-unit growth coefficient (TGC), feed intake, feed efficiency (FE), mortality, retained nitrogen (RN), recovered energy (RE), nitrogen retention efficiency (NRE), energy retention efficiency (ERE), chemical body composition and histological scores of the distal intestine for Diets 1 to 5 (containing graded levels of DON from the same source of corn) were analyzed using linear and quadratic orthogonal polynomial contrasts. An additional pair of independent orthogonal contrasts were used to compare
57 the treatment means of the abovementioned parameters for Diet 4 (2.0 ppm DON from highly contaminated corn) vs. Diet 6 (1.9 ppm DON from moderately contaminated corn) and for Diet 5 (2.6 ppm DON) vs. Diet 7 (control, pair-feeding). The significance level using the Bonferroni adjustment for four contrasts was P<0.0125. Data on the apparent digestibility coefficients (ADC) were analyzed using the Tukey's honestly significant difference test with P<0.05 used to detect significant differences among the means.
3.4 - Results
3.4.1 - Growth performance Live weight gain, growth rate (TGC), feed intake, feed efficiency and mortality of fish fed the experimental diets are reported in Table 3.3, while the corresponding growth curves are illustrated in Figures 3.1 and 3.2. Weight gain and feed efficiency offish fed Diets 1, 2, 3, 4 and 5 are presented in Figures 3.3 and 3.4 respectively. Weight gain and growth rate (TGC) decreased linearly (PO.0001) with increasing dietary levels of DON ranging from 0.3 ppm (Diet 1) to 2.6 ppm (Diet 5). There was a significant effect of feeding diets containing increasing levels of DON on the linear (P<0.0001) and quadratic (P<0.0125) decrease of feed intake and feed efficiency. No observed differences between treatments were found with regard to behaviour, colouration or respiration during an external examination offish in the growth trial prior to sample collection. Mortality was not significantly affected (P>0.0125) by diets containing increasing concentrations of DON. No significant differences (P>0.0125) with regard to weight gain, TGC, feed intake, feed efficiency and mortality were observed in fish fed a diet containing 2.0 ppm DON from the highly contaminated source of corn (Diet 4) compared to fish fed a diet
58 containing a similar concentration of DON (1.9'ppm) from the moderately contaminated corn (Diet 6). Weight gain and feed intake offish receiving the highest dietary level of DON (Diet 5, 2.6 ppm) were not significantly different (P>0.0125) from their pair-fed counterparts (Diet 7). However, fish fed Diet 7 had significantly better TGC (P<0.01) and feed efficiency (P<0.0001) than fish fed Diet 5, indicating that the control diet resulted in an improved growth rate and better utilization of feed for growth when fed at the same feed intake level as a diet containing 2.6 ppm DON. Significant linear decreases in RN, RE, ERE (PO.0001) and NRE (PO.001) were associated with increasing dietary levels of DON (Table 3.4). In agreement with the findings on growth and feed efficiency, there were no significant differences (P>0.0125) in RN, RE, ERE or NRE between fish fed diets containing similar concentrations of DON from the two different sources of contaminated corn (Diet 4 and Diet 6). Fish in the pair- feeding treatment (Diet 7) had significantly higher RN (PO.01), NRE and ERE (PO.0001) than fish fed Diet 5.
3.4.2 - Apparent digestibility The ADC values for crude protein and gross energy of Diets 1, 3, 4 and 6 are presented in Table 3.5. Diets containing increasing, graded levels of DON from the same source of corn (Diets 1, 3 and 4) did not result in significantly different (P>0.05) ADC values for CP and GE. Similarly, no significant effects (P>0.05) of diets containing
similar levels of DON from different sources of corn (Diet 4 vs. Diet 6) on the ADC of
CP and GE were observed.
59 3.4.3 - Histological examination Post-mortem gross internal examinations revealed subcapsular hemorrhage of the liver in some of the fish fed the diets containing the three highest levels of DON, specifically Diet 3 (1 .4 ppm DON), Diet 4 (2.0 ppm DON) and Diet 5 (2.6 ppm DON). No substantial pathological changes in the distal intestine offish fed the experimental diets were observed, however, considerable morphological changes of the liver, particularly in fish fed Diet 5, were noted (Figures 3.5 to 3.8). Interestingly, a lesion in the pancreas of one fish fed the diet containing the highest concentration of DON (2.6 ppm) was discovered during the histological examination of the tissues (Figure 3.9).
3.4.4 - Carcass composition While the carcass contents of water, lipid, ash and GE were not affected (P>0.0125) in fish fed the low, graded concentrations of DON present in Diets 1 to 5, body protein content decreased linearly (PO.01) with increasing dietary levels of DON (Table 3.6). The carcass composition offish fed Diet 4 was not significantly different (P>0.0125) compared to that offish fed Diet 6. Comparison of the carcass composition offish fed Diet 5 (2.6 ppm DON) and Diet 7 (control, pair-feeding) revealed that the pair- fed fish had a significantly higher (PO.01) CP content than fish fed the contaminated diet. No significant differences (P>0.0125) in the water, ash, CP, lipid and GE contents of fish fed Diet 5 and fish in the pair-feeding treatment (Diet 7) were noted.
60 Table 3.3 - Growth, feed intake, feed efficiency ratio and mortality of rainbow trout (initial average weight = 24.3 g/fish) fed the experimental diets for 56 days. Diet (ppm)DON (g/fish)Gain TGC5 Feed(g/fish)intake (gain/feed)FE5 Mortality(%)
1 0.3 96.2 0.305 82.6 1.16 0 2 0.8 77.6 0.265 66.9 1.16 5 3 1.4 61.9 0.226 56.5 1.09 0 4 2.0 53.4 0.205 49.2 1.09 0 5 2.6 38.9 0.162 43.6 0.89 8
Significance0 Linear PO.0001 PO.0001 PO.0001 PO.0001 N.S.f Quadratic N.S. N.S. PO.0125 PO.0125 N.S.
6 1.9 62.1 0.228 55.5 1.12 0 7 0.3 47.9 0.191 36.9 1.30 8 Significance*1 Diet 4 vs. Diet 6 N.S. N.S. N.S. N.S. N.S. Diet 5 vs. Diet 7 N.S. PO.01 N.S. PO.0001 N.S. S.E.M.6 2/7 0.010 2? O03 4 a TGC ¦= thermal-unit growth coefficient. b FE = feed efficiency. c Significance = significance of the orthogonal linear and quadratic contrasts of dependent variables across Diets 1, 2, 3, 4 and 5. d Significance = significance of the orthogonal contrasts of dependent variables for Diet 4 vs. Diet 6 and Diet 5 vs. Diet 7. e S.E.M. - standard error mean. fN.S. = not statistically significant (P > 0.0125).
61 Table 3.4 - Retained nitrogen, recovered energy, nitrogen retention efficiency and energy retention efficiency of rainbow trout (initial average weight = 24.3 g/fish) fed the experimental diets for 56 days. / Diet DON RNa RED NREC ERE° (PPm) (g/fish) (KJ/fish) (%IN) (% IE) 1 0.3 2.4 772 41.9 37.6 2 0.8 1.9 615 41.3 37.1 3 1.4 1.5 483 38.7 34.4 4 2.0 1.2 439 37.1 36.5 5 2.6 0.9 293 31.2 27.1 Significance* Linear PO.0001 PO.0001 PO.0001 PO.001 Quadratic N.S.h N.S. N.S. N.S.
6 1.9 1.5 523 38.2 38.1 7 0.3 1.2 392 47.9 43.0
Significance Diet 4 vs. Diet 6 N.S. N.S. N.S. N.S. Diet 5 vs. Diet 7 PO.01 N.S. PO.0001 PO.0001 S.E.M.g 0.1 37 1.2 1.6 a RN = retained nitrogen. b RE = recovered energy. 0NRE (% IN) = nitrogen retention efficiency (% ingested nitrogen). d ERE (% IE) = energy retention efficiency (% ingested energy). e Significance = significance of the orthogonal linear and quadratic contrasts of dependent variables across Diets 1, 2, 3, 4 and 5. f Significance = significance of the orthogonal contrasts of dependent variables for Diet 4 vs. Diet 6 and Diet 5 vs. Diet 7. g S.E.M. = standard error mean. 'N.S. = not statistically significant (P > 0.0125).
62 140 -•-Diet 1 (0.3 ppm) 120 -\ -?-Diet 2 (0.8 ppm) -•-Diet 3 (1.4 ppm) ^ 100 JS ge -*-Diet 4 (2.0 ppm) 5S. 80 -*-Diet 5 (2.6 ppm) S 60
28 56 Day Figure 3.1 - Growth curves of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from a naturally contaminated source of corn (n=3 for each diet).
80 Diet 5 (2.6 ppm) 70 Diet 7 (0.3 ppm, pair-feeding) 60 JS I 50 ¦** 40 JS OS I 30 20 H
10
0 0 28 56 Day Figure 3.2 - Growth curves of rainbow trout (initial average weight = 24.3 g/fish) fed a diet containing 2.6 ppm DON and a pair-feeding treatment of the control diet (n=3 for each diet).
63 110? 100- .e ¡? 90- 80- 70- ce DX) 60- .SI0 50- 40- 30- 20- 0.0 0.5 1.0 1.5 2.0 2.5 3.0 DON (ppm) Figure 3.3 - Weight gain of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 0.3 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from naturally contaminated corn (standard error bars; n=3 for each diet).
1.25- .8 1.20- 1.15- 1.10- 1.05- c QJ 1.00- "3 0.95- 0.90- i 0.85- 0.80- 0.0 0.5 1.0 1.5 2.0 2.5 3.0 DON (ppm)
Figure 3.4 - Feed efficiency of rainbow trout (initial average weight = 24.3 g/fish) fed diets containing 03 (control), 0.8, 1.4, 2.0 and 2.6 ppm DON from naturally contaminated corn (standard error bars; n=3 for each diet).
64 Table 3.5 - Apparent digestibility coefficients (ADC) for Diets 1, 3, 4 and 6 fed to rainbow trout (initial average weight = 8.5 g/fish) calculated over two collection periods (n=4 for each diet). Diet DON ADC (ppm) CP" GEt- 1 0.3 84a 74a 3 1.4 86a 75a 4 2.0 85a 74a 6 L9 85^ 74^ S.E.M.C 0.01 0.02 H.S.D.d 427 836 a CP = crude protein. b GE = gross energy. c S.E.M. = standard error mean. dH.S.D. = Tukey's honestly significant difference (PO.05). Means in the same column sharing the same superscript are not significantly different.
65 Figure 3.5 - Liver of a rainbow trout fed Diet 1 (control, 0.3 ppm DON) showing normal hepatic and sinusoidal architecture (H&E stain; bar = 55.59 µ??).
lüi m
¡Êà H tuMJm
si a
b.59u
Figure 3.6 - Liver of a rainbow trout fed Diet 3 (1.4 ppm DON) showing congestion and subcapsular edema with a fibrinous network (arrow) (H&E stain; bar = 55.59 µ??).
66 mm%
%
fin È
*7"V* am W ì J Mi w : «* m m ® i h "WW t Figure 3.7 - Liver of a rainbow trout fed Diet § (2.6 ppm DON) showing multifocal areas of fatty infiltration (arrows) (H&E stain; bar = 50.71 µ??). - * . '. %fw I- *;* * · * ·' '* ·· * Figure 3.8 - Liver of a rainbow trout fed Diet 5 (2.6 ppm DON) showing phenotypically altered hepatocytes; pyknosis and karyoh/sis (arrow) (H&E stain; bar = 50.42 µ??). 67 Figure 3.9 - Pancreas of a rainbow trout fed Diet 5 (2.6 ppm DON) showing hydropic degeneration in the islet of Langerhan (arrow head) (H&E stain; bar : 50.71 um) 68 Table 3.6 - Chemical body composition of the whole carcass of rainbow trout fed the experimental diets for 56 days. ~lWet (ppm)DON H¡0(%) CP1(%) Lipid(%) Ash(%) (KJ/g)GE6 1 0.3 72.0 15.3 10.1 2.3 . 7.7 2 0.8 72.2 15.3 9.9 2.3 7.6 3 1.4 72.5 15.1 9.5 2.4 7.4 4 2.0 72.1 14.7 10.5 2.3 7.7 5 2.6 73.3 14.9 8.9 2.5 7.2 Significance0 Linear N.S.h PO.01 N.S. N.S. N.S. Quadratic N.S. N.S. N.S. N.S. N.S. 6 1.9 71.5 15.0 10.9 2.3 7.9 7 0.3 71.9 15.8 9.9 2.3 7.6 Significance Diet 4 vs. Diet 6 N.S. N.S. N.S. N.S. N.S. Diet 5 vs. Diet 7 N.S. PO.01 N.S. N.S. N.S. S.E.M.e 047 016 049 O1OJ 0.19 a CP = crude protein. b GE = gross energy. c Significance = significance ofthe orthogonal linear and quadratic contrasts of dependent variables across Diets 1, 2, 3, 4 and 5. d Significance = significance of the orthogonal contrasts of dependent variables for Diet 4 vs. Diet 6 and Diet 5 vs. Diet 7. e S.E.M. = standard error mean. h N.S. = not statistically significant (P > 0.0125). 69 3.5 - Discussion The experimental diets used in this study were formulated to be isonitrogenous and isoenergetic and to meet all known nutritional requirements of rainbow trout based on NRC (1993) recommendations. Relative to previous work, the diets contained low concentrations of DON which could potentially be encountered in practice. Findings of recent mycotoxin surveys of animal feed ingredients indicated that nearly 60 % of samples analyzed contained detectable levels of DON, with average concentrations between 0.2 and 2.7 ppm depending on year, commodity type and geographical region of origin (Rodrigues, 2008; Rodrigues and Griessler, 2009). The dietary levels of DON used in this study, which ranged from 0.3 ppm (control) to 2.6 ppm, are in close accordance with these findings and therefore likely represent the concentrations of DON encountered in feed ingredients used in the formulation of commercial fish feeds. Furthermore, the use of a naturally contaminated source of DON, as opposed to a purified or artificial form, further enhances the relevance of the findings of the present study to salmonid aquaculture production. Significant reductions in growth performance were closely correlated with increasing levels of DON, suggesting that rainbow trout in this study were highly sensitive to DON present in the experimental diets. Specifically, significant linear decreases in weight gain and growth rate (TGC) and significant quadratic decreases in feed intake and feed efficiency (gain:feed) were associated with graded, increasing levels of DON ranging from 0.3 ppm (Diet 1, control) to 2.6 ppm (Diet 5) (Table 3.3; Figures 3.1, 3.3 and 3.4). Previously, Woodward et al. (1983) reported that diets containing graded levels of DON ranging from 1.0 to 12.9 ppm from artificially contaminated corn 70 caused progressively greater reductions in weight gain, feed intake and feed efficiency of juvenile rainbow trout. However, the effects ofthe control diet and diets containing low levels of DON (i.e. 1 and 2 ppm) on fish performance were variable. Potential differences between the findings of Woodward et al. (1983) and those of the current study with regard to the sensitivity of rainbow trout to DON were assessed by adjusting DON intake in terms of metabolic body weight and days (Figure 3.10). This comparison indicates that relative to the findings of Woodward et al. (1983), rainbow trout in the current study were more sensitive to low dietary concentrations of DON based on weight gain. Similarly, based on the results of the current study and those of Smith et al. (1997), rainbow trout appear to be more sensitive to DON than starter pigs. Conversely, when compared, the findings of Woodward et al. (1983) and Smith et al. (1997) suggest that rainbow trout and immature pigs respond similarly to DON (Figure 3.10). •mm I a .S h 2 i» î ìt 1•PN 'S ¦33 Bi fe h 250 .9 A R2 = 0.9903 UD 1 tao £ R2 = 0.9855 I .000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 DON (mg/kg average MBW/day) ? Current study ¦ Woodward et al., 1 983 A Smith et al., 1 997 Figure 3.10 - Comparison of the sensitivity of rainbow trout from two different studies and starter pigs to DON. 71 The increased sensitivity of rainbow trout to DON observed in the current work relative to the findings of Woodward et al. (1983) may be due to several factors including differences in the nutritional and health status offish prior to exposure, differences in environmental conditions and differences in the source of DON (Rotter et al., 1996). Some experiments suggest that for a given DON intake, consequences are greater when a naturally contaminated ingredient is used than when purified DON is added to the diet due to the potential synergistic and/or additive effects that may result from the presence of multiple mycotoxins (Speijers and Speijers, 2004). In the present study, naturally contaminated corn containing primarily DON, but also low levels of 15-ADON (1.5 ppm) and ZON (2.1 ppm), was used as the source of contamination. In contrast, corn from an artificially contaminated crop was used as the dietary source of DON in the study by Woodward et al. (1983). However, as indicated in Table 3.2, Diets 1 to 5, which contained increasing levels of DON from 0.3 (control) to 2.6 ppm, respectively, contained only trace amounts of 15-ADON and ZON which did not exceed 0.4 ppm. Furthermore, it was noted that the corn used in the study by Woodward et al. (1983) also contained 4 ppm ZON and a trace of 7-deoxyvomitoxin in addition to DON, suggesting that differences in the analyzed mycotoxin content of the DON sources used in the two studies had little or no effect on the observed differences in the performance of the fish. It is highly likely that differences in growth rates (TGC) offish in the two studies contributed to differences in the observed sensitivities of the fish to DON. The calculated TGC of rainbow trout fed the control diet in the study by Woodward et al. (1983) was 0.209, compared to a TGC of 0.305 for fish fed the control diet in the current study. A higher growth rate offish in the current study compared to that of Woodward et al. 72 (1983) may have resulted in more pronounced effects of DON on the performance parameters of the fish. Results of the current study indicate that relative to pigs, rainbow trout are much more sensitive to dietary DON (Figure 3.10). Generally, among terrestrial animals, the order of decreasing species sensitivity to DON is considered to be swine > mice > rats > poultry ~ ruminants (Rotter et al., 1996). Reduced feed intake of growing pigs has been attributed to dietary DON concentrations as low as 1 to 2 ppm, whereas, conversely, the performance of poultry and ruminants was not significantly affected by 8 and 6 ppm DON, respectively (Hamilton et al., 1985; Trenholm et al., 1985). Considerable differences also exist among fish species with regard to the effects of DON and other trichothecene mycotoxins on performance. In contrast to the findings of the current study and that of Woodward et al. (1983), Manning et al. (2005) observed no significant differences in the feed consumption, growth, hematocrit values or liver weights of juvenile channel catfish with an initial average weight of 6.8 g/fish fed diets containing up to 10 ppm DON from either a purified or naturally contaminated source. Differences in species sensitivity to DON are likely primarily the result of differences in metabolism and pharmacokinetics. The transformation of DON via intestinal or ruminai microbial activity to de-epoxy DON (DOM-I), the principal product of DON detoxification in animals, is correlated with a loss of cytotoxicity and has been well documented in rats, poultry, cattle and swine (Kollarczik et al., 1994; Yoshizawa et al., 1983; Côté et al., 1986; He et al., 1992; Dänicke et al., 2004). The sensitivity of swine to DON relative to poultry and ruminants can be explained, in part, by the rapid and efficient absorption, extensive systemic distribution and poor metabolism of DON in pigs (Prelusky et al., 73 1985; Friend et al., 1986; Prelusky et al., 1988). Similarly, rapid absorption and poor metabolism may partially account for the greater sensitivity of rainbow trout to DON relative to other fish, particularly warm water species (e.g. channel catfish) and terrestrial animals. Interestingly, Guan et al. (2009) recently attributed the variability in sensitivity to DON among several species offish to a differential ability ofthe intestinal microbes to transform DON to its less toxic metabolite, DOM-I . The DON-related decrease in feed intake observed in this study has been extensively documented in terrestrial species and is closely related to the effect of DON on brain neurotransmitter concentrations (Fitzpatrick et al., 1988; Smith, 1992; Prelusky and Trenholm, 1993). Oral dosing or consumption of DON has been correlated with significantly elevated brain concentrations of serotonin (5-hydroxytryptamine, 5-HT) in rats and pigs (Fitzpatrick et al., 1988; Swamy et al., 2002). Although the response of brain monoamines to DON has not been determined in aquatic species, serotonin has previously been implicated as an appetite suppressor in fish (De Pedro et al., 1998; Lin et al., 2000; Höglund et al., 2007), suggesting that the effect of DON on regional brain neurochemistry contributed to the reduction in feed intake offish exposed to the increasing dietary concentrations of DON in Diets 1, 2, 3, 4 and 5. Proximate analysis of carcass composition revealed a significant linear decrease in body crude protein (CP) content with increasing levels of DON. Conversely, no significant changes in the moisture, lipid, ash or gross energy (GE) carcass concentrations were associated with the reduction in protein content (Table 3.6). Significant linear decreases in retained nitrogen and recovered energy and in the efficiency of protein and energy utilization were associated with increased levels of DON from 0.3 ppm (Diet 1, 74 control) to 2.6 ppm (Diet 5). It is well-established that the toxicity of trichothecenes is partially explained by their ability to disrupt eukaryotic protein synthesis, particularly in highly proliferating cells and tissues, via binding to the 60S ribosomal subunit and interfering with peptidyl transferase activity (Ueno, 1984; Feinberg and McLaughlin, 1989). Chowdhury and Smith (2004) found that the fractional rate of hepatic protein synthesis was significantly reduced in laying hens fed a diet containing 11.9 ppm DON and 1.1 ppm 15-ADON. Likewise, protein synthesis significantly decreased in the kidneys, spleen and ileum of pigs fed chronically or acutely with diets containing 5.7 ppm DON from naturally contaminated wheat (Dänicke et al., 2006). Reductions in the protein synthesis rates of specific organs and subsequent utilization of free amino acids for energy directed to metabolism may account for the linear decreases in protein and energy retention and crude protein body content without corresponding changes in other carcass composition parameters. A pair-feeding treatment was incorporated into the growth trial in order to account for effects of DON solely related to the reduction in feed intake, a confounding factor in many studies. Notably, significant differences in growth rate (TGC), feed efficiency, protein and energy utilization efficiencies and carcass crude protein content offish exposed to the highest level of DON (2.6 ppm) and fish pair-fed the control diet were observed (Tables 3.3, 3.4 and 3.6; Figure 3.2). These findings suggest that the effect of DON on the performance of rainbow trout is not simply the result of lowered feed intake associated-with its consumption, but rather, is related direct or indirect deleterious effects on nutrient metabolism. 75 An apparent digestibility trial was conducted in an attempt to gain further insight into the mechanism(s) by which DON impairs the performance of rainbow trout. No significant differences in the apparent digestibility coefficients (ADC) of crude protein and energy of rainbow trout fed diets containing 0.3 (Diet 1, control), 1.4 (Diet 3) or 2.0 ppm (Diet 4) were found, suggesting that low concentrations of dietary DON do not impair the digestibility of nutrients in rainbow trout (Table 3.5). These observations are similar to those of Dänicke et al. (2004) who reported rather small changes in nutrient digestibility in pigs fed diets containing DON despite substantial effects on growth. Histological examination indicated morphological changes in some of the livers offish fed diets containing low levels of DON. Notably, the liver of a fish fed Diet 3 (1.4 ppm) showed subcapsular edema, indicating an alteration in vascular pressure (Figure 3.6). Edema can be caused by a lack of plasma protein (Schick and Greenbaum, 1945) and may therefore be related to the inhibitory effect of DON on protein synthesis. Additionally, two liver samples obtained from fish fed Diet 5 (2.6 ppm DON) showed focal areas of severe fatty infiltration and phenotypical alterations ofthe hepatocytes, in particular enlarged nuclei (Figures 3.7 and 3.8, respectively). Histopathological evidence of liver dysfunction was noted in prepuberal gilts fed diets containing 0.2, 3.1, 6.1 and 9.6 ppm DON. A dose-dependent decrease in glycogen was associated with increasing dietary levels of DON, while the thickness of interlobular connective tissue septum in liver cells was significantly increased in pigs fed diets containing 6.1 and 9.6 ppm DON for 35 days. Hepatocytes ofpigs fed diets containing 6.1 and 9.6 ppm DON also developed extensive, smooth endoplasmic reticulum, exhibited loss of bound ribosomes and acquired an increased number of autophagic and fatty vacuoles. A rough 76 endoplasmic reticulum with intact ribosomes is necessary to produce lipoproteins, which are important in the transport of triglycerides. Thus, the loss of ribosomes may result in the accumulation of fat inside hepatocytes (Tiemann et al., 2006). Sahu et al. (2008) found necrosis of hepatocytes in the livers of adult male Sprague-Dawley rats following a single intraperitoneal injection of a relatively large dose of DON (10 mg/kg body weight). An interesting lesion in the pancreas of one fish fed the diet containing the highest level of DON (2.6 ppm) was observed (Figure 3.9). Pathological effects of DON on the pancreas may affect the production of insulin, which is essentially an anabolic hormone in fish. Insulin decreases the plasma amino nitrogen content and regulates individual amino acid and protein turnover in fish. Generally, it functions to increase the incorporation of amino acids into muscle and liver by enhancing protein synthesis, while suppressing gluconeogenesis from amino acids (Stoskopf, 1993). Therefore, the effect of DON on the pancreas offish offers another potential mechanism by which DON may affect protein synthesis and warrants further investigation. It is important to note that limitations exist in studying the effects of mycotoxins on animals, primarily with regard to the quantification of mycotoxins in feedstuffs. Mycotoxins are not evenly distributed within an ingredient batch, but rather occur in hot spots, making errors in analysis due to sampling highly probable. Different analytical techniques (e.g. ELISA and HPLC) vary in their detection limits and accuracy, which further complicates matters. The accuracy of mycotoxin detection often depends on the matrix (i.e. feedstuff) being analyzed. It is likely more difficult to accurately determine the mycotoxin profile of a complex matrix, such as fish feed (which contains a 77 combination of several ingredients), as compared to a simpler matrix, such as grain. Additionally, some mycotoxins of importance to animal performance, such as fusaric acid, known to be present in Canadian-produced grains and act synergistically with DON, are not routinely analyzed (Smith, 1992). Furthermore, masked mycotoxins (e.g. DON glucosides) may also have a potential role in animal mycotoxicoses. These glucose conjugates are not found using routine detection methods, but can undergo hydrolysis to precursor toxins in the digestive tract ofthe animal, resulting in detrimental effects (Berthiller et al., 2005; Sasanya et al., 2008). Zhou et al. (2007) reported an increase in DON concentrations of up to 88 % when barley samples were treated with trifiuoroacetic acid prior to analysis. Based on these issues and concerns and given the current state of mycotoxin analysis, it is necessary to consider the analyzed mycotoxin content of feedstuffs as only an approximation of the true hazard they pose to animal productivity, performance and health. 3.6 - Conclusion Low, graded levels of DON ranging from 0.3 to 2.6 ppm from naturally contaminated corn resulted in highly significant decreases in growth, feed intake, feed efficiency and protein and energy utilization of rainbow trout. Furthermore, significant differences in growth, feed efficiency and in protein and energy utilization between fish receiving a diet containing 2.6 ppm and fish pair-fed the control diet indicated that decreases in the performance of rainbow trout associated with the consumption of DON- contaminated feed is related to direct or indirect deleterious effects on the nutrient metabolism offish and not strictly the result of reductions in feed intake. This finding 78 was further supported by evidence of histopathological changes, particularly in the liver, of some fish fed diets containing 1.4 and 2.6 ppm DON. The findings of this study indicate that rainbow trout are extremely sensitive to low levels of DON from naturally contaminated ingredients. The basis ofthe high sensitivity of rainbow trout to DON (and the cause of pathological changes induced by low levels of DON) remains to be identified. Furthermore, the highly linear decrease in weight gain ofthe fish exposed to the range of low DON concentrations tested appears to suggest that any degree of DON contamination of salmonid feeds may have the potential to result in depression of growth performance and significant economic impacts. Consequently, risk management strategies for DON and other naturally occurring feed- borne mycotoxins could become critical as the inclusion of plant protein sources in aquaculture feeds continues to increase. 79 4 - GENERAL DISCUSSION Increased utilization of plant ingredients as components of aquaculture feeds has markedly increased the potential for exposing farmed fish to naturally occurring mycotoxins. The Fusarium mycotoxin, deoxynivalenol (DON), is a particular concern in animal agriculture due to its ubiquitous presence and high prevalence in feedstuffs worldwide and its deleterious effects on animal performance and productivity (D'Mello and Macdonald, 1997). Despite substantial research efforts devoted to characterizing the effects of DON on terrestrial farmed species, few studies have examined the impact of DON on farmed fish. Woodward et al. (1983) previously determined the effect of diets containing 0 to 12.9 ppm DON from an artificially infected source of corn on the weight gain, feed intake and feed efficiency of rainbow trout. However, according to the findings of recent mycotoxin surveys of various agricultural commodities from different geographical regions, the range of DON concentrations used in the study by Woodward et al. (1983) are likely much higher than those encountered in finished aquaculture feeds (Rodrigues, 2008; Rodrigues and Griessler, 2009). Furthermore, the response offish to low levels of DON (i.e. 0 to 3 ppm) was variable in the aforementioned study. Therefore, given the limitations ofthe existing work, the objective of this study was to determine the effect of low, commercially representative, levels of DON on growth parameters, apparent nutrient digestibility and health (assessed by histological examination) of rainbow trout. The findings of the current study suggest that rainbow trout are extremely sensitive to low dietary concentrations of DON from naturally contaminated corn. Diets containing graded levels of DON ranging from 0.3 (control) to 2.6 ppm resulted in 80 significant reductions in weight gain, growth rate, feed intake, feed efficiency, body crude protein content, retained nitrogen, recovered energy and nitrogen and energy utilization efficiency of rainbow trout fingerlings. Significant differences in growth rate, feed efficiency, carcass crude protein content and nitrogen and energy utilization efficiency were also observed between fish fed the diet containing 2.6 ppm DON and fish pair-fed the control diet, suggesting that the decrease in performance of rainbow trout fed diets containing DON was not solely related to lowered feed intake, a confounding factor in many studies including that of Woodward et al. (1983). Rather, the effects of DON on fish performance were likely related, at least in part, to direct or indirect deleterious effects of DON on nutrient metabolism. Results of the digestibility trial indicated that low levels of DON do not affect the apparent digestibility of crude protein and energy in rainbow trout. It was therefore determined that reductions in nutrient digestibility did not contribute to the observed decreases in weight gain, growth rate and feed efficiency correlated with increasing levels of DON. It should be emphasized that the histopathological examination of tissues was not a major focus of this project and thus, samples were not scored and statistically analyzed. However, morphological changes of the livers offish fed diets containing 1.4 and 2.6 ppm DON were observed. Similar findings were described by Tiemann et al. (2006) who observed dose-dependent histopathological evidence of liver dysfunction in prepuberal gilts fed diets containing 0.2 to 9.6 ppm DON. Notably, hepatocytes of pigs fed diets containing 6.1 and 9.6 ppm DON developed extensive, smooth endoplasmic reticulum, exhibited loss of bound ribosomes and acquired an increased number of autophagic and fatty vacuoles. Fatty infiltration was also noted in the liver of a fish fed the diet containing 2.6 ppm DON. It is 81 well-established that DON inhibits protein synthesis via binding to the 60S ribosomal subunit (Ueno, 1984; Feinberg and McLaughlin, 1989). Tiemann et al. (2006) suggested that the accumulation of fatty vacuoles was related to the loss of intact ribosomes, which play important roles in the regulation of triglyceride metabolism. Additionally, in the present study, subcapsular edema was noted in the liver of a fish fed the diet containing 1 .4 ppm DON. A lack of plasma protein has been previously cited as a potential cause of edema, which, given the inhibitory effect of DON on protein synethesis, would seem to be a logical explanation (Schick and Greenbaum, 1945). These preliminary findings warrant further, in depth investigation regarding the mechanism of action of DON in fish. The use of naturally contaminated grain as a mycotoxin source is thought to improve the applicability of the findings to commercial production since mycotoxins are rarely found in isolation within any given ingredient. However, the presence of multiple mycotoxins in the feed may result in synergistic or additive effects, increasing the degree of adverse impacts on the performance and/or health ofthe animal (Speyers and Speijers, 2004). The experimental diets used in the current study contained traces of 15-ADON and ZON in addition to the increasing, low levels of DON, meaning that, in theory, some of the effects of the diets on the performance offish could have resulted from interactions of the detected mycotoxins. However, the concentrations of 15-ADON and ZON were minimal (< 0.4 ppm) and fairly constant across the experimental diets relative to the gradually increasing level of DON. The presence of ZON was likely inconsequential as toxicological synergism between DON and ZON has not been previously demonstrated in swine or mice (Côté et al., 1985; Forseil et al., 1986). 82 The presence of other mycotoxins in experimental and commercial feeds cannot be discounted. Current mycotoxin analysis/screening protocols do not cover all known mycotoxin and their metabolites. For example, the fusaric acid content of the experimental diets was not analyzed in the current study, nor was it addressed in the former study by Woodward et al. (1983). DON and fusaric acid have been demonstrated to elevate the brain serotonin concentration in pigs (Smith and MacDonald, 1991; Prelusky, 1993), albeit through different mechanisms (Chaouloff et al., 1986; Cavan et al., 1988). Smith et al. (1997) attributed decreases in weight gain of starter pigs to a toxicological synergism between DON and fusaric acid. Similarly, conjugates of DON, commonly referred to as masked mycotoxins, are not routinely analyzed. DON glucosides, products of DON metabolism in plants, are not detected in routine analysis, but may ultimately be hydrolyzed to the parent toxin in the digestive tract of the animal. Berthiller et al. (2005) found that DON-3-glucoside can be produced in significant amounts in cereals artificially and naturally contaminated with DON. An increase in the detected DON concentration of up to 88 % after treatment of barley samples with trifluoroacetic acid was noted by Zhou et al. (2007). Therefore, low detected levels of the parent compound (i.e. DON) may sometimes be misleading and underestimate the potential hazard a given mycotoxin poses to animal health and productivity. Given the high sensitivity of rainbow trout to DON in the current study, as well as the potential for co-contamination of aquaculture feeds with multiple mycotoxins, proper procedures must be established to minimize the risk of exposing cultured salmonid species to mycotoxin contaminated feeds. Preventative strategies, particularly mycotoxin analysis of ingredients and the implementation of regulated guidelines for the maximum 83 acceptable mycotoxin content offish feeds, are necessary in attaining this objective. Methods of mycotoxin analysis typically employed by laboratories usually involve chromatographic separation coupled with a suitable detection method such as high performance liquid chromatography (HPLC) and mass spectrometry (MS). More recently, significant progress in the simultaneous detection of different classes of mycotoxins has been achieved using liquid chromatography tandem-mass spectrometry (LC-MS/MS). However, such technologies are expensive and require specialized equipment and/or skills, making them impractical for feed manufacturers (Zheng et al., 2006; Krska and Molinelli, 2007). Thus, rapid tests, such as the lateral flow test and Enzyme Linked Immunosorbent Assay (ELISA), which offer a fast, simple and inexpensive technique of on-site mycotoxin screening of ingredients, have become increasingly important in feed production. These methods help to determine the effectiveness of quality control measures, determine legal compliance of ingredients and feeds and keep products moving quickly through marketing channels. ELISA test kits, in particular, are high throughput assays with low sample volume requirements and often less sample preparation requirements compared to reference methods such as HPLC (Zheng et al., 2006). Despite the advantages of ELISA, especially in an industrial setting, it commonly results in an overestimation of the true mycotoxin content of a sample due to matrix interference (Schuhmacher et al., 1997). Additionally, insufficient validation of ELISA methods or kits often limits their use to specific matrices (i.e. corn, wheat, etc.) for which they have been validated (Zheng et al., 2006). Given the high sensitivity of rainbow trout to DON as determined in this study, as well as the increased reliance on a 84 wide variety of plant sources and processed plant products, these limitations of ELISA screening could be a problem for fish feed manufacturers and producers alike. A small scale survey of the DON content of corn gluten meal samples used in the formulation ofpast experimental diets was conducted using a commercially available ELISA kit (AgraQuant® DON Assay, Romer Labs, Inc., Union, MO, USA). This kit provided quantitative determination of DON concentration within a range of 0.25 to 5.0 ppm. In total, 17 samples of corn gluten meal obtained between 1992 and 2008 were tested. The results of this analysis are presented in Table 4.1. Four of the samples, which were obtained from larger batches of corn gluten meal currently being used in experimental feed formulation, were also sent to Romer Labs Singapore for analysis by HPLC. A comparison of the DON content of these samples determined by the ELISA kit and by HPLC is shown in Table 4.2. Comparison of the four samples analyzed by both ELISA and HPLC indicates that ELISA analysis gave a higher DON content than did HPLC analysis. Discrepancies in the determination of mycotoxin content between methods could be a potential problem for fish feed manufacturers and suppliers offish feed ingredients. The results of the present growth trial highlight the importance of accurate quantitation of mycotoxins at very low levels in fish feed ingredients. Additionally, regardless of the analytical method used, sampling of ingredients presents an additional concern in mycotoxin analysis due to the homogenous distribution of mycotoxins. Therefore, the analyzed mycotoxin content of feed ingredients for use in salmonid feeds should be considered to be only an estimate ofthe mycotoxin risk and used with additional preventative and precautionary quality control measures. 85 Table 4.1 - Concentration of DON (ppm) in samples of corn gluten meal used as a feed ingredient of experimental diets for rainbow trout determined using ELISA8. DON Standard Sample number Sample ID Year (ppm) deviation 1 JH-17 1992 0.35 0.06 2 JH-03 1994 NDb 0.04 3 JH-16 1995 0.40 0.10 4 JH-02 1995 0.28 0.07 5 JH-06 1995 ND 0.08 6 JH-05 1996 0.27 0.03 7 JH-Il 1996 ND 0.05 8 JH-08 1997 0.48 0.06 9 JH-07 1997 0.32 0.08 10 JH-09 1998 0.28 0.07 11 JH-04 1998 0.31 0.17 12 JH-12 1998 0.36 0.10 13 JH-1 5 2003 0.38 0.08 14 JH-1 3 2004 0.36 0.08 15 JH-10 2005 0.40 0.05 16 JH-01 2006 0.52 0.07 17 JH- 14 2008 033 0.07 a Limit of detection = 0.2 ppm; Limit of quantitation = 0.25 ppm; Range of quantitation : 0.25 - 5.0 ppm. b ND = not detectable. Table 4.2 - Comparison of the concentration of DON (ppm) in four samples determined using an ELISA8 kit and HPLCb. Sample number Sample ID Year ft TSA jjpT r— Î JH- 13 2ÖÖ4 036 ÖÖ7 2 JH-10 2005 0.40 0.21 43 JH-01JH-14 20062008 0.52033 0.240.10 a Limit of detection = 0.2 ppm; Limit of quantitation = 0.25 ppm; Range of quantitation : 0.25 -5.0 ppm. b Limit of detection = 0.05 ppm 86 Further work is necessary to investigate and determine the mechanisms of action of DON in rainbow trout and other fish species. However, based on the research presented in this thesis, it is evident that rainbow trout are unable to tolerate low levels of DON from a naturally contaminated ingredient without significant effects on growth performance, feed efficiency and health. Without intervention, these effects have the potential to result in substantial financial losses to the aquaculture industry. Therefore, mycotoxin regulation and screening programs should be considered as necessary and integral components of fish feed production. 87 5 - REFERENCES Abbas, H.K., Mirocha, C.J., Tuite, J., 1986. Natural occurrence of deoxynivalenol, 15- acetyl-deoxynivalenol, and zearalenone in refusal factor corn stored since 1972. Appi. Environ. Microbiol. 51, 841 -843 . Abramson, D., 1998. Mycotoxin formation and environmental factors. In: Sinha, K.K., Bhatnagar, D. (Eds.) Mycotoxins in Agriculture and Food Safety. Marcel Dekker, Inc., New York, NY, USA. 255-277. Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R., White, M.R., Brown, P.B., 1998. Evaluation offish-meal free diets for rainbow trout, Onchorhynchus mykiss. Aquaculture Nutrition 4, 255-262. Alexis, M.N., Papaparaskeva-Papoutsoglou, E., Theochari, V., 1985. Formulation of practical diets for rainbow trout {Salmo gairdneri) made by partial or complete substitution of fish meal by poultry by-products and certain plant by-products. Aquaculture 50, 6 1 -73 . AOAC, Official Methods of Analysis. 1995. Official Methods of Analysis, sixteenth edition. Association of Official Analytical Chemists 1, 16-17. Arukwe, A., Grotmol, T., Haugen, T.B., Knudsen, E.R., Goks0yr, A., 1999. A fish model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites. Sci. Total Environ. 236, 153-161. Bergsjo, B., Matre, T., Nafstad, I., 1992. Effects of diets with graded levels of deoxynivalenol on performance in growing pigs. Zentralbl Veterinarmed A 39, 752-758. 88 Berthiller, F., Dall'Asta, C, Schuhmacher, R., Lemmens, M., Adam, G., Krska, R., 2005. Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry. J. Agrie, and Food Chem. 53, 3421-3425. Binder, E.M., Tan, L.M., Chin, L.J., Handl, J., Richard, J., 2007. Worldwide occurrence of mycotoxins in commodities, feeds and feed ingredients. Anim. Feed Sci. Technol. 137,265-282. Brown, D.W., McCoy, C.P., Rottinghaus, G.E., 1994. Experimental feeding ofFusarium moniliforme culture material containing fumonisin Bi to channel catfish, Ictalurus punctatus. J. Vet. Diagn. Invest. 6, 123-124. Bunner, D.L., Morris, E.R., 1988. Alteration of multiple cell membrane functions in L-6 myoblasts by T-2 toxin: an important mechanism of action. Toxicol. Appi. Pharmacol. 92, 113-121. Carlson, D.B., Williams, D.E., Spitsbergen, J.M., Ross, P.F., Bacon, C.W., Meredith, F.I., Riley, R.T., 2001. Fumonisin Bi promotes anatoxin Bi and JV-methyl-JV'- nitro-nitrosoguanidine-initiated liver tumors in rainbow trout. Toxicol. Appi. Pharmacol. 172,29-36. Carter, C.G., Hauler, R.C., 2000. Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo salar L. Aquaculture 1 85, 299-3 1 1 . CAST, 2003. Mycotoxins: risks in plant, animal and human systems. Task Force Report No. 1 1 6. Council for Agricultural Science and Technology, Ames, IA. 89 Cavan, K.R., MacDonald, E.J., Smith, T.K., 1988. Potential for dietary amino acid precursors of neurotransmitters to overcome neurochemical changes in acute T-2 toxicosis in rats. J. Nutr. 1 18, 901-907. CCAC, 1984. Canadian Council on Animal Care. Guide to the Care and Use of Experimental Animals, Ottawa, Ontario, Canada. Chaouloff, F., Laude, D., Merino, D., Surrurrier, B., Elghozi, J.L., 1986. Peripheral and central short-term effects of fusaric acid, a dopamine beta-hydroxylase inhibitor on tryptophan and serotonin metabolism in the rat. J. Neural Transm. 65, 219- 232. Cheng, Z.J., Hardy, R.W., 2004. Nutritional value of diets containing distiller's dried grains with solubles for rainbow trout, Oncorhynchus mykiss. Journal of Applied Aquaculture 15, 101-113. Chi, M.S., Mirocha, C.J., Weaver, G.A., Kurtz, H.J., 1980. Effect of zearalenone on female white Leghorn chickens. Appi. Environ. Microbiol. 39, 1026-1030. Cho, C.Y., 1982. Effect of dietary protein and lipid levels on energy metabolism of rainbow trout {Salmo gairdneri). Proceedings of the 9 Symposium on Energy Metabolism of Farm Animals, Norway. European Association for Animal Production Publication 29, 176-179. Cho, C.Y., Kaushik, S.J., 1990. Nutritional energetics in fish: energy and protein utilization in rainbow trout {Salmo gairdneri). World Rev. Nutr. Diet 61, 132- 172. 90 Cho, C.Y., Bayley, H.S., Slinger, S.J., 1974. Partial replacement of herring meal with soybean meal and other changes in a diet for rainbow trout {Salmo gairdneri). J. Fish Res. Board Can. 31, 1523-1528. Cho, C.Y., Hynes, J.D., Wood, K.R., Yoshida, H.K., 1991. Quantitation offish culture wastes by biological (nutritional) and chemical (limnological) methods; the development of high nutrient dense (HND) diets. In: Cowey, C.B., Cho, CY. (Eds.) Nutritional Strategies and Aquaculture Waste. University of Guelph, Guelph, Ontario, Canada. 37-50. Chowdhury, S.R., Smith, T.K., 2004. Effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on performance and metabolism of laying hens. Poult. Sci. 83, 1849-1856. Coppock, R.W., Mostrom, M.S., Sparling, CG., Jacobsen, B., Ross, S.C., 1990. Apparent zearalenone intoxication in a dairy herd from feeding spoiled acid- treated corn. Vet. Hum. Toxicol. 32, 246-248. Côté, L.M., Buck, W., Jeffery, E., 1987. Lack of hepatic microsomal metabolism of deoxynivalenol and its metabolite, DOM-I. Food Chem. Toxicol. 25, 291-295 Côté, L.M., Dahlem, A.M., Yoshizawa, T., Swanson, S.P., Buck, W.B., 1986. Excretion of deoxynivalenol and its metabolite in milk, urine, and feces of lactating dairy cows. J. Dairy Sci. 69,2416-2423. Coulombe Jr., R.A., 1993. Symposium: biological action of mycotoxins. J. Dairy Sci. 76,880-891. Dänicke, S., Valenta, H., Doli, S., 2004. On the toxicokinetics and the metabolism of deoxynivalenol (DON) in the pig. Arch. Anim. Nutr. 58, 169-180. 91 Dänicke, S., Goyarts, T., Doli, S., Grove, N., Spolders, M., Flachowsky, G., 2006. Effects of the Fusarium toxin deoxynivalenol on tissue protein synthesis in pigs. Toxicol. Lett. 1 65, 297-3 11. De Pedro, N., Pinillos, M.L., Valenciano, L, Alonso-Bedate, M., Delgado, M.J., 1998. Inhibitory effect of serotonin on feeding behavior in goldfish: involvement of the CRF. Peptides 19,505-511. Desjardins, A.E., Hohn, T.M., McCormick, S.P., 1993. Trichothecene biosynthesis in Fusarium species: chemistry, genetics and significance. Microbiological Reviews 57, 595-604. Dill-Macky, R., Jones, R.K., 2000. The effect of previous crop residues and tillage on Fusarium head blight of wheat. Plant Dis. 84, 71-76. D'Mello, J.P.F., Maçdonald, A.M.C., 1997. Mycotoxins. Anim. Feed Sci. Technol. 69, 155-166. Drochner, W., Schollenberger, M., Götz, S., Lauber, U., Tafaj, M., Piepho, H.P., 2006. Subacute effects of moderate feed loads of isolated Fusarium toxin deoxynivalenol on selected parameters of metabolism in weaned growning piglets. J. Anim. Physiol. Anim. Nutr. 90, 421-428. Ehrlich, K.C., Daigle, K.W., 1987. Protein synthesis inhibition by 8-oxo-12,13- epoxytrichothecenes. Biochim. Biophys. Acta 923, 206-213. Encarnaçao, P., de Lange, C, Bureau, D.P., 2006. Diet energy source affects lysine utilization for protein deposition in rainbow trout {Oncorhynchus mykiss). Aquaculture 261, 1371-1381. 92 Eriksen, G.S., Pettersson, H., 2004. Toxicological evaluation of trichothecenes in animal feed. Anim. Feed Sci. Technol. 114, 205-239. FAO, 2008. The state of world fisheries and aquaculture 2008. Food and Agriculture Organization of the United Nations, Rome. Electronic Publishing Policy and Support Branch. Communication Division. FAO. FAO Globefish. Fishmeal Market Report for January 2010. (http://www.globefish.org/dynamisk.php4?id=4782) Feinberg, B., McLaughlin, CS., 1989. Biochemical mechanism of action of trichothecene mycotoxins. In: Beasley, V.R. (Ed.) Trichothecene Mycotoxicosis: Pathophysiologic Effects Vol. I. CRC Press, Inc., Boca Raton, Florida, USA. 27- 35. Fink-Gremmels, J., 2008. Biological effects of zearalenone. In: Oswald, L, Tarami, I. (Eds.) Mycotoxins in Farm Animals. Transworld Research Network, India. 1 97- 218. Fioramonti, J., Dupuy, C, Dupuy, J., Bueno, L., 1993. The mycotoxin, deoxynivalenol, delays gastric emptying through serotonin-3 receptors in rodents. J. Pharmacol. 266, 1255-1260. Fitzpatrick, D.W., Boyd, K.E., Watts, B.M., 1988. Comparison of the trichothecenes deoxynivalenol and T-2 toxin for their effects on brain biogenic monoamines in the rat. Toxicol. Lett. 40, 241-245. Forsell, J.H., Witt, M.F., Tai, J.H., Jensen, R., Pestka, J.J., 1986. Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone. Food Chem. Toxicol. 24, 213-219. 93 Forsyth, D.M., Yoshizawa, T., Morooka, N., Tuite, J., 1977. Emetic and refusal activity of deoxynivalenol to swine. Appi. Environ. Microbiol. 34, 547-552. Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant- derived alternate fish feed ingredients and their effect in fish. Aquaculture 1 99, 197-227. Friend, D.W., Trenholm, H.L., Thompson, B.K., Fiser, P.S., Hartin, K.E., 1986. Effect of feeding diets containing deoxynivalenol (vomitoxin)-contaminated wheat or corn on the feed consumption, weight gain, organ weight and sexual development of male and female pigs. Can. J. Anim. Sci. 66, 765-775. Gelderblom, W.C.A., Kriek, N.P.J., Marasas, W.F.O., Thiel, P.G., 1991. Toxicity and carcinogenicity of the Fusarium moniliforme metabolite, fumonisin Bi, in rats. Carcinogenesis 13, 433-437. Gelineau-van Waes, J., Starr, L., Maddox, J., Alemán, F., Voss, K.A., Wilberding, J., Riley, R.T., 2005. Maternal fumonisin exposure and risk for neural tube defects: mechanisms in an in vivo mouse model. Birth Defects Research Part A 73, 487- 497. Ghosh, S., Strum, J.C., Bell, R.M., 1997. Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling. FASEB J. 1 1, 45-50. Goel, S., Lenz, S.D., Lumlertdacha, S., Lovell, R.T., Shelby, R.A., Li, M., Riley, R.T., Kemppainen, B.W., 1994. Sphingolipid levels in catfish consuming Fusarium moniliforme corn culture material containing fumonisins. Aquat. Toxicol. 30, 285-294. 94 Gogal Jr., R.M., Smith, B.J., Kalnitsky, J., Holladay, S.D., 2000. Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds. Cytometry 39, 310-318. Gomes, E.F., Rema, P., Kaushik, S.J., 1995. Replacement offish meal by plant proteins in the diet of rainbow trout {Oncorhynchus mykiss): digestibility and growth performance. Aquaculture 130, 177-186. Griessler, K., Encarnaçào, P., 2009. Fumonisins - mycotoxins of increasing importance in fish! In: Aquaculture Asia Magazine. Guan, S., He, J., Young, J.C., Zhu, H., Li, X.Z., Ji, C, Zhou, T., 2009. Transformation of trichothecene mycotoxins by microorganisms from fish digesta. Aquaculture 290, 290-295. Hamilton, R.M.G., Thompson, B.K., Trenholm, H.L., Fiser, P.S., Greenhalgh, R., 1985. Effects of feeding white Leghorn hens diets that contain deoxynivalenol (vomitoxin)-contaminated wheat. Poult. Sci. 64, 1840-1852. Hardy, R.W., 1996. Alternate protein sources for salmon and trout diets. Anim. Feed Sci. Technol. 59, 71-80. He, P., Young, L.G., Forsberg, C. 1992. Microbial transformation of deoxynivalenol (vomitoxin). Appi. Environ. Microbiol. 58, 3857-3863. Hesketh, P.J., Gándara, D.R., 1991. Serotonin antagonists: a new class of antiemetic agents. J. Natl. Cancer Inst. 83, 613-620. Höglund, E., Sorensen, C, Bakke, M.J., Nilsson, G.E., 0yvind, 0., 2007. Attenuation of stress-induced anorexia in brown trout (Salmo trutta) by pre-treatment with dietary L-tryptophan. Br. J. Nutr. 97, 786-789. 95 Howard, P.C., Eppley, R.M., Stack, M.E., Warbritton, ?., Voss, K.A., Lorentzen, R.J., Kovach, R.M., Bucci, T.J., 2001. Fumonisin Bi carcinogenicity in a two-year feeding study using F344 rats and B6C3F1 mice. Environ. Health Perspect. 109, 277-282. Hussein, H.S., Brasel, J.M., 2001. Toxicity, metabolism and impact of mycotoxins on humans and animals. Toxicology 167, 101-134. Iordanov, M.S., Pribnow, D., Magun,, J.L., Dinh, TH., Pearson, J.A., Chen, S.L.Y., Magun, B., 1997. Ribotoxic stress response: activation of the stress-activated protein kinase JNKl by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the a-Sarcin/Ricin loop in the 28S rRNA. MoI. Cell. Biol. 17, 3373-3381. Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B., Laroche, M., 1995. Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss. Aquaculture 133, 257-274. Ketola, G.H., 1983. Requirement for dietary lysine and arginine by fry of rainbow trout. J. Anim. Sci. 56, 101-107. Kiang, D.T., Kennedy, B.J., Pathre, S.V., Mirocha, C.J., 1978. Binding characteristics of zearalenone analogs to estrogen receptors. Cancer Research 38, 361 1-3615. Kiessling, K.H., 1986. Biochemical mechanism of action of mycotoxins. Pure & Appi. Chem. 58, 327-338. 96 King, R.R., McQueen, R.E., Levesque, D., Greenhalgh, R., 1984. Transformation of deoxynivalenol (vomitoxin) by rumen microorganisms. J. Agrie. Food Chem. 32, 1181-1183. Kollarczik, B., Gareis, M., Hanelt, M., 1994. In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs. Nat. Toxins 2, 105-110. Koski, P., 1985. Studies on the pathology caused by trichothecenes {Fusarium mycotoxins) in farmed rainbow trout {Salmo gairdneri). In: Chelkowski, J. (Ed.) Fusarium Mycotoxins, Taxonomy and Pathogenicity. Elsevier Science Publishing Company Inc., New York, NY, USA. 1 34. Krska, R., Molinelli, ?., 2007. Mycotoxin analysis: state-of-the-art and future trends. Anal. Bioanal. Chem. 387, 145-148. Krska, R., Welzig, E., Boudra, H., 2007. Analysis of Fusarium toxins in feed. Anim. Feed Sci. Technol. 137, 241-264. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Cortón, J.C, Safe, S.H., Van der Saag P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139, 4252-4263. Laskin, J.D., Heck, D.E., Laskin, D.L., 2002. The ribotoxic stress response as a potential mechanism for MAP kinase activation in xenobiotic toxicity. Toxicol. Sci., 289- 291. Leathwood, P.D., 1987. Tryptophan availability and serotonin synthesis. Proc. Nutr. Soc. 46, 143-156. 97 Leung, M.C.K., Díaz-Llano, G., Smith, T.K., 2006. Mycotoxins in pet food: a review on worldwide prevalence and preventative strategies. J. Agrie. Food. Chem. 54, 9623-9635. Lim, C, Yildirim-Aksoy, M., 2008. Distillers dried grains with solubles as an alternative protein source in fish feeds. 8th International Symposium on Tilapia in Aquaculture, 67-82. Lin, X., Volkoff, H., Narnaware, Y., Bernier, N.J., Peyon, P., Peter, R.E., 2000. Brain regulation of feeding behavior and food intake in fish. Comparative Biochemistry and Physiology Part A 126, 415-434. Lucky, Z.K., 1997. Methods for the diagnosis of fish diseases. Amerind Publishing Co., New Delhi, India. Lumlertdacha, S., Lovell, R.T., 1995. Fumonisin-contaminated dietary corn reduced survival and antibody production by channel catfish challenged with Edwardsiella ictaluri. J. Aquat. Anim. Health 7, 1-8. Lumlertdacha, S., Lovell, R.T., Shelby, R.A., Lenz, S.D., Kemppainen, B.W., 1995. Growth, hematology, and histopathology of channel catfish, Ictalurus punctatus, fed toxins from Fusarium moniliforme. Aquaculture 130, 201-218. Manning, B., 2005. Mycotoxins in aquaculture. In: Diaz, D. (Ed.) The Mycotoxin Blue Book. Nottingham University Press, Nottingham, UK. 139-156. Manning, B.B., Li, M.H., Robinson, E.H., 2003. Response of channel catfish to diets containing T-2 toxin. J. Aquat. Anim. Health 15, 229-238. 98 Manning, B.B., Terhune, J.S., Li, M.H., Robinson, E.H., Wise, D.J., Rottinghaus, G.E., 2005. Exposure to feedborne mycotoxins T-2 toxin or ochratoxin A causes increased mortality of channel catfish challenged with Edwardsiella ictaluri. J. Aquat. Anim. Health 1 7, 1 47- 1 52 . Marasas, W.F.O., Bamburg, J.R., Smalley, E.B., Strong, F.M., Ragland, W.L., Degurse, P.E., 1969. Toxic effects on trout, rats and mice of T-2 toxin produced by the fungus Fusarium tricinctum. Toxicol. Appi. Pharmacol. 15, 471-482. Marquardt, R.R., 1996. Effects of molds and their toxins on livestock performance: a western Canadian perspective. Anim. Feed Sci. Technol. 58, 77-89. Matthews, J., Celius, T., Halgren, R., Zacharewski, T., 2000. Differential estrogen receptor binding of estrogenic substances: a species comparison. J. Steroid Biochem. MoI. Biol. 74, 223-234. Merrill Jr., A.H., Sullards, M.C., Wang, E., Voss, K.A., Riley, R.T., 2001. Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins. Environmental Health Perspectives 109, 283-289. Morgavi, D.P., Riley, R.T., 2007. An historical overview of field disease outbreaks known or suspected to be caused by consumption of feeds contaminated with Fusarium toxins. Anim. Feed Sci. Technol. 137, 201-212. Moss, M.O., 1991. The environmental factors controlling mycotoxin formation. In: Smith, J.E., Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 37-56. 99 Moyano, F.J., Cardenete, G., De la Higuera, M., 1992. Nutritive value of diets containing a high percentage of vegetable proteins for trout, Oncorhynchus mykiss. Aquat. Living Resour. 5, 23-29. Munkvold, G.P., 2003. Cultural and genetic approaches to managing mycotoxins in maize. Annu. Rev. Phytopathol. 41, 99-1 16. Nagatsu, T., Hidaka, H., Kuzuya, J., Takeya, K., Umezawa, H., Takeuchi, T., Suda, H., 1970. Inhibition of dopamine beta-hydroxylase by fusaric acid (5-butylpicolinic acid) in vitro and in vivo. Biochem. Pharmacol. 19, 35-44. National Research Council (NRC), 1993. Nutrient Requirements of Fish. National Academy Press, Washington, D.C. Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiù, ?., Elliott, M., Farrell, A.P., Forster, L, Gatlin, D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. PNAS 106, 15103-15110. Noga, E.J., 1996. Fish Disease: Diagnosis and Treatment. Mosby-Year Book, Inc., St. Louis, MO, USA. Ohanian, J., Ohanian, V., 2001. Sphingolipids in mammalian cell signaling. Cell MoI. Life Sci. 58, 2053-2068. Pestka, J.J., 2007. Deoxynivalenol: toxicity, mechanisms and animal health risks. Anim. Feed Sci. Technol. 1 37, 283-298. Pestka, JJ., Smolinski, A.T., 2005. Deoxynivalenol: toxicology and potential effects on humans. J. Toxicol. Environ. Health Part B 8, 39-69. 100 Pestka, J.J., Zhou, H.R., Moon, Y., Chung, Y.J., 2004. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox. Toxicol. Lett. 153, 61-73. Petrinec, Z., Pepeljnjak, S., Kovacic, S., Krznaric, A., 2004. Fumonisin Bi causes multiple lesions in common carp (Cyprinus carpio). Atsch. Tierarztl. Wochenschr. 358-363. Pitt, J.I., Leistner, L., 1991. Toxigenic Pénicillium Species. In: Smith, J.E., Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 325-340. Placinta, CM., D'Mello, J.P.F., MacDonald, A.M.C., 1999. A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim. Feed Sci. Technol. 78, 21-37. Poston, H.A., Coffin, J.L., Combs Jr., G.F., 1982. Biological effects of dietary T-2 toxin on rainbow trout, Salmo gairdneri. Aquat. Toxicol. 2, 79-88. Prelusky, D.B., 1993. The effect of low-level deoxynivalenol on neurotransmitter levels measured in pig cerebral spinal fluid. J. Environ. Sci. Health Part B 28, 731-761. Prelusky, D.B., Trenholm, H.L., 1993. The efficacy of various classes of anti-emetics in preventing deoxynivalenol-induced vomiting in swine. Nat. Toxins 1, 296-302. Prelusky, D.B., Veira, D.M., Trenholm, H.L., 1985. Plasma pharmacokinetics of the mycotoxin deoxynivalenol following oral and intravenous administration to sheep. L Environ. Sci. Health B20, 603-624. 101 Prelusky, D.B., Veira, D.M., Trenholm, H.L., Hartin, K.E., 1986. Excretion profiles of the mycotoxin deoxynivalenol, following oral and intravenous administration to sheep. Fund. Appi. Toxicol. 6, 356-363. Prelusky, D.B., Hartin, K.E., Trenholm, H.L., Miller, J.D., 1988. Pharmacokinetic fate of 14C-labeled deoxynivalenol in swine. Fundam. Appi. Toxicol. 10, 276-286. Prelusky, D.B., Yeung, J.M., Thompson, B.K., Trenholm, H.L., 1992. Effect of deoxynivalenol on neurotransmitters in discrete regions of swine brain. Arch. Environ. Contam. Toxicol. 22, 36-40. Riley, R.T., Hinton, D.M., Chamberlain, W.J., Bacon, C.W., Wang, E., Merrill Jr., A.H., Voss, K.?., 1994. Dietary fumonisin B¡ induces disruption of sphingolipid metabolism in Sprague-Dawley rats: a new mechanism of nephrotoxicity. J. Nutr. 124, 594-603. Riley, R.T., Enongene, E., Voss, K.A., Norred, W.P., Meredith, F.I., Sharma, R.P., Spitsbergen, J., Williams, D.E., Carlson, D.B., Merrill Jr., A.H., 2001. Sphingolipid perturbations as mechanisms for fumonisin carcinogenesis. Environmental Health Perspectives 109, 301-308. Rizzo, A.F., Atroshi, F., Ahotupa, M., Sankari, S., Elovaara, E., 1994. Protective effect of antioxidants against free radical-mediated lipid peroxidation induced by DON or T-2 toxin. Zentralbl Veterinarmed A. 2, 81-90. Rodrigues, I., 2008. Biomin Mycotoxin Survey Program 2008. In: Biomin Newsletter. Biomin Holding GmbH, Vol. 7. Rodrigues, I., Griessler, K., 2009. Biomin's Mycotoxin Survey - Quarterly Report. In: Mycotoxin Report. Biomin Holding GmbH. 102 Ross, P.F., Rice, L.G., Osweiler, G.D., Nelson, P.E., Richard, J.L., Wilson, T.M., 1992. A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates. Mycopathologia 117, 109-114. Rotter, B.A., Prelusky, D.B., Pestka, JJ., 1996. Toxicology of deoxynivalenol (vomitoxin). J. Toxicol. Environ. Health Part A 48, 1-34. Sahu, S.C., Garthoff, L.H., Robl, M.G., Chirtel, S.J., Ruggles, D.I., Flynn, T.J., Sobotka, T.J., 2008. Rat liver clone-9 cells in culture as a model for screening hepatotoxic potential of food-related products: hepatotoxicity of deoxynivalenol. J. Appi. Toxicol. 28, 765-772. Santin, E., 2005. Mould growth and mycotoxin production. In: Diaz, D. (Ed.) The Mycotoxin Blue Book. Nottingham University Press, Nottingham, UK. 225-234. Sasanya, J.J., Hall, C, Wolf-Hall, C., 2008. Analysis of deoxynivalenol, masked deoxynivalenol and Fusarium graminearum pigment in wheat samples, using liquid chromatography-UV-mass spectrometry. J. Food Prot. 71, 1205-1213. Schaafsma, A.W., Tamburic-Llinic, L., Miller, J.D., Hooker, D.C., 2001. Agronomic considerations for reducing deoxynivalenol in wheat grain. Can. J. Plant Pathol. 23, 279-285. Schick, B., Greenbaum, J.W., 1946. Edema with hypoproteinemia due to a congenital defect in protein formation. J. Pediatrics 27, 241-245. Schollenberger, M., Müller, H.M., Rüfle, M., Suchy, S., Plank, S., Drochner, W., 2006. Natural occurrence of 16 Fusarium toxins in grains and feedstuffs of plant origin from Germany. Mycopathologia 161, 43-52. 103 Schuhmacher, R., Krska, R., Weingaertner, J., Grasserbauer, M., 1997. Interlaboratory comparison study for the determination of the Fusarium mycotoxins deoxynivalenol in wheat and zearalenone in maize using different methods. Fresenius J. Anal. Chem. 359, 510-515. Scott, P.M., 1989. The natural occurrence oftrichothecenes. In: Beasley, V.R. (Ed.) Trichothecene Mycotoxicosis: Pathophysiologic Effects Vol. I. CRC Press, Inc., Boca Raton, Florida, USA. 1-26. Scott, P.M., 1997. Multi-year monitoring of Canadian grains and grain-based foods for trichothecenes and zearalenone. Food Additives and Contaminants 14, 333-339. Shotwell, O.L., 1991. Natural occurrence of mycotoxins in corn. In: Smith, J.E., Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 325-340. Smith, G.W., Constable, P.D., Haschek, W.M., 1996. Cardiovascular responses to short- term fumonisin exposure in swine. Fund. Appi. Toxicol. 33, 140-148. Smith, T.K., 1992. Recent advances in the understanding ofFusarium trichothecene mycotoxicoses. J. Anim. Sci. 70, 3989-3993. Smith, T.K., MacDonald, E.J., 1991. Effect of fusaric acid on brain regional neurochemistry and vomiting behavior in swine. J. Anim. Sci. 69, 2044-2049. Smith, T.K., Sousadias, G., 1993. Fusaric acid content of swine feedstuffs. J. Agrie. Food Chem. 41, 2296-2298. Smith, T.K., McMillan, E.G., Castillo, J.B., 1997. Effect of feeding blends oí Fusarium mycotoxin-contaminated grains containing deoxynivalenol and fusaric acid on growth and feed consumption of immature swine. J. Anim. Sci. 75, 2184-2191. 104 Smith, T.K., Diaz, G., Swamy, H.V.L.N., 2005. Current concepts in mycotoxicoses in swine. In: Diaz, D. (Ed.) The Mycotoxin Blue Book. Nottingham University Press, Nottingham, UK. 235-248. Speijers, G.J.A., Speijers, M.H.M., 2004. Combined toxic effects of mycotoxins. Toxicol. Lett. 153, 91-98. Spring, P., Fegan, D.F., 2005. Mycotoxins - a rising threat to aquaculture. Nutritional biotechnology in the feed and food industries. Proceedings of Alltech' s 21st Annual Symposium, Lexington, Kentucky, USA, 22-25 May 2005. Spring, P., Burel, C, 2008. Effect of mycotoxins in aquaculture. In: Oswald, I., Tarami, I. (Eds.) Mycotoxins in Farm Animals. Transworld Research Network, India. 71- 90. Stoskopf, M.K., 1993. Clinical physiology. In: Stoskopf, M.K. (Ed.) Fish Medicine. W.B. Saunders Company, Philadelphia, PA, USA. 48-57. Strange, R.N., 1991. Natural occurrence of mycotoxins in groundnuts, cottonseed, soya, and cassava. In: Smith, J.E., Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 341-362. Sudakin, D.L., 2003. Trichothecenes in the environment: relevance to human health. Toxicol. Lett. 143, 97-107. Suneja, S.K., Wagle, D.S., Ram, G.C., 1989. Effect of oral administration of T-2 toxin on glutathione shuttle enzymes, microsomal reductases and lipid peroxidation in rat liver. Toxicom 27, 995-1001. 105 Swamy, H.V.L.N., Smith, T.K., MacDonald, EJ., Boermans, HJ., Squires, EJ., 2002. Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on swine performance, brain regional neurochemistry, and serum chemistry and the efficacy of a polymeric glucomannan mycotoxin adsorbent. J. Anim. Sci. 80, 3257-3267. Swanson, S.P., Corley, R.A., 1989. The distribution, metabolism and excretion of trichothecene mycotoxins. In: Beasley, V.R. (Ed.) Trichothecene Mycotoxicosis: Pathophysiologic Effects Vol. I. CRC Press, Inc., Boca Raton, Florida, USA. 37- 61. Sweeney, MJ., Dobson, A.D.W., 1998. Mycotoxin production by Aspergillus, Fusarium and Pénicillium species. Int. J. Food Microbiol. 43, 141-158. Tacón, A.G.J., Metían, M., 2009. Fishing for feed or fishing for food: increasing global competition for small pelagic forage fish. Ambio 38, 294-302. Tiemann, U., Brüssow, K.P., Küchenmeister, U., Jonas, L., Kohlschein, P., Pöhland, R., Dänicke, S., 2006. Influence of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected enzymatic and histological parameters of liver in gilts. Food Chem. Toxicol. 44, 1228-1235. Toflefsen, K.E., Mathisen, R., Stenersen, J., 2002. Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology 126, 14-22. 106 Trenholm, H.L., Thompson, B.K., Hartin, K.E., Greenhalgh, R., McAllister, A.J., 1985. Ingestion of vomitoxin (deoxynivalenol) contaminated wheat by non-lactating dairy cows. J. Dairy Sci. 68, 1000-1005. Tuan, N.A., Manning, B.B., Lovell, R.T., Rottinghaus, G.E., 2003. Responses ofNile tilapia (Oreochromis niloticus) fed diets containing different concentrations of moniliformin or fumonisin Bi. Aquaculture 217, 515-528. Ueno, Y., 1984. Toxicological features of T-2 toxin related trichothecenes. Fundam. Appi. Toxicol. 4, Sl 24-Sl 32. Ueno, Y., 1991. Biochemical mode of action of mycotoxins. In: Smith, J.E., Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 437-453. United States Department of Agriculture Market News (USDA). National weekly feedstuff wholesale prices for October 6, 2009. (www.ams.usda.gov/mnreports/ms_gr8 5 2.txt) Voss, K.A., Gelineau-van Waes J.B., Riley, R.T., 2006. Fumonisins: current research trends in developmental biology. Mycotoxin Research 22, 61-69. Voss, K.A., Smith, G.W., Haschek, W.M., 2007. Fumonisins: toxicokinetics, mechanism of action and toxicity. Anim. Feed Sci. Technol. 137, 299-325. Wang, E., Norred, W.P., Bacon, C.W., Riley, R.T., Merrill, A.H., 1991. Inhibition of sphingolipid biosynthesis by fumonisins: implications for diseases associated with Fusarium moniliforme. J. Biol. Chem. 266, 14486-14490. 107 Wang, E., Ross, P.F., Wilson, T.M., Riley, R.T., Merrill, A.H., 1992. Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium monili/orme. J. Nutr. 122, 1706-1716. Weaver, G.A., Kurtz, HJ., Behrens, J.C., Robison, T.S., Seguin, B.E., Bates, F.Y., Mirocha, C.J., 1986. Effect of zearalenone on the fertility of virgin dairy heifers. Am. J. Vet. Res. 47, 1395-1397. Wei, CM., McLaughlin, CS., 1974. Structure-function relationship in the 12,13- epoxytrichothecenes - novel inhibitors of protein synthesis. Biochem. Biophys. Res. Comm. 57, 838-844. Whitlow, L.W., Hagler Jr., W.M., 2002. Mycotoxins in feeds. In: Feedstuffs: The Weekly Newspaper for Agribusiness. Vol. 74. Woodward, B., Young, L.G., Lun, A.K., 1983. Vomitoxin in diets for rainbow trout (Salmo gairdneri). Aquaculture 35, 93-101. Yildirim, M., Manning, B.B., Lovell, R.T., Grizzle, J.M., Rottinghaus, G.E., 2000. Toxicity of moniliformin and Fumonisin Bi fed singly and in combination in diets for young channel catfish Ictalurus punctatus. J. World Aquaculture Society 31, 599-608. Yoshizawa, T., 1991. Natural occurrence of mycotoxins in small grain cereals (wheat, barley, rye, oats, sorghum, millet, rice). In: Smith, J.E. and Henderson, R.S. (Eds.) Mycotoxins and Animal Foods. CRC Press, Inc., Boca Raton, Florida, USA. 301-324. 108 Yoshizawa, T., Takeda, H., Ohi, T., 1983. Structure of a novel metabolite from deoxynivalenol, a trichothecene mycotoxin, in animals. Agrie. Biol. Chem. 47, 2133-2135. Young, L.G., McGirr, L., Valli, V.E., Lumsden, J.H., Lun, A. 1983. Vomitoxin in corn fed to young pigs. J. Anim. Sci. 57, 655-664. Zheng, M.Z., Richard, J.L., Binder, J., 2006. A review of rapid methods for the analysis of mycotoxins. Mycopathologia 161, 261-273. Zhou, B., Li, Y., Gillespie, J., He, G.Q., Horsely, R., Schwarz, P., 2007. Doehlert matrix design for optimization of the determination of bound deoxynivalenol in barley grain with trifluoroacetic acid (TFA). J. Agrie. Food Chem. 55, 10141-10149. 109