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%0$503"5%&-6/*7&34*5²%&506-064& %ÏMJWSÏQBS Université Toulouse 3 Paul Sabatier (UT3 Paul Sabatier)
1SÏTFOUÏFFUTPVUFOVFQBS Cheat Sophal le mercredi 8 juillet 2015 5JUSF
Individual and combined effects of the trichothecenes deoxynivalenol and nivalenol ex vivo and in vivo on pig intestinal mucosa
²DPMFEPDUPSBMF et discipline ou spécialité ED SEVAB : Pathologie, Toxicologie, Génétique et Nutrition 6OJUÏEFSFDIFSDIF UMR 1331 TOXALIM, Research Centre in Food Toxicology, INRA-INP-ENV-UPS de Toulouse %JSFDUFVS USJDF T EFʾÒTF
Kolf-Clauw Martine
Jury:
Couderc Bettina, Professeur Desfontis Jean-Claude, Professeur, Rapporteur Feidt Cyril, Professeur, Rapporteur Oswald Isabelle, Directeur de Recherches Raymond-Letron Isabelle, Maître de Conférences HDR Acknowledgements
First of all, I would like to express my sincere thanks to all the members of the thesis committee who had taken their busiest time to be present at my thesis defense, Prof.
Bettina Couderc; the reporters, Prof. Jean-Claude Desfontis and Prof. Cyril Feidt; Dr.
Isabelle Oswald and Dr. Isabelle Raymond-Letron.
I wish to extend my deepest appreciation and thanks to Prof. Martine Kolf-Clauw for her gentle, kind and generous, yet robust supervision on the thesis works. She has always given me the constructive critical ideas alongwith during the three years aiming at improving knowledge, ability and inspiration for toxicological assessment. Being as similar as mother and son, she introduced a well-equipped apartment with a good surrounding environment and provided extra household stuffs for being used during the stay.
I wish to extend my deepest appreciation to Dr. Isabelle Oswald for accepting to take part in her research team, team 5, and her very strong recommendation letter for obtaining the scholarship, resulting in today’s thesis. She often provides me a global point of view in the research field in mycotoxins and technical methods that are appropriable and applicable for being used in home country. Besides, she welcomes for any further collaborations in the research field between home country, Cambodia and host country, France; thank you for your glance at our country with high consideration in improving higher education in such a developing country.
I also wish to extend my deepest appreciation and thanks to Dr. Isabelle Raymond-Letron for her suggestion in the field of histopathology. In spite of the fact that there had been plenty piles of working microscopic slides of her own on her desk; still she was able to share her time to explain me the principal concepts by illustrating my own treated slides under a microscope.
I also extend my deepest appreciation to Prof. Sovan Lek, an initiator who has brought the
TECHNO1 scholarship project and the others from ERASMUS MUNDUS programme, for opening a way to this world of research. Through these projects, mutual enrichment and
1 better understanding between the EU and South-East Asia should be fostered at higher education level. Although this works may not be immediately applied in my home country due to the research facilities, I have a broader vision for long term development in this field as well as the collaboration. Prof. Bernard Salles, director of Toxalim is acknowledged for his informative e-mail to reach the acceptance from Dr. Isabelle Oswald. Prof. Claude Maranges, head of the doctoral school, SEVAB, is grateful for his authorization to the doctoral program. Dominique Pantalacci, secretary of the SEVAB, is appreciated for her well management and coordination in the doctoral course works. Amelie Sigaudo, European project manager and her colleagues are appreciated for their well coordination.
I wish to extend my deepest appreciation and thanks to my colleagues and friends throughout the team, the Toxalim, the ENVT, the UPS and the Alliance Française de Toulouse, who have so generously provided their constructive critical suggestions and encouragement for this work and also for the French language study.
I also wish to acknowledge the physical, mental and time contributions of the team and the others: Philippe Pinton for his qRT-PCR technical and experimental animal support Anne-Marie
Cossalter for animal care and experimental animal support, Joëlle Laffitte and Soraya
Tadrist for their helpful assistance, Olivier Lafaix and Juliette Cognie for helping in the loops experiments, Celine Bleuart and Isabelle Pardo for training in tissue staining and technical assistance for immunohistochemistry, Faouzi Lyazhri for statistical advice, Jean-
Denis Bailly for his constructive idea and opinion on the thesis oral presentation and on the researches in the future.
I also appreciate the kind suggestions and helpful manner from Imourana Alassane-Kpembi, a closed friend. An unforgettable first lunch from Selma Snini at the cantine, a kindness in accommodation installation from Anwar EL Mahgubi, kindly regards from Patricia Cano, Alix
Pierron, Julie Seeboth, Yann Biggy, Stela Desto, Brankica Aleksic, Sylviane Bailly, Olivier Puel,
Arlette Querin, Marie-Rose Trumel, Pascal Gourbeyre, Delphine Payros, Joanna Tannous,
Sabria Mimoun, Rhoda El Khoury, Joya Makhlouf, Amaranta Aliciacus, Laura Costes, Leticia
Murate, Viviane Maruo, Huong Le Thanh and everyone from the team are appreciated and
2 acknowledged. This works successfully achieved depend on a very warm and comfortable working environment. Thank you for allowing me in this best condition!
I am grateful to Cambodian brothers, sisters in France for their welcome and encouragement and those in Cambodia for their informative communication, especially those from Royal
University of Agriculture.
Finally, I wish to thank my daughter Arunvatei Phal, my son Duongpisedth Phal, my wife
Sereirumny Huot and my parents for their patience, support and encouragement during this process. Without them, this thesis might not be completed; thank you for being with me!
Grâce au Cambodge, Merci la France!!
Toulouse, July 08, 2015
Sincerely yours,
Sophal CHEAT
3 List of documents
- Book chapter: Mycotoxin outbreak in animal feed, 15 - Paper 1: Nivalenol has a greater impact than deoxynivalenol on pig jejunum mucosa in vitro on explants and in vivo on intestinal loops, 68 - Paper 2: Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes, 86
4 Acknowledgements, 1 List of documents, 4 List of publications and communications, 6 List of abbreviations, 8
Introduction, 9 Chapter I: General background and objective of the work, 9 1.1. Context of the study, 9 1.1.1. Mycotoxins, 9 1.1.2. Pig intestine as a target for DON and NIV, 10 1.1.3. Sensitive biomarkers of mycotoxins in small intestine, 12 1.2. Mycotoxin outbreak in animal feed (book chapter) , 15 1.3. Objective of the PhD thesis, 59 1.4. The PhD thesis structure, 59 Chapter II: Materials and Methods, 60 2.1. Biological models, 60 2.1.1. Explant model, 60 2.1.2. Loops model, 62 2.1.3. Animal experiments, 64 2.2. Histological assessments, 65 2.2.1. Morphometry, 65 2.2.2. Immunohistochemistry, 65 2.3. Gene expression (qRT-PCR) , 66 Chapter III: Results and discussion, 68 3.1. Paper 1: Nivalenol has a greater impact than deoxynivalenol on pig jejunum mucosa in vitro on explants and in vivo on intestinal loops, 68 3.2. Paper 2: Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes, 86 3.3. General discussion, 112 3.4. General conclusion, 116 3.5. Perspectives, 116
References, 118 Appendix, 140
5 List of publications and communications
Publications
1). Cheat S , Oswald IP, Kolf-Clauw M. Mycotoxin outbreak in animal feed. In: Carol A. Wallace and Louise J. Manning, editors. Foodborne diseases: Case studies of outbreaks in agri-food industries. The CRC Press, UK; 2015?. Accepted . 2). Cheat, S .; Gerez, J.R.; Cognié, J.; Alassane-Kpembi, I.; Bracarense, A.P.F.L.; Raymond-Letron, I.; Oswald, I.P.; Kolf-Clauw, M. Nivalenol Has a Greater Impact than Deoxynivalenol on Pig Jejunum Mucosa in Vitro on Explants and in Vivo on Intestinal Loops. Toxins 2015 , 7, 1945- 1961. 3). Cheat S , Pinton P, Lafaix O, Cossalter AM, Cognie J, Raymond-Letron I, Oswald IP, Kolf- Clauw M. Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes. J. Appli. Toxicol., in submission
Posters
1). Cheat S , Pinton P, Lafaix O, Cossalter AM, Cognie J, Raymond-Letron I, Oswald I, Kolf- Clauw M. Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes. The poster presented at Gordon Research Conference: Mycotoxins and Phycotoxins, Massachusetts (USA), 14-19 June 2015
2). Cheat S , Younanian M, Lafaix O, Cognie J, Raymond-Letron I, Cossalter AM, Oswald IP, Kolf-Clauw M. Effects of mycotoxin exposure on proliferation and apoptosis in the porcine intestinal mucosa. The poster presented at: - European Society for Health Pig Management (ESHPM), Sorrento (Italy), 7-9 May 2014 - Journées “Mycotoxines”. Montpellier (France), 5-6 June 2014 - The 8 th conference of “the world Mycotoxin Forum”, Vienna (Austria), 10-12 November 2014
3). Cheat S , Lafaix O, Younanian M, Raymond-Letron I, Cognie J, Kolf-Clauw M. Development of jejunal loop model: effect of mycotoxins on proliferation and apoptosis in porcine enterocytes. The poster presented at: - “Journées de l’école Doctorale SEVAB 2013”, Toulouse (France), 12 November 2013
6 - Société Française de Toxicologie (SFT), Paris (France) 14-15 November 2013 - “Journées des Scientifiques Toxalim”, Toulouse (France), 17-18 December 2013
4). Younanian M, Lafaix O, Cheat S, Kolf-Clauw M. Effects of single and multiple mycotoxin- contaminated diets on proliferation and apoptosis in the porcine intestinal mucosa. Poster presentation. Merial Veterinary Scholars Program (MVSP), Michigan (USA), August 2013.
7 List of abbreviations
bw : Body weight CO2 : Carbon dioxide Ctrl : Control °C : Degree Celsius DNA : Deoxyrebonuceic acid DON : Deoxynivalenol EC : European Commission EU : European Union ENN : Enniatin B1 ENVT : Ecole Nationale Vétérinaire de Toulouse EFSA : European Food Safety Authority Fa : Fraction affected FB: Fumonisin mycotoxin Fig : Figure g : Gram GGT : Gamma-glutamyl transferase GLM : General Linear Model g/L : Gram per liter h : Hour HE : Hematoxylin-eosin IEC : Intestinal epithelial cell kg : Kilogram L : Liter mg.kg -1 : Milligram per kilogram mg : Milligram µM : Micromole µL : Microliter µm : Micrometer mM : Millimole mRNA : Messenger ribonucleic acid n : number of sample nM : Nanomole NIV : Nivalenol nL : Non-loop segment P : Probability value PhD : Doctor of Philosophy qRT-PCR : Quantitative Real-Time Polymerase Chain Reaction SEVAB : Sciences Ecologiques, Vétérinaires, Agronomiques & Bioingénieries T-2 : T-2 toxin TDI : Tolerable daily intake TECHNO1 : Engineering Technology Education and Research Exchanges Toxalim : Research Center in Food Toxicology UPS : Université Paul-Sabatier U/L : Unit per litre WME : Williams’ culture media
8 Introduction
The today’s growing world population requires the earth to produce more sufficient and safety food to feed the entire world. In this context, food and feed safety play an important role for both animal and human being to assure that they are toxin free for consumption. Even though, toxigenic fungi often grow on edible plants, thus grains and processed grains which are used as food or feed stuffs often contaminated with mycotoxins worldwide. This contamination can occur due to inappropriate agricultural practice, storage and the climate change. As a result, common mycotoxin-contaminated agricultural commodities are of potential concerns for human and animal health when unintentional ingestion or once those stuffs heavily contaminated, destroy action must be taken that lead to economic loss.
Chapter I: General background and objective of the work
1.1. Context of the study
The contaminants in food, feed, drinking water and environment resulting from industrial, agricultural practices or climate changes, and occurring either as single or as mix forms, raise concerns for adverse effects after long-term and low-doses exposure. Generally, human and animals are exposed to these compounds primarily via food, and consequently intestinal tissue is the first physiological barrier tissue potentially highly exposed to these contaminants (Pinton and Oswald 2014; Bouhet and Oswald 2005). Among the major contaminants, mycotoxins, the natural food/feed contaminants, can represent an important risk factor for human and animal health. After ingestion, mycotoxins will cause lowered performance, induce various pathologies on humans and animals depending on the type, duration and level of exposure. However, the signs are not directly recognized in relation to mycotoxins due to their effects on the immune system, the gut barrier or the oxidative status of the animals (Pestka and Smolinski 2005; Pestka et al. 2004).
1.1.1. Mycotoxins
Mycotoxins are toxic secondary metabolites produced by fungi in response to stress although they are not essential to fungal growth. They can occur before or under post-harvest conditions, and that up to 72% of one mycotoxin and to 38% of co-contamination with two or more mycotoxins in agricultural commodities, particularly cereals and grains had been found to be contaminated worldwide which result in potential deterioration of human and animal health (Streit et al. 2013;
9 Schatzmayr and Streit 2013; Wild and Gong 2010; Rai and Varma 2010; Oswald et al. 2005). They are mainly produced by fungi in the Aspergillus, Penicillium and Fusarium genera. More than 300 mycotoxins are identified, and this number is increasing, especially because of the masked mycotoxins, plant metabolites or food processes of mycotoxins and emerging mycotoxins, not routinely determined mycotoxins. Among those, the major produced mycotoxins and the most often occurring mycotoxins, particularly in swine feed, are aflatoxins, ochratoxins (A) fumonisins, zearalenone and trichothecenes i.e. deoxynivalenol (Berthiller et al. 2013; Galaverna et al. 2009; Creppy 2002; Miller 1998). The trichothecence mycotoxins are extremely prevalent and broadly produced by Fusarium genus, the genus that is capable of forming a variety of secondary metabolites (Pinton and Oswald 2014). The trichothecenes are divided into two groups: Type A, including T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol (DAS), while the Type B including fusarenon-X (FUS-X), nivalenol (NIV) and deoxynivalenol (DON) and its 3-acetyl and 15-acetyl derivatives (3- and 15-ADON). The limit levels of DON of 0.2 to 1.75 mg/kg are for cereals and derived products for the human consumer. In feed, the maximum levels depend on the species, from 0.9 to 5 mg/kg for complementary and complete feedstuffs have been recommended (EC 2006). NIV has also been established a tolerable daily intake (TDI) of 1.2 µg/kg b.w. per day in grains and grain-based foods for human while the concerns in animal remained underestimated (EFSA 2013).
Co-occurrence of DON and NIV has been shown in grains and grain products; with DON usually present at higher concentrations than NIV (Rasmussen et al. 2003). Recently, mycotoxins contaminated maize in South-East of France has been reported with 100% and those with more than one mycotoxins (Trichothecenes, zearalenone, fumonisins) (Airfaf 2015). A large scale data survey has indicated that DON and NIV are present in 57% and 16%, respectively, of food samples collected in the European Union (Schothorst and van Egmond, 2004). This occurrence brought up a concern for European Commission. In consequence, EFSA was asked to assess the co-occurrence of nivalenol with deoxynivalenol in feed and food, as signs of toxicity in animal (pigs) can be observed (EFSA 2013). Therefore, our study has been designed to give scientific data contributing to human or animal risk health assessment of DON and NIV.
1.1.2. Pig intestine as a target for DON and NIV
Pig is the most sensitive species to DON followed by rodent > dog > cat > poultry >ruminants (Pestka and Smolinski 2005) also a species suggested by European Commission (EC 2000) to be sensitive to NIV. Pig digestive physiology is very similar to human (Kararli 1995), and this species
10 can be regarded as the most relevant animal model for extrapolating to human (Rothkötter et al. 2002; Nejdfors et al. 2000; Rotter et al. 1996). Following the ingestion of contaminated food/feed or toxic chemicals, the gastrointestinal tract intrinsically able to resist or regulate those stuffs. Its principal functions when subject to those contaminants include storage, propulsion, digestion, absorption, secretion, barrier activity and elimination (Haschek et al. 2010). Small intestine, a segment of gastrointestinal tract, responsible for secretion and absorption of nutrient (95%), water uptake regulations and moreover, the intestinal epithelium acts as a selective and effective barrier to biotransforms xenobiotics, exclusion of potential pathogens in luminal contents (bacteria and nonabsorbed compounds) out of the body (Gelberg 2012; Vereeke et al. 2011; Haschek et al. 2010; Oswald 2006). Small intestinal mucosa morphology consists of surface epithelium, crypts/glands, lamina propria , and a thin layer of muscle separating the mucosa and submucosa. It can be artificially divided into two zones: villi for absorptive and enzyme release functions; and crypts for secretion and replacement (Haschek et al. 2010).
The intestinal epithelium is a specialized monolayer of columnar cells lining the gut lumen (Fig.1). Four main types of differentiated cell types are present: goblet cells, Paneth’s cells, enterocytes and enteroendocrine cells with multi-functional roles as the most crucial role of barrier to the passage from the external environment into the organism (van der Flier and Clevers 2009; Groschwitz and Hogan 2009). The process of cell shedding under homeostatic conditions must be tightly regulated to preserve the integrity of the intestinal barrier, and proliferative regeneration after cytotoxic damage, appear to originates at crypt site (Vereeke et al. 2011; Booth and Potten 2000). Meanwhile, different cellular outputs from a crypt and induction of the stem cells to favor the forms of differentiation, displacement or apoptosis may be required due to the changes in the tissue environment (Booth and Potten 2000). Apoptosis is an organized process of programmed cell death. A process of apoptosis that is induced by a loss of anchorage, is seen as the main mechanism by which epithelial cells are eliminated at the villus tip. The epithelial proliferation and turnover, under inflammatory conditions (Vandenbroucke et al. 2011, Bracarense et al. 2012), are accelerated, result in increasing the risk of barrier leakage.
11
Figure 1. Schematic overview of the crypt-villus architecture and the cellular composition of the small intestine. Adapted from Zachary and McGavin (2012) and Vereecke et al. (2011)
1.1.3. Sensitive biomarkers of mycotoxins in small intestine
Pig intestinal epithelial cells serve as a dynamic barrier. An increasing number of studies suggest that they are targets for deoxynivalenol (DON) and other Type B trichothecenes (Pinton et al. 2009; Pinton and Oswald 2014; Hooper and Macpherson 2010), and for testing the effects of mycotoxins (Table 1.). The jejunum is selected for this study because it is the most absorptive part (95% of nutrient absorption), with the highest villi, and the longest segment of the small intestine (Gelberg 2012; Haschek et al. 2010) (Fig. 2.). Several endpoints can be used as biomarkers of toxicity and have been described with DON and other trichothecenes (Table 2.).
12
Fig ure 2. Schematic diagram of the topographic histology of the digestive tube, adapted from Gelberg (2012)
Table 1. Pig digestive effects induced by DON, NIV and other trichothecenes Modes Segment Toxins Concentrations Effects References Induced atrophy, induced histological lesions; decreased Jejunum, 1.5 mg/kg feed; In vivo DON villus height in jejunum; Gerez et al. 2015 ileum 4 weeks reduced crypt depth in jejunum and ileum 2.3 mg/kg feed; In vivo Jejunum DON Induced histological lesions Lucioli et al. 2013 5 weeks Jejunum, 4 mg/kg feed; Enhanced permeability; In vivo DON Xiao et al. 2013 ileum 30 days damaged villi Jejunum, 3 mg/kg feed; 5 Induced atrophy; fused and Bracarense et al. In vivo DON ileum weeks decreased villi height 2012 3 mg/kg feed; Dänicke et al. In vivo Jejunum DON Increased crypt depth 10 weeks 2012 DON, 15- 2.29 mg/kg Induced atrophy, histological In vivo Jejunum Pinton et al. 2012 ADON feed; 4weeks lesions; fused villi, necrosis
In vivo intestines NIV 2.5, or 5 mg/kg; Gastrointestinal erosions Hedman et al.
13 3 weeks 1997
Induced histological lesions; cubic epithelial cells; induced Jejunal Kolf-Clauw et al. Ex vivo ENN, T-2 0.3–30 nM; 4h cellular debris, lysis of villi explant 2013 and oedema in the lamina propria DON, 3- Induced histological lesions, Jejunum ADON Ex vivo 10 µM; 4h lyses of enterocytes; flattened Pinton et al. 2012 explant 15- and coalescent villi ADON Flattened and coalesced villi; Jejunal 0.2-5 µM; 4h edema and necrosis in the Kolf-Clauw et al. Ex vivo DON explant and 8h lamina propria ; pyknotic 2009 nuclei in enterocytes
Effects on proliferation and apoptosis have been described in vivo, ex vivo and in vitro (Table 2.). The biomarker endpoints used here were Ki-67 and Caspase-3 for jejunal epithelial cell proliferation and apoptosis, respectively. The Ki-67 was suggested and reported as a reliable marker of mitosis because it is expressed during mitosis in all mammalian species from rodents to humans, and its half-life is very short. Moreover, it is an endogenous marker that does not have any adverse effects on living cells (Markovits et al. 2013; Kee et al. 2002; Scholzen and Gerdes 2000; Endl and Gerdes 2000). It is reported that one of the most important biochemical markers of apoptosis is activation of caspase-3. Besides, a central event in the process of apoptosis is the activation of the death protease caspase-3, and protease assays are mostly used to measure caspase-3 activity in the lysate of a whole population of treated cells. Consequenly, the caspase-3 is considered to play a pivotal role at the final step producing DNA fragmentation in the process of apoptosis (Werner and Steinfelder 2008; Budihardjo et al. 1999; Arends and Wyllie 1991).
Table 2. Small intestinal and cellular proliferation and apoptosis from different modes of mycotoxin exposures Modes Cells Toxins Concentrations Effects References Pig jejunum, 4 mg/kg feed; Inhibited cell In vivo DON Wu et al. 2014 ileum 37 days proliferation Pig jejunum, 4 mg/kg feed; Epithelial cell In vivo DON Xiao et al. 2013 ileum 30 days proliferation
14 Epithelial apoptosis; Pig jejunum, 3 mg/kg feed; 5 Bracarense et al. In vivo DON Decreased ileum weeks 2012 proliferation in crypt Pig jejunal Decreased epithelial Kolf-Clauw et al. Ex vivo T-2 0.3–10 nM; 4h explant cell proliferation 2013 Ex vivo Pig jejunum DON 10 µM; 4h Induced apoptosis Pinton et al. 2012 Pig jejunal 0.2-5 µM; 4h Apoptotic cells in Kolf-Clauw et al. Ex vivo DON explant and 8h lamina propria 2009 Apoptosis, NIV In vitro Rat IEC-6 DON, NIV 0.5–80 µM; 24h Bianco et al. 2012 greater than DON J774A.1 murine Apoptosis, NIV Marzocco et al. In vitro DON, NIV 10-100 µM; 24h macrophages greater than DON 2009
1.2. Mycotoxin outbreak in animal feed (book chapter)
The book chapter was accepted to publish in Carol A. Wallace and Louise J. Manning, editors. Foodborne diseases: Case studies of outbreaks in agri-food industries. The CRC Press, UK; 2015?
15 Mycotoxin Outbreak in Animal Feed
Sophal CHEAT 1,2,3,4 , Isabelle P. OSWALD 1,3 , Martine KOLF-CLAUW 2,3
1 Université de Toulouse, INP, UMR1331, Toxalim, F- 31000 Toulouse, France
2 Université de Toulouse, INP-ENVT, Ecole Nationale Vétérinaire de Toulouse, F- 31076 Toulouse, France
3 INRA, UMR1331, Toxalim, Research Center in Food Toxicology, F- 31076 Toulouse, France
4 Faculty of Animal Science and Veterinary Medicine, Royal University of Agriculture, Phnom Penh,
Cambodia
Introduction
1 Mycotoxin Occurrence
1.1 Origins of Mycotoxin and Occurrence
1.1.1 Fungal Species
1.1.2 Toxinogenesis: Conditions Favoring Mycotoxin Production
1.1.3 Mycotoxins in Feed
1.2 Mycotoxin Contamination of Feedstuffs
2 Mycotoxins and Pathologies
2.1 Mycotoxins and Livestock Diseases
2.1.1 Aflatoxins
2.1.2 Deoxynivalenol
2.1.3 Fumonisins
2.1.4 Ochratoxins
2.1.5 Zearalenone
2.2 Diagnoses of Mycotoxicosis
2.3 Field Cases
3 Mycotoxins and Animal Productivity
3.1 Feed Intake
3.2 Animal Reproduction
3.3 Animal Immunomodulation
16 3.4 Economic Impacts of Mycotoxin
3.4.1 Feed Securities
3.4.2 Tissue Residues
4 Strategies for Prevention and Management
4.1 Agricultural Practices
4.2 Feed Storage and Processing
4.3 Decontamination
4.3.1 Physical Methods
4.3.2 Chemical Methods
4.4 Mycotoxin-Detoxified Agents
4.4.1 Binders
4.4.2 Biotransforming Agents
Conclusions
References
17 Introduction
In recent years, the occurrence of mycotoxins is increasing in food and feed especially because of new technologies such as the production of bioethanol and biodiesel, and the increasing availability of new by- products suitable for inclusion in animal feeds from these technical processes. For example, distillers dried grains are known to contain unexpected high concentrations of mycotoxins (EFSA 2012). Climate change trends could affect fungal infection of crops and the degree of mycotoxin production particularly resulting in higher pre-harvest levels of mycotoxins. This poses both economic and health risks (Wu et al. 2011).
Mycotoxins, secondary metabolites produced by fungi, have been found to contaminate up to 72% of one mycotoxin and 38% of co-contamination with two or more mycotoxins in agricultural commodities
(n=17,316) worldwide, particularly cereals and grains, resulting in potential deterioration of human and animal health (Streit et al. 2013; Schatzmayr and Streit 2013; Wild and Gong 2010; Rai and Varma 2010;
Oswald et al. 2005). The remaining forms of extractable conjugated or non-extractable bound mycotoxins are present in the plant tissue, but are currently neither routinely screened for in food nor regulated by legislation, thus they may be considered as “masked” as the analysis of the mycotoxin content of samples containing these compounds leads to their underestimation (Berthiller et al. 2013). Meanwhile, there are a number of mycotoxins that either have a low incidence or are unknown that may (re-) emerge or be (re-) introduced into the food chain (Van der Fels-Klerx et al. 2009). Furthermore, mycotoxin co-occurrence, in particular the interaction effects, could impact animal health at already low doses (Streit et al. 2012).
Following the findings and reports, the European Food Safety Authority (EFSA) has raised concern for both human and animal health. The toxicological syndromes in human, caused by ingestion of mycotoxins, include growth impairment, induction of cancer (Bryden 2007), impaired intestinal health and pathogen fitness, resulting in altered host pathogen interactions (Antonissen et al. 2014) and decreased resistance to infectious diseases (CAST 2003). The Fusarium contamination of cereals and related products also causes feed-borne intoxication especially in farm animals (Fink-Gremmels and Malekinejad 2007).
1. Mycotoxin Occurrence
1.1 Origins of Mycotoxin and Occurrence
18 1.1.1 Fungal Species
The fungal species (Table 1-1) most often implicated in the field cases of mycotoxicoses belong to the
Aspergillus , Alternaria , Claviceps , Fusarium , Penicillium and Stachybotrys genera (Mili ćevi ć et al. 2010).
Other genera including Chaetomium, Cladosporium, Diplodia, Myrothecium, Phoma, Phomopsis,
Pithomyces also contain mycotoxic fungi (Moss 1991). Aspergillus, Fusarium and Penicillium are the genera of most concern, particularly in Europe, (Creppy 2002; Miller 1998). The major toxins (Figure 1-1) produced by these three genera include aflatoxins, ochratoxins, trichothecenes, zearalenone (ZEA) and fumonisins
(Miller 1998). Bayman and co-workers (2002) reported various metabolites of different species of
Aspergillus , including A. alliaceus , A. auricomus , A. carbonarius , A. ochraceus , A. glaucus , A. melleus , and
A. niger that belong to ochratoxin family . The most important toxigenic Fusarium species occurring in grains and animal feedstuffs are F. culmorum, F. equiseti, F. graminearum, F. langsethiae, F. moniliforme, F. poae and F. sporotrichioides . A variety of Fusarium fungi, that are common soil fungi, are routinely found on cereals grown in the temperate regions of America, Europe and Asia. They produce a number of different mycotoxins of the class of trichothecenes in addition to ZEA. Trichothecene mycotoxins can be classified into two types: Type A including T-2 toxin and HT-2 toxin, diacetoxyscirpenol (DAS) and neosolaniol, and
Type B including deoxynivalenol (DON), nivalenol (NIV) and fusarenon X (FX) as determined by Sugita-
Konsihi and Nakajima (2010) and Creppy (2002). Fusarium toxins have been shown to cause a variety of toxic effects in both experimental animals and livestock.
[Insert Table 1-1 here]
19 Table 1 -1. Mycotoxin occurrences found on cereal grains, their fungal species and clinical signs in farm animals (pig, poultry, ruminants and horse)
Cereal/food Mycotoxins Fungal species Mycotoxicosis Clinical signs commodity
Aspergillus Barley, Mortality, hemorrhage, liver damage, reduced productivity, increased flavus, maize, rice, Aflatoxins (B) Aflatoxicosis susceptibility to disease, immunosuppression, inferior egg shell and Aspergillus sorghum, carcass quality, demineralization, weight loss parasticus wheat
Barley, Immunosuppression, hepatic lesions, ovary damage, hemorrhage, beak Fusariotoxicosis, Deoxynivalenol Fusarium sp. maize, oats, and mouth necrosis, digestive-mucosa ulceration, ataxia, decreased egg mouldy corn toxicosis wheat production, decreased feed intake and weight gain
Fusarium Porcine pulmonary Respiratory distress and cyanosis (PPE), neurotoxicity (ELEM), vericilloides edema (PPE); Equine Fumonisins (B1) Maize hepatotoxicity, nephrotoxicity, cardiotoxicity, decreased feed intake, (moniliforme, leukoencephalomalacia depression, ataxia, blindness, hysteria F. proliferatum (ELEM)
Mainly affects proximal tubules of the kidneys in pigs and poultry;
Penicillium Barley, Rye, kidneys are grossly enlarged and pale; fatty livers in poultry, hepatic Ochratoxin A Porcine nephropathy verrucosum wheat lesions, mouth lesions, diarrhea, digestive-mucosa ulceration, ataxia, feed
refusal, demineralization, decreased egg production
20 Immunosuppression, embryonic mortality, swollen, reddened vulva,
Barley, reduced fertility, vaginal prolapse and sometimes rectal prolapse in pigs; Fusarium Zearalenone maize, Hyperestrogenism suckling piglets may show enlargement of vulvae; bind to estrogen graminearum wheat receptors, causing functional and morphologic changes in the responsive
reproductive organs
Adapted from Wawrzyniak and Waskiewicz (2014); Bryden, (2012); Sugita-Konsihi and Nakajima (2010); Mili ćevi ć et al. (2010)
21 [Insert Figure 1-1 here]
Deoxynivalenol Aflatoxin B1 Zearalenone
Ochratoxin A Fumonisin B1 Figure 1 -1. Chemical structures of the main occurring mycotoxins in animal feed
In addition to Aspergillus and Fusarium, Penicillium species have been reported to produce a variety of mycotoxins or toxic metabolites such as P. citrinum; producing citrinin, P. verrucosum; producing ochratoxin , P. patulum or P. urticae or P. griseofulvum, P. expansum ; producing patulin, P. crustosum; producing penitrem A, P. cyclopium or P. aurantiogriseum, P. camembertii ; producing cyclopiazonic acid,
P. roqueforti, P. camemberti or P. caseicola ; producing penicillin acid, roquefortine, isoflumigaclavines A,
B, PR toxin, and cyclopiazonic acid (Wawrzyniak and Waskiewicz 2014; Tannous et al. 2014; Bennett and
Klich 2003).
1.1.2 Toxinogenesis: Conditions Favoring Mycotoxin Production
The presence of fungi, the commodity composition, the agronomy practices, the harvest conditions, handling and storage practices all affect the ability of molds and fungi to produce mycotoxins (Bryden 2009).
Moisture and temperature are the two major influences on growth of mold and on production of mycotoxins
(Pitt and Hocking 2009). Higher moisture levels (20-25%) are usually required by pathogenic fungi that attack and spread into crops prior to harvest than for fungi to proliferate during storage (13-18%). In addition to moisture and temperature, other factors such as water activity within the interval 0.90-0.995 and at least
22 50% of CO 2 being available also affect the reduction of production of ochratoxin A (OTA), and particularly inhibit the growth of P. verrucosum and other molds in wheat grain (Battacone et al. 2010). For this reason, some mycotoxin contaminations are preferentially driven, prior to harvest (on field contamination) and others the optimum conditions cause contamination in storage. As a result most feedstuffs with moisture contents above 13% are susceptible to mold growth and mycotoxin formation (Magan 2006). Toxins are produced at temperatures ranging from 0°C to 35°C depending on the fungal species (Frisvad 1995). For instance, the temperatures for optimum growth and for OTA production by P. verrucosum have been shown to be with 15-35°C (Magan and Aldred 2005). In partially dried grain, the key mycotoxigenic molds are P. verrucosum (ochratoxin) in damp cool climates of Northern Europe, and A. flavus (aflatoxins), A. ochraceus
(ochratoxin) and some Fusarium species (fumonisins, trichothecenes) on temperate and tropical cereals
(Magan and Aldred 2007). Thus, the geographic-repartitioned temperatures indicate that globally there will be a differentiation between the fungal species that are active and thus the potential for contamination with the toxins they produce.
1.1.3 Mycotoxins in Feed
In general only a few mycotoxins, and in particular five mycotoxins/mycotoxin groups have received widespread attention among 300-400 mycotoxins known today (Schatzmayr and Streit 2013; Bennett and
Klich 2003; Hussein and Brasel 2001). These five mycotoxins/mycotoxin groups and their prevalence as a percentage of the 19,757 samples of feed and feed raw material tested (with the average contamination expressed as µg/kg feed or feed raw materials) are aflatoxins most often detected in South Asia (78% positives; 128 µg/kg), followed by South-East Asia (55%; 61 µg/kg). ZEA was most prevalent in North Asia with 56% positive samples containing an average of 386 µg/kg of the mycotoxin. North Asia is also the region where the occurrence of DON was highest (78%; 1,060 µg/kg). The highest average DON contamination however has been observed in North America, where positive samples (68%) contained an average of 1,418 µg/kg DON. Fumonisins were most frequently detected in South American samples (77%;
2,691 µg/kg). OTA prevalence and average contamination were highest in South Asia (55%; 20 µg/kg).
Eastern European samples frequently showed positive for OTA as well (49%), but the average contamination was much lower at 4 µg/kg feed (Schatzmayr and Streit 2013; Streit et al. 2013; Rodrigues and Naehrer
23 2012). In maize, peanuts, cotton seed, rice and spices, significant levels and types of aflatoxin can be found in the various countries in the world, and that producing aflatoxigenic species (Reddy et al. 2010).
Ochratoxin contamination can occur in wheat, barley, rye and grapes (Jørgensen 2005). In many situations, especially pre-harvest, the fungi that produce these toxins also produce other toxins and co-contamination may occur (Glenn 2007) as 38% of 17,316 samples analyzed contained two or more mycotoxins, suggesting that co-contamination is frequently observed in finished feed sample (Streit et al. 2013; Schatzmayr and
Streit 2013). A global survey of the incidence of mycotoxins (aflatoxin B1 (AFB1), DON, fumonisins B1
(FB1), B2 (FB2) and B3 (FB3), OTA, T-2 toxin and ZEA) in animal feedstuffs showed significant regional differences at low level of contamination, particularly between tropical and temperate areas (Rodrigues and
Naehrer 2011; Rodrigues and Griessler 2010).
Apart from those mentioned mycotoxins, orther mycotoxins or toxic metabolites have been reported such as ergot alkaloids, citrinin, patulin or its metabolites (clavacin, claviformin, expansin, mycoin c and penicidin),
DON metabolites (DOM-1, 3-epi-DON, DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, etc.), moniliformin, phomopsins, tremorgenic mycotoxins, glutinosin (a mixture of the macrocyclic trichothecenes verrucarin A and B), penicillin acid, roquefortine, isoflumigaclavines A and B, PR toxin, penitrems, janthitrems, lolitrems, aflatrem, paxilline, paspaline, paspalicine, paspalinine, paspalitrem A and B, cyclopiazonic acid, citreoviridin, luteoskyrin, rugulosin, rubroskyrin, etc. (Mili ćevi ć et al. 2010; Bennett and
Klich 2003).
1.2 Mycotoxin Contamination of Feedstuffs
Many factors (Figure 1-2) contribute to feed contamination and subsequent animal exposure (Bryden 2012).
Two mains factors are genetic modification of crops to resist fungal attack and feed handling on farm.
Mycotoxins can occur in the food chain in the field or/and during storage, and their occurrence increases when shipping, handling, and storage practices permitting mold growth (Reddy et al. 2010). Incorrect bulk handling and inferior storage conditions, such as prolonged time in storage and low dry matter content
(below 87%), influence contamination of feedstuffs. The on-farm occurrence of ZEA, DON and aflatoxin in forage, cereal grains and straw was investigated in Australia and the mycotoxins were found in all the
24 commodities, and highest occurrence being ZEA (Moore et al. 2008). Recent study raised concerns the excessive aflatoxin levels in maize from Bulgaria, Romania and Serbia (Schatzmayr and Streit 2013). In the survey, the lowest frequency of contamination was found in grains whereas grains are often considered the only source of mycotoxins when examining a field toxicosis. These results pointed out the potential risk of contamination of feedstuffs and forages rather than grain used in animal production. In addition, the contamination of straw, which may be used as a roughage source in ruminant and horse diets or as bedding for horse, poultry and pigs, may also be a source of mycotoxin exposure on farm (Katie 2013; Degen 2011).
[Insert Figure 1-2 here]
Environment Biology Harvesting
Crops
Storage Feed/food
Animals Human
health Public Diseases Animal products Pathology and reduced productivity - Conventional effect - Unconventional effect Figure 1 -2. Factors affecting mycotoxins occurrence in feed/food
25 2. Mycotoxins and Pathologies
The pathologies induced by mycotoxins and the field cases will be described in livestock excluding pets, whilst noting the sensitivity of the pets to some mycotoxins i.e. dogs and cats to Aflatoxin and dogs to OTA
(Bryden 2012).
2.1 Mycotoxins and Livestock Diseases
Livestock may be accidentally fed with contaminated feed resulting in animal disease or health problem because of the toxic effect of the mycotoxins. The susceptibility of species like pigs, poultry or ruminants to mycotoxins is different (Steyn et al. 2009), and these differential sensitivities might be due to the difference in absorption, metabolism, distribution, and elimination of the mycotoxins (Petska 2007). Presented below are the toxic effects of the five mycotoxins or mycotoxin groups that are considered to be most frequently occurring and worldwide the most significant ones (Schatzmayr and Streit 2013; Bryden 2012).
2.1.1 Aflatoxins
Three species of Aspergillus including A. flavus, A. parasiticus, and A. nomius that contaminate plants and plant products may produce aflatoxins (Reddy et al. 2010; JECFA 2001) with several chemical forms such as aflatoxin B1, B2, G1, G2, M1. AFB1 is the most frequently occurring, leading to AFB1’s metabolite in milk from lactating humans and animals that consume AFB1-contaminated food or feed: aflatoxin M1 (Alborzi et al. 2006). AFB1 is the most potent known carcinogen inducing hepatocarcinogenicity in both animals and in humans (JECFA 2001; IARC 1993). The absorption of aflatoxin is rapid before primary metabolism by liver microsomal enzymes to active or detoxified metabolites (Haschek et al. 2002). The reliable biomarkers of aflatoxin exposure are blood (aflatoxin-albumin adducts in serum) and urine (aflatoxin N7-guanine adducts)
(Wild and Turner 2002; JECFA 2001).
In poultry, aflatoxin contaminated feed ingestion is frequently observed as chronic toxicity (Bailly and
Guerre 2008). Doses of about 0.1 mg/kg, 0.3-0.5 mg/kg and 0.5-2 mg/kg feed in duck, turkey and chicken,
26 respectively, for long exposure of more than 2.5 months resulted in poor growth performances and productions, hemorrhage (Dalvi 1986), hepatic lesions, fibrosis and proliferation of biliary ducts (Bailly and
Guerre 2008). Similar signs have also been observed in ruminants such as reduced feed intake and milk production, diarrhea, biochemical changes in serum, hepatitis changes at necropsy such as congestion, hemorrhage, enlarged gall bladder and color changes showing cirrhosis and necrosis (Morgavi et al. 2008).
The decreases of growth rate and feed intake with increasing aflatoxin concentrations in the diet were reported in pig (Dersjant-Li et al. 2003). Adult animals are less sensitive to the toxic effect of aflatoxins than young animals (Barringer and Doster 2001). For aflatoxins, specific regulations have been set, particularly in feed for dairy cattle in a number of EU countries, ranging from 5ppb (the majorities of country) to 50 ppb for aflatoxin B1 in animal feed and from 0 or 0.01 ppb to 50 ppb for total aflatoxins (FAO, 2004).
2.1.2 Deoxynivalenol (DON)
DON is the main mycotoxin of the type B trichothecenes, which occurs predominantly in grains such as wheat, barley, and maize and less often in oats, rice, rye, sorghum and triticale (Sugita-Konsihi and
Nakajima 2010). DON is rapidly metabolized (Petska 2007) and de-epoxidation via gut microbiota may be extensive i.e. in pigs (Eriksen et al. 2002), cows (Annison and Bryden 1998) and rats, but not in chickens, horses and dogs (Swanson et al. 1988). It is suggested that pigs are most sensitive species to DON (Pestka
2010). The classical clinical signs or response following ingestion of DON are feed refusal and emesis; probably due to a neurochemical imbalance in the brain (Pestka and Smolinski 2005). It was reported that
DON stimulates the main structures involved in feed intake in pigs, and the anorexigenic effects of DON could be contributed by catecholaminergic and NUCB2/nesfatin-1 neurons (Gaigé et al. 2013).
Feed intake and body weight were reduced when the piglets were fed the diet contaminated with DON
2.29mg/kg feed for 4 weeks (Pinton et al. 2012). However, a chronic exposure to low doses of DON did not elicit important clinical signs such as body weight gain, hematology and biochemistry in piglets (Grenier et al. 2011). Feed refusal, reduced weight gain, and emesis have been observed in pigs while consuming a level of DON as low as 1 mg/kg, >2–5 mg/kg and >20 mg/kg feed, respectively (Haschek et al. 2002; Trenholm et al. 1984). DON has been found to decrease feed intake in ruminants at the levels of 10-20 mg/kg feed
27 (Osweiler 2000) whereas poultry tolerate up to 20 mg/kg DON in feed (Petska 2007). The resistance to DON explained by the detoxification by the intestinal microflora of DON to DOM-1 (He et al. 1992), poor absorption into plasma, rapid elimination in excreta and low-level residues in tissues in chicken (Prelusky et al. 1986). Although poultry can tolerate the toxin, in broiler chickens, 10 mg/kg DON in the diet has been found to impair nutrient cotransport in the jejunum and appeared to alter the gut function while not affecting the growth performance (Awad et al. 2004).
Pestka (2008) reported the lethal DON doses that affect histopathology range from hemorrhage or necrosis of the intestinal tract, necrosis in lymphoid tissues and bone marrow, to kidney and heart lesions. The clinical signs of ingestion of DON include gastrointestinal disorders, diarrhea, (Haschek et al. 2002) alteration in immune function (Bondy and Pestka 2000) and predisposition to infections by enteric pathogens (Bracarense et al. 2011). A lethal dose (LD50) for DON at 27 mg/kg body weight (subcutaneously) and 140 mg/kg body weight (orally) have been reported in 10-day old duckling and 1-day-old broiler chicks, respectively whereas acute exposure to relatively low doses at ≥50µg/kg body weight (intraperitoneally or orally) can cause vomiting in pigs. However, no lethal doses have been suggested in ruminants because of their rumen microflora metabolism which plays a significant role in DON detoxification (Petska 2008; Pestka 2007).
Furthermore, mild renal nephrosis, reduced thyroid size, gastric mucosal hyperplasia, increased albumin/alpha-globulin ratio, and sometimes mild changes in other hematological parameters in pigs have also been reported due to the effects of DON (JECFA 2001). Taken together, DON is not considered to be acutely toxic to farm animals, and the concern is economic losses due to poor performance (Morgavi and
Riley 2007).
2.1.3 Fumonisins
It has been shown that fumonisins at high doses cause porcine pulmonary edema and equine leukoencephalomalacia (Haschek et al. 2002). Porcine pulmonary edema syndrome is characterized by dyspnea, weakness, cyanosis and death. The reproduction and performance disturbance are not frequently observed (Taranus et al. 2008). Different states of interstitial and interlobular edema with pulmonary edema and hydrothorax at varying amounts of clear yellow fluid accumulate in the pleural cavity have been seen in
28 pig necropsy affected by fumonisins (WHO 2000). Fumonisins disrupt sphingolipid metabolism by inhibiting ceramide, an enzyme responsible for the acylation of sphinganine and sphingosine (Steyn et al.
2009). Available biomarkers of exposure to toxic levels of fumonisins in farm animals can be used through plasma, ratio of sphinganine/sphingosine in serum or urine (Voss et al. 2007) and the increase in serum and tissue of free sphingoid bases (Riley et al. 1994).
Equine leukoencephalomalacia is characterized by lethargy, head pressing and reduced feed intake, followed by convulsions and death after several days (Morgavi and Riley 2007), and the presence of liquefactive necrotic lesions in the cerebrum white matter, however, the grey matter may also be involved (WHO 2000).
In addition, abnormalities of histology in kidney and liver such as apoptosis and cell regeneration have also been found in horses and ponies, following minimum toxic doses of FB1 between 15 ppm and 22 ppm in feed, but not below 6 ppm (JECFA 2001). It has been reported, besides liver and lung, that fumonisins target pulmonary intravascular macrophages, kidney, pancreas, oesophagus and heart (Morgavi and Riley 2007;
WHO 2000). In poultry, diarrhea, increased liver weight and growth retardation have been reported when consuming the fumonisins-contaminated feed. In feed, pure FB1 at 10 ppm and fumonisins B1 at 30 ppm from F. verticillioides culture material have altered haematological parameters in broiler chicks (Morgavi and Riley 2007; JECFA 2001).
2.1.4 Ochratoxins
The kidney is the target organ of ochratoxins, particularly OTA that induces nephropathy (Marquardt and
Frohlich 1992). Ochratoxin has been detected in blood, milk including human, pork and other animal tissues
(Reddy et al. 2010). Ruminants are insensitive to OTA, because they largely convert OTA into ochratoxin α in the rumen (Jouany and Diaz 2005; Galtier and Alvinerie 1976) and OTA naturally occurring in feed is not regarded as a health threat. It is highly toxic, however, in monogastric farm animals for instance poultry and pigs. High doses of 20 mg/kg can reduce feed intake or cause death with friable livers and pulmonary edema in sheep and in lambs respectively (Morgavi et al. 2008).
29 Feed contaminated by ochratoxins caused poultry disease (Hamilton et al. 1982) and endemic disease in
Denmark (Krogh 1987). In poultry, the concentration of OTA at 0.3-4 mg/kg feed for several weeks and at 2-
10 mg/kg once was reported to induce chronic and acute toxicity, respectively with decreased growth rate, poor feed conversion ratio, low egg production, moist feces, degeneration and focal necrosis of liver (Bailly and Guerre 2008). Significant toxic effects have been shown in chicken fed a diet of about 4 mg/kg for degeneration of internal organs such as kidney, liver, lymphoid, changes of biochemical parameters, and also immunosuppression (Stoev 2008).
Ochratoxin showed a mild effect on microscopic lesions in 3-4 month pigs with level of about 200 µg/kg feed and 90 µg/kg feed in combination with penicillic acid, and a macroscopic kidney damage with 180
µg/kg feed in combination with penicillic acid. These results suggested that interaction effects of ochratoxin with other mycotoxins are very important to studied (Stoev 2008). Battacone et al. (2010) reported reduced feed intake and altered metabolism in pigs exposed to OTA, with several changes of serum blood parameters such as hyperproteinemia and azotemia, hypocholesterolemia and hypercalcemia.
Toxicological studies from animals lead to European Union Scientific Committee to postulate a limit level of
OTA intake below 5 ng/kg of body weight per day (Sweeney et al. 2000). However the EFSA revised this to
17 ng/kg of body weight per day due to its indirect effect on genotoxicity (EFSA 2006), and this revision has been currently applied (EFSA 2010).
2.1.5 Zearalenone (ZEA)
McErlean (1952) was the first to suggest that Fusarium graminearium caused probably hyperoestrogenism in pigs (Bryden 2012). Farm animals, particularly pigs, a very sensitive species, are reported to be affected by
ZEA acting by hyperrestrogenism at prepuberty (Gremmels and Malekinejad 2007; Osweiler 2000) and inducing vulvovaginitis and anestrus (Raisbeck et al. 1991). Dietary levels of ZEA 1-5 ppm and 3-10 ppm can induce vulvovaginitus and anestrus respectively while other clinical signs have been observed in ovariectomized sows, such as tenesmus. Vaginal and rectal prolapse, reduced litter size, fetal resorption, implantation failure, reduced libido and reduced plasma testosterone have been seen in young female pigs
30 (Osweiler 1986; Weaver et al. 1986). Alpha-zearalenol and β-zearalenol are major metabolites resulting from the interaction of ZEA with estrogen receptors. The metabolites elicit estrogenic activity in animals, particularly pigs, which correspond to their binding affinities for mammary, uteri and hypothalamic oestrogen receptors (Fink-Gremmels and Malekinejad 2007; Zinedine et al. 2007). ZEA is reported in the field outbreaks of estrogenic syndrome in pigs in Europe, Africa, North America, Africa, Australia and Asia
(Christensen 1979), and suspected to cause reproductive problems in grazing sheep (CAST 2003), ruminants
(Raisbeck et al. 1991). It was reported to disturb an induction of parturition program together with signs of estrogenism in newborn piglets (Alexopoulos 2001). However, cattle (Weaver et al. 1986), cycling mares
(Juhasz et al. 2001) and poultry (Haschek et al. 2002) have been reported more resistant to the estrogenic effects of ZEA at low doses similar to the natural contamination.
2.2 Diagnoses of Mycotoxicosis
In cases where animal productivity is reduced or in cases of disease, a mycotoxicosis may be suspected when outbreaks exhibit the following characteristics: the cause is not readily identifiable; the condition is not transmissible; syndromes may be associated with certain batches of feed; treatment with antibiotics or other drugs has little effect; and outbreaks may be seasonal as weather conditions may affect mold growth (Bryden
2012).
To ascertain that a mycotoxin is the underlying cause of a field problem it is necessary to demonstrate biologically effective concentrations of the toxin in the suspect feed. This needs a homogenous feed sample representative (Whitaker 2006). In making a diagnosis, not only mycotoxins must be considered for their unique effects, but also they must be evaluated for possible interactions with infectious agents and environmental stressors commonly encountered in animal production (Hamilton 1978). As animals may be fed with diets that contain low levels of more than one toxin, then field situations are often more complicated. For instant, aflatoxins, DON, ZEA and fumonisins can occur together in the same grain (CAST
2003). Moreover, many fungi simultaneously produce several mycotoxins, especially Fusarium species.
Toxicity resulting from the interaction between mycotoxins is usually additive and not synergistic as often suggested (CAST 2003). Thuvander et al. (1999) found that combination of NIV with T-2 toxin, DAS or
31 DON resulted in an additive effect. In addition, recent results in Caco-2 cells model showed that interaction between DON and NIV were synergistic at in vitro cell level (Alassane-Kpembi et al. 2013) and less than additive at ex vivo explant level (Kolf-Clauw’s personal communication). Besides, mycotoxin interaction is complex which varies according to the animal species, the toxin doses, the exposure duration as well as the end points measured (Grenier and Oswald 2011).
In any situation, keeping aware of the possible consequences of mycotoxin-contaminated feed when animal productivity is identified as being poor or simply unexplainable and the condition cannot be diagnosed as being caused by pathogenic agents.
2.3 Field cases
A disease outbreak in the field suspected to a specific mycotoxin in feedstuff can be very difficult to define since the signs of disease are generally subtle and unspecific (Morgavi and Riley 2007), and they usually do not cause acute disease. In the case of acute diseases, multiple interacting factors can modify the expression of toxicity (Hamilton et al. 1982). However, the incidence of acute disease, not associated with known infectious diseases and often involving mortality or evidence of acute toxicity, in farm animals indicated the presence of mycotoxins in feeds (Morgavi and Riley 2007).
Long before these toxins were discovered, aflatoxin, DON, ergot, fumonisins and ZEA, mold-contaminated feed was assumed to be the cause of animal disease in field outbreaks (Morgavi and Riley 2007). Feed contaminated with aflatoxin (corn and peanut meal) killed 12 yearling colts, 2-year-old horses, and a 7-year- old mare in Thailand and the southern USA, respectively. The similar autopsy and hematological and serum biochemical changes were found suggesting that horses are susceptible to aflatoxicosis (Smith and Girish
2008). In pigs, cases of pulmonary edema were reported during 1989 in the US. It was concluded that this was due to consumption of corn contaminated with Fusarium verticilloides, a fumonisins-produced fungus, ranging in levels from 20-360 mg/kg feed. This syndrome was experimentally reproduced in 3 of 4 piglets treated with FB1 at 10mg/kg feed, that were observed to have a slight pulmonary edema (Taranu et al. 2008).
32 Another study in pigs fed Fusarium culture material reported evidence of pulmonary edema was detected at a concentration of FB1 equivalent to 0.4 mg/kg body weight per day (JECFA 2001).
Fumonisins-caused equine leukoencephalomalacia has been reported in corn contaminated with fumonisins, and the fatal equine leukoencephalomalacia has been reproduced with a contamination of 10mg/kg feed.
Fumonisins B1 at the level of greater than 10mg/kg often encounter the field outbreak of equine leukoencephalomalacia since 1983 in many regions of the US including Indiana, and Southern Brazil (Smith and Girish 2008). Thus, 66 purebred Arabian horses in Arizona fed with the diet containing FB1 37-122 mg/kg showed the clinical feature such as hemorrhagic foci of the brain stem, liquefactive necrosis of cerebral white matter, histopathological changes including rarefied white matter with pyknotic nuclei and eosinophilic (Smith and Girish 2008). The other cases of equine leukoencephalomalacia outbreaks have also been reported in South Africa suspectedly caused by consumption of moldy maize, and the clinical signs were blindness and ataxia (Pienaar et al. 1981).
Mycotoxicosis from Stachybotrys (atra) , a saprophytic mold, was first reported in deer in Western Europe in
1977 and has been reported as frequent causes that lead to the death of horses, particularly in the Southern parts of France (Le Bars and Le Bars 1996). In Morocco in 1991, approximately 800 donkeys, mules and horses died from Stachybotryotoxicosis. In any cases of death, moldy straw was suspected. The symptoms of this mycotoxicosis ranging from mild cases: hyperesthesia (jump refusal in race horse), rhinitis, conjunctivitis to severe cases: oral mucosa necrosis, bleeding (from small injuries), tissue hemorrhage (Le
Bars and Le Bars 1996). Spontaneous porcine and avian nephropathy were frequently observed. The relative concentrations ranged from 91 to 310 ppm of OTA in feed samples in Bulgaria where the nephropathy occurrences in different batches of slaughtered pigs/chicks varied from 1-2% up to 90-100% (Stoev 2008).
Field outbreaks of porcine hyperestrogenism (vulvo-vaginitis) characterized by swelling and reddening of the vulva and teats in prepubertal gilts reported in South Africa, were caused by the ingestion of Fusarium graminearum infected maize contaminated with ZEA 10 mg/kg feed (Aucock et al. 1980).
33 In farm animal disease outbreaks, DON and other trichothecenes (including T-2 toxin and DAS) have been implicated in many areas of the world, and in pigs, cattle and chickens, the most commonly observed effect is feed refusal (Morgavi and Riley 2007; Haschek et al. 2002; Osweiler 2000). In suspected field outbreaks of DON in farm animals or human, β-glucuronidase is a useful urinary biomarker since it hydrolyzes DON- glucuronide to DON (Meky et al. 2003).
3. Mycotoxins and Animal Productivities
The most difficult consequence after mycotoxin contamination of animal feed supply chain is the reduction of the animal productivity, but not acute disease episodes (Bryden 2012). The poor growth performance of animal which is not due to the nutritional content in feed, sometimes related to a low dose ingestion of toxin which resulted in metabolic disturbances and/or may be accompanied by pathological change (Haschek et al.
2002). The effects of mycotoxins may considerably vary according to the type of mycotoxins involved and the animal species. Pig is considered as the most sensitive species to DON toxic effects followed by rodent > dog > cat > poultry > ruminants (Pestka and Smolinski 2005). Pig has been shown to be also the most sensitive species to other mycotoxins, like NIV (EFSA 2013), ZEA (Fink-Gremmels 2008), fumonisins
(Morgavi and Riley 2007) whereas horses are also the species most sensitive to fumonisins (Smith and
Girish 2008). An overview of the animal performances affected by mycotoxins, particularly pig and poultry
(ruminants are more resistant) have highlighted that the effects of trichothecenes on feed intake may be influenced by factors such as age, body weight, sex, feeding period, feeding system and diet, specifically in pigs (Eriksen 2003). Furthermore, the genetic heterogeneity of these species may often lead to large individual differences and inconsistent results comparing to rodents.
3.1 Feed Intake
Growth retardation, commonly induced after repeated ingestion of a contaminated feed (chronic mycotoxicosis), is usually the first sign. However, Hedman et al. (1997a) study on NIV in young male pigs
(pathogen-free Swedish Landrace x Yorkshire) exposed to 2.5 or 5 mg purified NIV/kg feed in the diet for 3 weeks revealed no sign of altered feed intake or body weight and no vomiting or clinical problem. Feed
34 intake was found to be reduced in pig fed a diet containing 5.8mg NIV/kg feed or more but not in pigs fed
2.9 mg NIV/kg feed. In animal studies, DON (in pigs) and ergot alkaloids (in chickens) are the mycotoxins most often involved in feed refusal, which probably involve neurochemical mechanisms (Brake et al. 2000;
Bakau et al. 1998). It has also been demonstrated that chronic dietary exposure to DON causes impaired weight gain, anorexia, decreased nutritional efficiency and immune dysregulation (Haschek et al. 2002;
Trenholm et al. 1984). Moreover, it is estimated that the diet contaminated with low level of aflatoxin, fumonisins and DON, depressed the growth rate and feed intake. For instance in the diet, each increasing mg/kg (0.2-4mg/kg) of aflatoxin leads to 16% depressed in pigs and 5% in broilers; and DON (1-20mg/kg feed) leads to 8% depressed marginal growth rate reduction (MGR) in pigs while broilers showed no response to DON concentrations below 16 mg/kg feed. In the same consequence, fumonisin impacted less on the growth rate that was estimated to be at 0.4% and 0%/mg per kg for pigs and broilers, respectively
(Battacone et al. 2010; Dersjant-Li et al. 2003).
3.2 Animal Reproduction
Mycotoxin ingestion may affect reproductive efficiency of both males and females with impairment of metabolic function (Diekman and Green 1992). ZEA may increase abortion, stillbirths and neonatal mortality in pregnant gilts and sows. These two stages of swine, gilt and sow, particularly the prepubertal gilts are the most sensitive ones (Kanora and Maes 2009). ZEA at 22 mg/kg diet cause alterations in the reproductive tract of swine such as in the uterus, and affects follicular and embryo development (Tiemann and Dänicke
2007). Litter size may be decreased by ergot alkaloids (Kanora and Maes 2009; Bryden 1994).
3.3 Animal Immunomodulation
It is pointed out that mycotoxins are immunomodulators, mostly immunosuppressive (Richard 2008) and are known to increase the susceptibility of animals to infectious disease (CAST 2003). Pestka et al. (2004) showed that dose frequency and duration of exposure to DON will determine whether it has an immunostimulatory or immunosuppressant effect including apoptosis and differential gene expression in immune cells. The experimental result in pig showed that DON could be immunotoxic with a lowest-
35 observed-adverse effect level of 0.12 mg/kg body weight per day (Petska 2010) or chronic exposure to low doses of DON or FB especially in the combination (Grenier et al. 2011; Pinton et al. 2008). NIV has also been observed to adversely affect immune system by decreasing the cellularity of the spleen in animals exposed to 5 mg NIV/kg feed (Hedman et al. 1997b). Negative effect of OTA has been reported consisting in severe immunosuppression in laying hens fed a diet contaminated at 1mg/kg of feed for 60 days (Battacone et al. 2010).
Ingestion of toxins can reduce the effectiveness of vaccination programmes and this failure during aflatoxicosis is related to the immunotoxicity. The toxin impairs the immune function by decreasing cell- mediated immunity and by inducing an inflammatory response (Meissonnier et al. 2008; Oswald et al. 2005).
Bracarense et al. (2011) postulated that the chronic ingestion of low dose of mycotoxins alters the intestine
(induces tissue lesions, modulate the immune cell count as well as the cytokine synthesis and decreases the expression of proteins involved in cell adhesion) and thus may predispose animals to infections by enteric pathogens. In agreement to that, studies in pigs have revealed that fumonisins have increased susceptibility to intestinal infection with Escherichia coli (Oswald et al. 2003).
3.4 Economic Impacts of Mycotoxin
The economic effects of mycotoxin contamination are profound, and crops with large amounts of mycotoxins often have to be destroyed or diverted into animal feed resulting in reduced growth rates, illness, and death (Reddy et al. 2010). Iheshiulor et al. (2011) suggested that there are multiple criteria for assessing the economic impact of mycotoxins on animal agriculture and human including loss of animal life, livestock production and forage crops and increased cost of health care, veterinary care regular costs and research costs focusing on relieving the impact and severity of the mycotoxin problems. Moreover, the impact can be on feed safety as well as security.
3.4.1 Feed Securities
36 Between humans and animals and at the same time with the world population growing, competition is increased for food commodities as developing countries raise their appetite for animal products (Woodward
2010). The threat from fungal invasion of crops is also likely to increase due to the climate change (Garrett et al. 2006). In consequence, fungal infection of crops can also be facilitated by the change in insect population dynamics due to the climate change (Wu et al. 2011). It is obvious that mycotoxins remain a threat to global food supplies as a product of plant disease (Strange and Scott 2005) and the degree of risk depends on the country or region in which crops are grown. These concerns are due to the biological complexity involving predicting changes in ambient temperature, relative humidity, carbon dioxide levels, interrelationships between different fungal genera and different crops in different geographical locations (Bryden 2012).
3.4.2 Tissue Residues
Animals consuming mycotoxin-contaminated feeds can produce meat and milk that contains potentially toxic residues (Reddy et al. 2010). Contamination of animal products with mycotoxins or their metabolic products has been a crucial issue and poses problems in public health (Boudergue et al. 2009). There is no evidence for tissue accumulation of DON (Eriksen et al. 2003; JECFA 2001) and its transfer into milk
(Keese et al. 2008) due to its rapid metabolism and de-epoxidation via gut microbiota (Eriksen et al. 2002).
However, aflatoxin residues are most likely contained in milk, especially the hydroxylated metabolite of
AFB1, aflatoxin M1 in milk representing from 1 to 6% of dietary intake of aflatoxins (Fink-Gremmels
2008). In several European countries, OTA has been found in kidneys, blood, muscle tissues and liver of the pigs (Van Egmond and Speijers 1994). Petterrson (2004) showed the half-life of OTA in chickens and pigs as being of 140-180 h and approximately 4 h, respectively. However the tissue deposition of toxins, for instance OTA, is different between poultry and pig due to their pharmacokinetics (Petterrson 2004).
Consumption of pork has been a significant source of human exposure to OTA (Jørgensen and Petersen
2002) and the presence of this mycotoxin in animal-derived products (Gareis and Scheuer 2000).
Not all residues of mycotoxins such as trichothecenes, fumonisins and ZEA are a threat to public health.
Experimental animals were fed with very high levels of the toxins, and very low levels were actually found in the animal tissues (Petterrson 2004; Pestka 1995). Fumonisins are able to disrupt sphingolipid metabolism
37 (Steyn et al. 2009), and as the result, sphinganine accumulation in tissues initiates a cascade of events that may cause toxicity, especially of the liver and kidneys, and carcinogenicity (Voss et al. 2007). When extrapolating or predicting tissue residues, caution should be made as there are often insufficient data on which to anticipate the outcome of any field toxicosis (Pestka 1995).
4. Strategies for Prevention and Management
Reducing the concentration of mycotoxins in feed or a given crop is possible with the preventive methods.
However, concentrations exceeding the limit prescribed in regulations may still occur in spite of all efforts
(Schatzmayr and Streit 2013). Because of the stability of these compounds, reduction is a difficult task (Cole
1986). Therefore, it is important to limit the mycotoxin contamination of food and feed materials prior to harvest. The best approach is minimizing mycotoxin production itself by harvesting the grain at maturity and low moisture and then storing it at cool and dry conditions. This is difficult to achieve during a wet season harvest or in countries with a warm and humid climate (Huwig et al. 2001). Prevention and reduction of the occurrence and the impact of mycotoxins requires an integrated understanding of crop biology, agronomy, fungal ecology, harvesting methods, storage conditions, feed processing and detoxification strategies
(Schatzmayr and Streit 2013; Bryden 2009). Prevention involves first reduction of mycotoxin levels in feed/foodstuffs and then further increasing the intake of diet components through supplementation such as vitamins, antioxidants, suphur-containing amino acids, trace elements and substances that are known to prevent carcinogenesis (Creppy 2002; Nahm 1995).
However, no single treatment in degrading or removing toxins and retaining the nutritional and functional qualities of the treated commodity has proved completely successful since grains may be naturally contaminated with multi-mycotoxins and their distinct chemical characteristics (Bryden 2012; Park and Price
2001).
4.1 Agricultural Practices
38 Good agricultural practice such as crop rotation and tillage, ploughing after wheat and maize, reducing plant density and adequate irrigation, can all minimize the likelihood of mycotoxin formation (Schatzmayr and
Streit 2013). For example, DON level was low (from 15.1ppm to 1.2ppm) in the wheat crop following maize as a result of one year rotation between crops (Jouany 2007; Schaafsma et al. 2001). Plant protection products such as microbial antagonists are a promising alternative to conventional fungicides and are also regarded as biological control. For example Bacillus subtilis strains have been reported to inhibit the growth of fusaria (Jouany 2007) and atoxigenic A. flavus strains successfully reduce aflatoxin contamination in treated crops (Cole and Cotty 1990). Genetic modification of fungi and crops is considered as the best solution. For example, quantitative trait loci for Fusarium resistance have been identified in wheat, and often coincide with genes controlling morphological plant characteristics (Miedaner et al. 2006). However, other varietal characteristics that do not seem to relate to fungal resistance may also have an impact on mycotoxin levels (Anderson 2007).
4.2 Feed Storage and Processing
Grains have to be dry to minimize mycotoxin production. Whilst grain may go into storage at a uniform temperature, over a period the grain mass will cool at different rates in the center compared to the periphery.
Storage of grain at an appropriate moisture content, generally below 13% moisture, regular inspection of grain for temperature, wet spots and insect infestation will limit the potential for fungal development in feeds and feedstuffs. The rapid turnover of feed in high volume animal units will also reduce the risk of feed contamination, because there will be less time for fungi to grow and to produce toxins (Magan et al. 2010).
Good and Hamilton (1981) demonstrated that poultry performance can be improved significantly by increasing feed turnover or decreasing feed residence time on farm simply by reducing the amount of feed per delivery. Thirty five years later it is still good practice to match feed deliveries and storage time to usage to minimize residence period.
Animal feed pelleting, milling or processing affect mycotoxin levels in finished feed. It is reported that most mycotoxins are heat-resistant within the range of common food processing temperatures (80-121 °C), so little or no reduction in overall toxin levels occurs as a result of normal cooking conditions such as frying
39 and boiling (Kabak 2009). However, AFB1 and AFB2 levels can be reduced by cooking and/or processing of feed up to 41% of their initial levels (Furtado et al. 1981). Processing time and temperature affect the decomposition rate of fumonisins B1 and B2 which start at 150 to 175 °C. It is known that 90% of contamination was them were lost after 60 min treatment (Jackson et al. 1996). DON and ZEA are reported as being stable compounds and not known to be destroyed by thermal processing (Visconti et al. 2004;
Gajecki 2002). Over 50% of OTA in wheat was decomposed at 100°C after 120 min (Boudra et al. 1995).
DON, NIV, ZEA and fumonisins have been shown almost free from wet milling starch of maize and at the same time DON and NIV are recovered in steep water whereas fumonisins and ZEA are found mainly in the gluten fraction (Meister and Springer 2004).
The risk of mycotoxin feed contamination would be minimized by ensuring moisture control and undertaking regular monitoring and where necessary turning of feed materials. It is also important to note that the amount of reduction of mycotoxins relies heavily on the methods of processing e.g. cooking conditions, such as temperature, time, water and pH, as well as the type of mycotoxins and their concentration in the food or feedstuff.
4.3 Decontamination
Pre- and post-harvest technologies have been investigated to avoid or minimize mycotoxicosis. These are physical methods, chemical methods and detoxifying agents (Jouany 2007; Kabak et al. 2006; CAST 2003;
Ramos and Hernandez 1997; Park 1993).
4.3.1 Physical Methods
Physically, DON, ZEA, fumonisins and aflatoxins in grains can be reduced by implementing washing procedures using water and sodium carbonate solution or saturated sodium chloride solution. The DON level could be reduced by 65-69% in barley and maize wash (Kabak et al. 2006) while a 54% reduction of patulin was observed in apple during washing and handling stages (Acar et al. 1998). A 66% reduction of OTA has also been reported in hard wheat containing 618µg/kg toxin by using standard milling techniques (Kabak et
40 al. 2006). Cleaning the kernel surface, thus removing the more heavily contaminated particulate matter, have been proved effective in reducing mycotoxin concentrations when there is light to medium contamination of the feed. However, this depends on the level of fungal penetration of the endosperm (Kabak et al. 2006). In many situations once maize is contaminated with fumonisins, removing the screenings will also remove the greatest portion of the grain (Bryden 2012). A simple and the most widely used method for improving animal performance is the dilution of mycotoxin-contaminated grain with uncontaminated grain by physical mixing (Bryden 2012).This practice is not allowed in some countries e.g. Europe.
4.3.2 Chemical Methods
Chemically, the reduction of aflatoxin levels in feed by ammoniation detoxification is an approved procedure for the detoxication of aflatoxin-contaminated feed in some US States as well as in Senegal, France, and the
UK (Park and Price 2001). It can be also applied to fumonisin concentrations in maize (Norred et al. 1991).
Calcium hydroxide monoethylamine, ammonia and ozone can destroy some mycotoxins (Huwig et al. 2001;
Lemke et al. 1999; McKenzie et al. 1997; Park 1993). Nevertheless, the ineffectiveness of these agents against other mycotoxins and the possible deterioration of the animal health by excessive residual ammonia being present in the feed are the main disadvantages of using this detoxicant. Feed additives like antioxidants, sulphur-containing amino acids, vitamins, and trace elements can also be useful as detoxicants
(Nahm 1995).
4.4 Mycotoxin-Detoxified Agents
Mycotoxin-detoxified agents are assumed to detoxify the contaminated feedstuffs during passage through the digestive tract by adsorbing and/or degrading the mycotoxins (Döll and Dänicke 2004). However, their efficacy is determined by the chemical structure of both the detoxifying agent and the mycotoxin
(Avantaggiato et al. 2005). Besides, the effect of specific mycotoxins and detoxifying agents differ for each type of animal species. Therefore, the evaluation of the efficacy of detoxifying agents against the different mycotoxins presents in feeds is undertaken separately for poultry, swine, ruminants, rodents and other species (Boudergue et al. 2009).
41
Furthermore, potential mycotoxin-binding agents such as bleaching clays, synthetic cation or anion exchange zeolites, bentonite, lucerne, spent canola oil and hydrated sodium calcium aluminosilicate (HSCAS) have been investigated to reduce toxicity in poultry and livestock (Oguz 2011; Jouany 2007; Huwig et al. 2001).
Phillips et al. (2002) reported that HSCAS is a high affinity adsorbent for aflatoxins, capable of forming a very stable complex with the toxin and hence reducing its bioavailability, but is less effective with other mycotoxins.
4.4.1 Binders
The efficiency of mycotoxin binders differs considerably depending primarily on the chemical structure of both the adsorbent and the toxin (Huwig et al. 2001). A yeast cell wall derived glucomann prepared from
Saccharomyces cerevisiae has been shown in vitro to efficiently adsorb aflatoxin, ZEA and fumonisins
(Jouany 2007). The cell wall harboring proteins, lipids and polysaccharides exhibits numerous different and easily accessible adsorption centers and different mechanisms such as hydrogen bonds, ionic or hydrophobic interaction. It has been suggested that on one gram of cell wall it is possible to bind 2.7 mg ZEA (Huwig et al. 2001). Different adsorbents added to mycotoxin-contaminated diets provide hope of being effective in the gastro-intestinal tract in a prophylactic rather than in a therapeutic manner. This would make them good candidates as feed additives. Natural clay products and synthetic polymers are used as inorganic-mycotoxin- adsorbing agents. Sodium bentonite has been reported to adsorb AFB1 and FB, while zeolites affect OTA,
NIV, DAS, T-2 toxin, ZEA and AFB1 (Boudergue et al. 2009). At the same time, activated charcoal which is formed by pyrolysis of organic materials is reported to adsorb aflatoxins, fumonisins, OTA and trichothecenes (Huwig et al. 2001); while activated carbon showed relevant ability in binding DON and NIV
(Avantaggiato et al. 2005). Bentonites, zeolites and aluminosilicates are the most common feed additives that are effective in binding aflatoxins. Furthermore, cholestyramine was proven to be an effective binder for fumonisins ( in vivo ) and ZEA using an in vitro -dynamic gastrointestinal model (Avantaggiato et al. 2005).
The utilization of mycotoxin-binding adsorbents is the most applied way of protecting animals against the harmful effects of decontaminated feed (Ramos et al. 1996).
42 4.4.2 Biotransforming Agents
Isolating microorganisms and/or enzymes that will degrade or metabolize a mycotoxin rendering it nontoxic takes considerable effort (Schatzmayr et al. 2006). For instance, trichothecenes can be detoxified by stabilized bacterium (Eubacterium : BBSH 797 and LS100 are two potential microorganisms), i.e. a feed additive that removes the epoxide group in vivo (Boudergue et al. 2009; Fuchs et al. 2002) and as such are regarded as an approach to mycotoxin decontamination (Molnar et al. 2004). It has been reported that interesting results were obtained by applying Flavobacterium aurantiacum NRRL B-184 , the soil bacterium, to significantly remove aflatoxins from several substrates, including animal feeds, and it was found safe for use with chicks (Boudergue et al. 2009). The mixture of aflatoxin-contaminated substance and
Flavobacterium aurantiacum were incubated at 28°C for 12 hours and it was observed that all of the aflatoxin G was removed, as well as a part of aflatoxin B, which was diminished. Moreover, the strain
Nocardia asteroids reduced AFB1 by biotransformation to another fluorescent product (Boudergue et al.
2009). Recently, fumonisin esterase (FUMzyme ®), an enzyme-based feed additive produced from a genetically modified strain of Komagataella pastoris , which is intended to degrade fumonisin mycotoxins found as contaminants in feeds for growing pigs has been approved by EFSA. Metabolites of fumonisin B1 produced by the action of the active agent have a lower toxicity than the parent compound. Thus, it is considered safe for consumers of pigs and pork product (EFSA FEEDAP Penel 2014).
Other suggestions of animal feed additive have been made for Trichosporon mycotoxinivorans, a yeast strain showing the potential to detoxify or degrade OTA and ZEA (Molnar et al. 2004). ZEA is degraded by the yeast to be carbon oxide and other non-toxic metabolites while OTA is detoxified with the cleavage of the phenylalanine moiety from the isocumarin derivate ochratoxin α. This metabolite has been described to be non-toxic or at least 500 times less toxic than the parent compound (Molnar et al. 2004). However, with transformation of mycotoxins by microorganisms, their application in detoxification of animal feeds has been limited. This may be due to the lack of information about mechanisms of transformation, toxicity of transformation products, effects of the transformation reactions on nutritional values of the feeds as well as the safety towards animals (Boudergue et al. 2009). Fermentation procedures with microorganisms can be used to detoxify mycotoxin contaminated feed. However, these have not yet been used in practice though the
43 number of corresponding patents has increased (Duvick and Rood 2000). The microorganism conversion is generally slow and incomplete (Karlovsky 1999; Sweeney and Dobson 1998).
Conclusions
Mycotoxins are presenting in farming, feed manufacturing and food production, and many are harmful to animals and if not fatal they can importantly impact on their performances and productivity. In order to reduce the exposure of animals and humans to mycotoxins, necessary practices such as the prevention of the occurrence of mycotoxins in the first place, ensuring the consistent quality of raw feed materials, and the implementing and verifying the effectiveness of control and test procedures should be fully considered.
However, high concentrations of those mycotoxins may still exist in food and feed ingredients spite the best of efforts. It is also difficult to predict the concentration of the mycotoxin precisely in a large bulk lot because of the heterogeneity of their distribution in commodities i.e. clumping can occur, and the large variability associated with the overall mycotoxin isolation procedure. Therefore, it is crucial to continuously monitor food and feed ingredients with fast and reliable screening methods so that the extent of contamination can be fully and accurately determined and effective measures can be adopted at the relevant stages in the supply chain to mitigate such contamination should it occur.
Drying and moisture control during storage is generally well understood as a control measure, in addition to identification of various crop genotypes that are resistant to mycotoxigenic fungus infection and the potential use of biocompetitive agents in different biological control strategies to prevent the pre-harvest mycotoxins particularly aflatoxin contamination of crops.
Comprehensive food safety programs targeting both market vendor and local farmers may prevent or minimize the outbreaks of mycotoxin, especially aflatoxins. An occurrence of mycotoxins in agricultural commodities may continue to remain as a on the health and an economic issue caused in part by the complexity of the problem of masked mycotoxins, emerging mycotoxins and the problem of mycotoxin mixtures.
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58 1.3. Objectives of the PhD thesis
The thesis was aimed to: - Develop an intestinal “loop” model for studying mycotoxins toxicology (the model in between in vivo and ex vivo ) - Compare the individual or combined effect of DON and NIV on jejunum segment at different concentrations in in vivo and ex vivo models - Identify and characterize the most sensitive and/or relevant biomarkers or the endpoints at different level of tissue to be used in each model
1.4. The PhD thesis structure
The thesis includes of three chapters: - Chapter I General background and objective of the work - Chapter II Materials and methods - Chapter III Results and discussion: paper 1, paper 2 and general discussion
In Chapter III, the first published article is mainly based on the concordant results found at 4-h incubation between explant and loops model of the effect of individual DON and NIV. The second in-submitting article is based on the dose-response interaction analysis in loops and the confirmed concordant results found between 24-h-incubation loops and 28-day animal experiment of DON or DON combined with NIV.
59 Chapter II: Materials and Methods
To achieve the thesis objectives, three biological experimental models (Fig. 3.) were used and various samplings were performed on pigs. The 3 experimental pig models are (i) Explant ( ex-vivo ), (ii) Loops surgical model ( in vivo ) and (iii) animal experiment ( in vivo ).
Fig ure 3. The diagram illustrates the 3 biological models from pig and contaminations dosages used
2.1. Biological models
2.1.1. Explant model (Kolf-Clauw et al. 2009)
Six pigs were used with 3 treatments at different levels of DON and NIV and DON+NIV (1:1) at a concentration of 0, 1, 3, and 10 µM in the culture medium. In total, 72 paraffin blocks (3 explants per block) were made. The methods for explant culture, toxin exposure, histological scoring,
60 histological processing, immunohistochemistry, architectural changes, proliferative and apoptosis indices are described in paper1. Sampling process is shown in Fig. 4.
- Explant procedures Six 4-to-5-month-old crossbred piglets, housed in the animal facility of the INRA Toxalim, Research center in food toxicology, Toulouse, France, fed ad libitum with free access to water were used to obtain explants of jejunal tissue. The procedures of explant culture were performed as previously described in Kolf-Clauw et al. (2009) and Girard et al. (2005) with slight modifications. In brief, feed was deprived for 6 h prior to the explant procedures. A 5-cm middle jejunum segment was collected in prewarmed PBS added with 200 U/mL penicillin and 200 µg/mL streptomycin (Sigma, Saint-Quentin Fallavier, France). The jejunum was opened longitudinally, without removing external tunica muscularis and washed for 10 min at 39 °C, in culture medium contained with 200 U/mL penicillin and 200 µg/mL streptomycin. Sterilized biopsy punch (Stiefel laboratories LTD., Sligo, Ireland) was used to dissect the segment into 6 mm-piece diameter explants. All these operations were achieved in less than 1 h after the pig had been euthanized.
The pigs jejunal explants were incubated villi up in 6-well culture plates (Nunclon, Sigma) for 4 h in the complete cell culture medium, Williams E at 37 °C prewarmed and gassed under CO 2- controlled atmosphere, 95% O 2/5% CO 2 with orbital shaking. The medium (WME), contains 100 U/mL penicillin, 100 µg/mL streptomycin and 50 µg/mL gentamicin and supplemented with D- glucose (2.5 g/L) and 30 mM Alanine-Glutamine (Sigma). Three explants per well were incubated on 1-cm 2 biopsy sponge, in standard culture conditions.
61
Fig ure 4. The process of explants sampling. (A) Sampling jejunum segment. (B) Punching jejunum segment opened. (C) Explants treated with the mycotoxins solutions. (D) Paraffin blocks of included explants. (E) A microtome for slicing the explant blocks. (F) The slides ready for viewing under a microscope.
2.1.2. Loops model (Gerdts et al. 2001)
Five pigs were used for surgery of the jejunal loops; three pigs were sampled for 4h and two pigs 24h after injection of mycotoxin into loops. Eleven loops were made, nine of which treated with different concentrations and two of which were control loops. Each pig, one normal intestinal segment (non-loop) was also sampled. Each loop was injected with 3 mL mycotoxin solution of DON and NIV and DON+NIV (1:1) at a concentration of 0, 1, 3 and 10 µM. In total, 70 paraffin blocks (3 pieces of intestinal segment per block) were made. The methods for loops incubation, toxin exposure, histological processing, immunohistochemistry, architectural changes, proliferative and apoptosis indices are described in paper 1 and 2.
- Loops surgical processes and procedures Five 2-month-old Large White female pigs weighing 20-25 kg, maintained in warmed compartments with straw, fed with pelleted stock diet (Sevryplus-SANDERS) were used. Animals were fasted for 6 h prior to surgery but the pigs had full access to water. The loop surgery was performed as described in Girard-Misguich et al. (2011) and Gerdts et al. (2001). In short, for
62 anaesthesia, premedication was started with an intra-muscular (IM) injection of 2 mg/kg bw Xylazine (Rompun ® 2%, Bayer-Schering AG, Leverkusen, Germany) with 20 mg/kg bw ketamine (Imalgene ® 1000, Merial SAS, Villeurbanne, France). The animals were intubated using a cuffed endotracheal tube (Rüsch, Waiblingen, Germany) and anaesthesia was maintained with closed circuit of Isoflurane 3% and oxygen and ventilated with a veterinary anesthesia ventilator (Model 2000, Hallowell EMC, Pittsfield, MA, USA) in volume-controlled mode (100% oxygen).
After cleaning and disinfection of the abdomen, a midline abdominal incision was made and a 100- 150 cm long segment of intestine was exteriorized and surgically prepared in the jejunum, where Peyer’s Patches could rarely be individualized. Eleven loops, each 10-12 cm in length with 2-3 cm interloop in between were produced from one pig through the mesentery without damaging grossly visible mesenteric arcades and thus maintaining full blood supply for both loops and interloop segments (Fig. 5.). An end-to-end anastomosis was made to maintain the intestinal flow from duodenum to jejunum and ileum in the gut besides the isolated loops. Before producing the loops, ingesta in the prepared jejunal segments were removed by flushing 2 times with a warm 100 mL physiological water solution. The exteriorized jejunum was frequently moistened with sterile physiological serum. The created-jejunum segments were in situ maintained and the midline abdominal incision was closed with U-shape interrupted sutures (peritoneum and muscles or abdominal wall) and the continuous sutures (skin). Pigs were treated with 0.15 mg/kg IM analgesic buprenorphine (Buprecare, Animalcare, Dunnington York, UK) immediately after the operation had been finished. The surgery lasted for almost 2 h per pig.
After 4 h (3 pigs) and 24 h (2 pigs) post-surgery, the pigs were intravenously (IV) euthanized by barbiturate overdose to collect created jejunum-loop segments. Jejunal loops were quickly collected and washed twice with physiological serum prior to be fixed within 10% formalin.
63 Fig ure 5. Surgical creation of intestinal segment (A) and loops (B/I). The intestinal segment at 100-150 cm long was separated from the jejunum, which was then rejoined with an end-to-end anastomosis (B/II). The intestinal segment was subdivided into 11 loops per pig. Each loop was 10-12 cm long with 2-3 cm interloops (B/III).
2.1.3. Animal experiment
Two different animal experiments each carried out at 2 different locations (Arvalis, Vendome and Toxalim, Toulouse) with 24 pigs (male and female) and with duration of 28 days per each, and a total of 48 pigs were sampled. Four treatments were designed with DON, DON+NIV and DON+. The animals were fed with naturally contaminated feed. The exposed groups were Control (Similar to non-contaminated feed), DON at 2.89 mg.kg -1 feed, DON 3.5 mg.kg -1 + NIV 0.72 mg.kg -1 feed and DON+ at 4.3 mg.kg -1 feed. Samples were taken from 9 organs such as duodenum, jejunum, ileum, Peyer’s patch jejunum, colon, thymus, liver and mesenteric lymph nodes. There were 432 samples embedded in paraffin block in total from 48 pigs. In the experiment conducted at Toxalim, pigs (n=24) were blood sampled at day0 (the first day) before feeding, at day7 and at day28 (the end of the experiment). The parameters (Fig. 6.) examined in vivo were growth performance, feed intake, clinical biochemistry: blood total protein, albumin, fibrinogen, Gamma-glutamyl transferase (GGT). The animal housing conditions and experimental protocols are described in paper 2. The results and discussions will be mainly focused excluding the DON+ treatment due to the realistic contamination of the mycotoxin.
64
Figure 6. The diagram showing parameters for sampling in the animal experiment.
2.2. Histological assessment
2.2.1. Morphometry
The jejunum segments from the three biological models were used for an assessment. The explants were assessed by a morphological scoring system. The loops (4h and 24 h) and the animal experiment were assessed by measuring villus height and crypt depth. The assessment and measurement protocols are described in paper 1.
2.2.2. Immunohistochemistry
The tissues from loops model (jejunum segment) and from animal experiment (jejunum and Peyer’s patches jejunum) were processed for histology and immunohistochemistry (for proliferation; Ki-67 and apoptosis; caspase-3). The procedures of this process are described in paper 1 and paper 2. The intestinal crypts were first chosen for proliferative enterocyte count, and then followed by total-cell
65 proliferation and total-cell apoptosis count, finally the proliferative enterocyte and apoptotic enterocyte counts at the tip of the villi.
2.3. Gene expression (qRT-PCR)
Since DON acts on intestinal immunomodulation and junction protein genes, we investigated the effects of DON by using qRT-PCR (quantitative Real-Time Polymerase Chain Reaction) analysis in loops model. In the jejunum segments, treated at 10 µM DON (4 h and 24 h), were determined the expression of mRNA encoding for IL-1α, IL-1β, IL-6, IL-8, IL-10, IL12-p40, IL-21, TNF-α, TGF- β, CCL-20, IFN-γ, EDN-2, NF-κB, ALP, TLR-1, TLR-2, TLR-5, TLR-9, PCNA, Lysozyme, ZO-1, E-cadherin, Occludin, Claudin-1, Claudin-2, Claudin-3, Claudin-4 (Table 3.). The cyclo A and β- actin were used as housekeeping genes to normalize the values obtained. The genes expression was calculated relative to the control treatment. The protocols were previously described in Cano et al. (2013). Briefly, the jejunum tissue pieces stored at -80 °C after fixing in liquid nitrogen, were extracted with Trizol Reagent (Extract all, Eurobio) for total RNA. RNA quality, concentration and integrity were assessed spectrophotometrically using Nanodrop ND1000 (Labtech International, Paris, France). Then, the steps of reverse transcription and real-time qPCR were performed as described in Meissonnier et al. 2008. To verify genomic DNA amplification signal, non-reverse transcripted RNA was used as non-template control. Finally, dissociation curves were analyzed and assessed for the specificity of qPCR products after the reactions. Primers were purchased from Invitrogen (Invitrogen, Life Technologies Corporation, Paisley, UK). The DDCt method was used to determined amplification efficiency and initial fluorescence.
Table 3 . List of genes used in qTR-PCR and theirs primer sequences (F: forward, 5' "3'; R: reverse, 3' "5'). Accession number Gene symbol Gene name Primer sequence (reference) F: TCAGCCGCCCATCCA NM_214029,1 IL-1α Interleukin 1 - alpha R: AGCCCCCGGTGCCATGT (Cano et al. 2013) F: ATGCTGAAGGCTCTCCACCTC NM_214055 IL-1β Interleukin 1 - beta R: TTGTTGCTATCATCTCCTTGCAC (Von der Hardt et al. 2004) F: TTCACCTCTCCGGACAAAACTG NM_214399 IL-6 Interleukin 6 R: TCTGCCAGTACCTCCTTGCTGT (Gourbeyre et al. 2014) F: GCTCTCTGTGAGGCTGCAGTTC NM_213867 IL-8 Interleukin 8 R: AAGGTGTGGAATGCGTATTTATGC (Grenier et al. 2011) F: TGAGAACAGCTGCATCCACTTC NM_214041 IL-10 Interleukin 10 R: TCTGGTCCTTCGTTTGAAAGAAA (Royaee et al. 2004) F: GGTTTCAGACCCGACGAACTCT NM_214013 IL12-p40 Interleukin 12 p40 R : CATATGGCCACAATGGGAGATG (Cano et al. 2013)
66 F: GGCACAGTGGCCCATAAATC MN_214415 IL-21 Interleukin 21 R: GCAGCAATTCAGGGTCCAAG (Kiros et al. 2011) F: ACTGCACTTCGAGGTTATCGG NM_214022 TNF-α Tumor necrosis factor - alpha R: GGCGACGGGCTTATCTGA (Meissonnier et al. 2008) F: GAAGCGCATCGAGGCCATTC NM_214015 TGF-β Transforming growth factor - beta R: GGCTCCGGTTCGACACTTTC (Meurens et al. 2009) F: GCTCCTGGCTGCTTTGATGTC NM_001024589 CCL-20 Chemokine (CCL20) R: CATTGGCGAGCTGCTGTGTG (Meurens et al. 2009) F: TGGTAGCTCTGGGAAACTGAATG NM_213948 IFN-γ Interferon - gamma R: GGCTTTGCGCTGGATCTG (Royaee et al. 2004) F: TACTTCTGCCACTTGGACATCATC ENST00000372587 EDN-2 Endothelin 2 R: GGCCGTAAGGAGCTGTCTGT (Gourbeyre et al. 2014) F: CCTCCACAAGGCAGCAAATAG ENSSSCT00000033438 NF-κB Nuclear factor-kappa-B R: TCCACACCGCTGTCACAGA (Present study) F: AAGCTCCGTTTTTGGCCTG ENSSSCT00000017732 ALP alkaline phosphatase R: GGAGGTATATGGCTTGAGATCCA (Gourbeyre et al. 2014) F: TGCTGGATGCTAACGGATGTC AB219564 TLR-1 Toll-like receptor 1 R: AAGTGGTTTCAATGTTGTTCAAAGTC (Arce et al. 2010) F: TCACTTGTCTAACTTATCATCCTCTTG AB085935 TLR-2 Toll-like receptor 2 R: TCAGCGAAGGTGTCATTATTGC (Gourbeyre et al. 2014) F: CCTTCCTGCTTCTTTGATGG NM_001123202 TLR-5 Toll-like receptor 5 R: CTGTGACCGTCCTGATGTAG (Meurens et al. 2009) F: CACGACAGCCGAATAGCAC AY859728 TLR-9 Toll-like receptor 9 R: GGGAACAGGGAGCAGAGC (Arce et al. 2010) F: GTTGATAAAGAGGAGGAAGCAGTT ENSSSCT00000032581 PCNA Proliferating cell nuclear antigen R: TGGCTTTTGTAAAGAAGTTCAGGTAC (Gourbeyre et al. 2014) F: GGTCTATGATCGGTGCGAGTTC ENSSSCT00000034939 Lysozyme Lysozyme R: TCCATGCCAGACTTTTTCAGAAT (Gourbeyre et al. 2014) F: ATAACATCAGCACAGTGCCTAAAGC AJ318101 ZO-1 Zonula occludens-1 R: GTTGCTGTTAAACACGCCTCG (Present study) F: ACCACCGCCATCAGGACTC ENSSSCG00000006369 E-cadherin E-cadherin R: TGGGAGCTGGGAAACGTG (Present study) F: AGCTGGAGGAAGACTGGATCAG U79554 Occludin Occludin R: TGCAGGCCACTGTCAAAATT Yamagata et al. 2004 F: GGGCAGATCCAGTGCAAAGT AJ318102 Claudin-1 Claudin-1 R: GGCTATTAGTCCCAGCAGGATG (Present study) F: CAGCATGAAATTTGAGATCGGA NM_001161638.1 Claudin-2 Claudin-2 R: GAGGAAATGATGCCCAAGTAGAGA (Present study) F: CTGCTCTGCTGCTCGTGCCC AY625258 Claudin-3 Claudin-3 R: TCATACGTAGTCCTTGCGGTCGTAG (Present study) F: CTGCTTTGCTGCAACTGCC AK233156 Claudin-4 Claudin-4 R: TCAACGGTAGCACCTTACACGTAGT (Present study) F: CCCACCGTCTTCTTCGACAT NM_214353 Cyclo A Cyclophilin A R: TCTGCTGTCTTTGGAACTTTGTCT (Curtis 2009) F: TCATCACCATCGGCAACG AY550069 β-actin beta actin R: TTCCTGATGTCCACGTCGC (Von der Hardt et al. 2004)
67
Chapter III: Results and discussion
3.1. Paper 1: Nivalenol has a greater impact than deoxynivalenol on pig jejunum mucosa in vitro on explants and in vivo on intestinal loops
68 Toxins 2015, 7, 1945-1961; doi:10.3390/toxins7061945 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Article Nivalenol Has a Greater Impact than Deoxynivalenol on Pig Jejunum Mucosa in Vitro on Explants and in Vivo on Intestinal Loops
Sophal Cheat 1,2,3,†,*, Juliana R. Gerez 1,2,4,†, Juliette Cognié 5, Imourana Alassane-Kpembi 1,2,6, Ana Paula F. L. Bracarense 4, Isabelle Raymond-Letron 7,8, Isabelle P. Oswald 1,2 and Martine Kolf-Clauw 1,2,*
1 Université de Toulouse, Institut National Polytechnique-Ecole Nationale Vétérinaire (INP-ENVT), Unité Mixte de Recherche UMR 1331 Toxalim, Research Center in Food Toxicology, 23 chemin des Capelles, F-31300 Toulouse, France; E-Mail: [email protected] (J.R.G.) 2 INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, 180 chemin de tournefeuille F-31027 Toulouse, France; E-Mails: [email protected] (I.A.K.); [email protected] (I.P.O.) 3 Faculty of Animal Science and Veterinary Medicine, Royal University of Agriculture, P.O. box 2696, Phnom Penh, Cambodia 4 Laboratory of Animal Pathology, Universidade Estadual de Londrina, 86057-990 Londrina, Brazil; E-Mail: [email protected] 5 Plate-forme CIRE Chirurgie et Imagerie pour la Recherche et l’Enseignement UMR 085 PRC, INRA, 37380 Nouzilly, France; E-Mail: [email protected] 6 Hôpital d’Instruction des Armées, Camp Guézo 01BP517 Cotonou, Benin 7 INP-ENVT, Université de Toulouse, F-31300 Toulouse, France 8 STROMALab UMR5273 UPS EFS INSERM U1031, 1 Avenue Jean Poulhes, 31403 Toulouse, France; E-Mail: [email protected]
† These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: [email protected] or [email protected] (S.C.); [email protected] (M.K.-C.); Tel.: +855-12555-572 (S.C.); +33-561-193-283 (M.K.-C.); Fax: +33-561-193-978 (M.K.-C.).
Academic Editor: Sven Dänicke
Received: 22 March 2015 / Accepted: 20 May 2015 / Published: 29 May 2015
Toxins 2015, 7 1946
Abstract: The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells) were more sensitive than enterocytes (epithelial cells) to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment.
Keywords: mycotoxins; jejunum explant; loops; deoxynivalenol; nivalenol; enterocytes; histomorphology
1. Introduction
Fungi of the Fusarium genus commonly contaminate cereals in the temperate climatic zones of the world and contribute to poor quality grains entering the feed and food chain. Among the mycotoxins produced by Fusarium, the large group of trichothecenes is extremely prevalent, particularly deoxynivalenol (DON) for which many exposure and toxicological surveys have been carried out or reviewed recently [1,2]. Nivalenol (NIV), classified with DON as a type B trichothecene, is a biologically active metabolite of DON, present in agricultural commodities [3,4]. A large-scale data survey has indicated that DON and NIV are present in 57% and 16%, respectively, of food samples collected in the European Union [5]. From their first discovery, there has been concern about the relationship between trichothecenes exposure and health damage based on both experimental toxicity and epidemiological data. Studies have shown that mycotoxins cause toxic effects in humans as well as in all animal species so far investigated, the pig being the most sensitive species [6]. Studies in laboratory and farm animals have revealed a complex spectrum of toxic effects. Experimentally, low to moderate acute oral exposure to trichothecenes cause vomiting, diarrhea and gastroenteritis, whereas higher doses cause severe damage to the lymphoid and epithelial cells of the gastrointestinal mucosa resulting in hemorrhage, endotoxemia and shock. Chronic exposure to trichothecenes can cause anorexia, reduced weight gain, diminished nutritional efficiency, neuroendocrine changes, and immune modulation. Although not as prevalent as
DON [7], NIV showed higher acute toxicity than DON in animal studies, with oral LD50 values in mice of 78 and 39 mg·kg−1 for DON and NIV, respectively [8]. NIV is of added concern for food safety but in vivo information for assessing the health risk remain scarce, and NIV toxicity is considered similar to
Toxins 2015, 7 1947
DON toxicity for protecting human health [9]. At the molecular level, DON and NIV, like other trichothecenes, display multiple inhibitory effects on the primary metabolism of eukaryotic cells including the inhibition of proteins, DNA and RNA synthesis [10]. This impairment leads to altered cell proliferation in tissues with high rates of cell turnover such as spleen, bone marrow, thymus and intestinal mucosa [11]. Following ingestion of contaminated feed or food, the intestine and intestinal mucosa can be exposed to a high concentration of food contaminants, such as mycotoxins [12]. However, only a few studies have investigated the effects of mycotoxins on this target, even though there is increasing evidence that intestinal epithelium is repeatedly exposed to mycotoxins at a higher concentration than other tissues [13]. Pigs receiving 3 mg/kg feed of DON for five weeks showed significant histopathological changes compared to control animals, such as atrophy and fusion of villi and reduction of the number of goblet cells and lymphocytes [14]. Little is known about the effects of NIV on the intestinal tract of pigs. Pigs receiving 2.5 or 5 mg NIV/kg of feed for three weeks, showed gastrointestinal erosions [15] and reduced enzymatic ability of the intestinal epithelium [16]. In the context of implementing the 3Rs, “Replace, Reduce, Refine” [17], in vitro and in vivo alternatives can be used to reduce the number of experimental animals. An intestinal explants-in vitro model and an intestinal loops-in vivo model have been developed for studying intestinal responses to pathogens [18,19]. The culture of intestinal explants allows preservation of the normal histological structure in vitro [20]. The pig jejunal explant model has previously been used to study the digestive effects of the mycotoxin DON [21–23], and to analyze the toxicity of mixtures of mycotoxins [24]. The present work was designed to compare the acute impacts of DON and NIV on pig jejunal mucosa. The above two models were used in parallel. First, a dose-response study with explants was carried out to estimate the toxic dose for DON and NIV on the mucosa, then, jejunal loops were injected with the two toxins at the selected toxic dose. In the two models, the results, assessed after 4-h of exposure, were concordant, showing a greater impact of NIV compared to DON on the intestinal mucosa.
2. Results
2.1. Explants Model
2.1.1. Histological Analysis before and after Incubation and Effect of DMSO
First, the effects of the culture and of DMSO on the histology of the jejunal explants were investigated. The explants were observed microscopically and scored from 22 (no lesion) to 0. Before incubation (T0), the scores were between 16 and 21 for all explants (Figure 1 panel I). The histological lesions observed were mild edema in the lamina propria and slight dilatation of the lymphatic vessels, resulting in an average score of 18 ± 2 (Figure 1 panels I and IIa). After incubation in Williams E Medium (WME) for 4 h, with or without DMSO, the scores did not differ significantly from those of the non-incubated explants (Figure 1I), although flattened villi were apparent after this incubation period (Figure 1IIb). The mean villus height was 141 ± 29 µm in the explants incubated with WME alone and 147 ± 41 µm in the explants incubated with DMSO and did not differ significantly from the T0 results. No statistically significant difference was observed between the different incubation groups, with 0.1% DMSO, or without DMSO (Figure 1I). The scores of control
Toxins 2015, 7 1948 explants with or without DMSO were grouped into a single 4-h culture control group for subsequent analyses (n = 84).
Figure 1. Histological scores of jejunal explants. (I) Explants exposed to different treatments: T0 (Time 0H, before culture ), T4/WME (4 h in William’s medium E), T4/WME + 0.1%DMSO (dimethylsulfoxyde), DON (deoxynivalenol) or NIV (nivalenol): 1, 3 and 10 µM. Values are mean ± SEM. (II) Effect of DON and NIV on the histological score after 4 hours of exposure. Values are mean ± SEM. a, b, c scripts are different at p ≤ 0.05 by Tukey’s test (II) (a) Jejunal explant uncultured (T0; n = 12). Slight dilatation of the lymphatic vessels (arrow), HE (hematoxylin-eosin), ×200; (b) explants exposed to WME with 0.1% DMSO (DMSO n = 42). Edema of the lamina propria and mild villus atrophy (arrow), HE ×200; (c) 3 µM DON-exposed explant. Moderate fusion and cubic epithelial cells (arrow) (HE, ×200); (d) 10 µM NIV-exposed explant. Fusion and atrophy of villi with severely flattened epitelium (arrow) and apical denudation of villi (dotted arrow) (HE, ×200).
Toxins 2015, 7 1949
2.1.2. Effect of Mycotoxins on the Histological Scores
Each treatment, DON (1–10 µM) and NIV (1–10 µM) induced a dose-dependent decrease in the histological scores of the jejunal explants after 4 h of exposure (p < 0.01) (Figure 1II). In the explants exposed to DON, the main morphological change was coalescence with moderate fusion of villi. Lesions included cubic epithelial cells instead of the cylindrical epithelial cells seen in the control, areas of edema in the lamina propria, villus atrophy and apical denudation of villi with focal loss of apical enterocytes (Figure 1IIc). In the group treated with NIV, the changes were similar to those of the group exposed to DON but both the flattening of the epithelial cells and apical denudation of the villi were more severe (Figure 1IId). The individual treatments with the mycotoxins DON and NIV resulted in a significant decrease of the histological score from doses of 3 µM and 1 µM, respectively. The corresponding scores were reduced to about 70% of the control explants by 3 µM and 10 µM DON or 1 µM NIV, to almost half the mean score of control explants (59% ± 6%) by the highest NIV concentration (Figure 1II). So, NIV showed greater toxicity than DON in the explant model, with a lowest observed effect concentration of 1 µM.
Figure 2. Jejunum morphology in a non-loop segment (a) and in a control loop (b) showing vascular changes in the submucosa (large arrow) and edema of the villi central lymphatic vessels (thin arrows); (c) Ki-67 immunostaining in a control loop, showing the methodology for morphometric and proliferation assessments.
Toxins 2015, 7 1950
2.2. Loops Model
2.2.1. Comparison of Loops Segments with Non-Loops Segments
The surgery induced vascular disorders in the loops, more pronounced in the serosa and submucosa layers, with moderate interstitial edema, congestion, focal blood extravasation and moderate focal dilation of the central lacteal in the villi tips (Figure 2b). The proliferation and apoptosis counts did not differ between the control loops and non-loops segments, and were subsequently used as endpoints after toxins exposure.
2.2.2. Effect of DON and NIV Exposure on Morphometry in the Loops
The mean crypt-depth to villus-height ratios after DON (0.99 ± 0.15) and NIV (1.01 ± 0.16) treatments were increased by 15%–20% compared to the control ratio (0.86 ± 0.11) without significant difference between DON and NIV (Figure 3).
Figure 3. Morphometric analysis of the jejunum loops: crypt-depth to villus-height ratio after DON and NIV exposure at 10 µM for 4 h. Mean values ± SEM expressed as % of the control group; a, b scripts are different at p ≤ 0.05; Tukey’s test; n = 3 to 6 loops, 30 well-oriented villi and crypts per loop.
2.2.3. Effect of DON and NIV Exposure on Proliferation in the Loops
At the villus tip, a significant decrease was observed in the total cells proliferation compared with control loops. NIV exposed loops showed a significant 30% decrease in the number of cells proliferating in the mucosa (p < 0.001), while DON-exposed loops showed a 13% decrease compared with the controls (Figure 4A). At the crypt level, proliferative index of crypt enterocytes was increased only after NIV treatment (p = 0.001, Figure 4B).
2.2.4. Effect of DON and NIV Exposure on Apoptosis in the Loops
At the villus tip, the number of total apoptotic cells was higher in NIV-exposed loops than in the controls. A tendency was detected for the enterocyte apoptotic index (p = 0.057) with mean values of
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2.36% ± 1.12%, 2.57% ± 1.52% and 3.43% ± 2.55% for the control, DON and NIV, respectively. The total-cell proliferation to total-cell apoptosis ratios at villus tip showed a significant decrease (p < 0.001) with values of 6.98 ± 1.84, 5.60 ± 1.65 and 3.89 ± 1.2 for the controls, DON and NIV, respectively. NIV reduced the total-cell proliferation to total-cell apoptosis ratio at the tip of the villus by 44% compared to the controls, while DON reduced this ratio by only 20% (Figure 4D). In lamina propria, the number of apoptotic cells was significantly increased by NIV (p < 0.001, Figure 4C).
200 (A) Global proliferation (total cell count) 200 (B) Proliferative index of crypt enterocytes
150 150 b a b a a c 100 100 % comparedto Ctrl 50 % comparedto Ctrl 50
0 0
200 (C) Lamina propria apoptosis 200 (D) Villus proliferation/apoptosis ratio
b 150 150 ab a a b 100 100 c
% compared to Ctrl 50 % compared to Ctrl 50
0 0 Ctrl DON NIV Ctrl DON NIV
Figure 4. Proliferation and apoptosis in the jejunum loops after DON and NIV exposure at 10 µM for 4 h. Mean values ± SEM expressed as % of the control group (Ctrl) (A) total cell proliferation at villus tip (upper one-third), p < 0.001; (B) proliferative index of crypt enterocytes, p = 0.001; (C) lamina propria apoptosis at villus tip (upper one-third), p < 0.001; and (D) total proliferating cells to total apoptotic cell ratio, at villus tip p < 0.001. a, b, c scripts are different at p ≤ 0.05; Tukey’s test; n = 3 to 6 loops, 20 villi/loop.
3. Discussion
Two alternative models were used in this study to analyze intestinal mucosal toxicity, a major target for xenobiotics. An explant model, previously shown to be sensitive [23,24], was first used to demonstrate dose-related toxicity following in vitro exposure to DON and NIV, and a higher toxicity of NIV. This result was then confirmed in vivo, using the intestinal loops model.
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3.1. The Jejunum Explants and Loops Alternative Models Reduce the Number of Animals
These two alternative models enabled to reduce the number of animals in experiments, according to the 3Rs recommendations, as the explants or loops are the experimental unit and not the whole animal. Pig intestinal explant culture represent a relevant model for investigating the effects of feed and food contaminants, due to the relevance of the pig model-species in relation to humans, and its high sensitivity to mycotoxins. In the current study, before analyzing the effects of the mycotoxins DON and NIV on the scores, the histological scoring was refined and demonstrated the absence of impact of DMSO up to 0.1% as solvent. This study illustrates for the first time, to the best of our knowledge, the use of intestinal loops in toxicology. However, the scoring system developed for the explants was not suitable to use in the loops model, because of the inter-loops variations brought about by the surgery-induced vascular changes (edema in the lamina propria). Proliferation and apoptosis were used to assess the in situ mucosal changes, being quantifiable biomarkers specifically affected in vitro and in vivo by the trichothecenes [14,25–28].
3.2. Acute Exposure to NIV More Toxic in Vitro on Jejunum Mucosa than DON
Our study revealed higher intestinal mucosa changes after acute exposure to NIV than to DON exposure, both in vitro and in vivo. Following in vitro 4-h single exposure, both trichothecenes induced a dose-related decrease of the explants scores, NIV showing a higher toxicity than DON. Significant changes were observed in explants exposed to 3 and 10 µM DON. The main histological changes were focal enterocyte desquamation, moderate atrophy and fusion of villi, in accordance with previous studies [21,23,27]. The 30% reduction of the histological score following 10 µM DON was similar to the results obtained by Basso et al. [21] who reported a 37% decrease of the histological score. In our experiment, all doses of NIV significantly affected the histological scores of the explants. NIV toxicity on intestinal cells in vitro has already been reported. For example, NIV decreased dose-dependently the viability of Caco-2 and IPEC-J2 cells [29,30]. In a previous study, the reduction of IEC-6 viability due to treatment with NIV was related to apoptosis induction [26]. The less severe toxic effect of DON, as compared to NIV, on intestinal explants is also in accordance with previous studies, which demonstrated that NIV exerted a stronger effect than DON on both intestinal and non-intestinal cell lines [26,28,29,31,32].
3.3. Acute Exposure to NIV More Toxic in Vivo on Jejunum Mucosa than DON
In the loops model, as in the explants study, the intestinal toxicity of NIV was higher than that of DON. At 10 µM, both DON and NIV increased the crypt-depth to villus-height ratios, reflecting intestinal damage in vivo, as described in conventional animal experiments [33]. At 10 µM, DON and NIV induced in vivo lamina propria apoptosis and decreased total cells proliferation at the villus tip in the loops model, while only NIV increased the enterocytes proliferation index at the crypt level. So, our study shows that the lamina propria cell populations are the most sensitive target of the jejunum mucosa, after DON and NIV exposure. The major cell populations of lamina propria, besides connective tissue cells, are immune cells, with high proliferative rate, recognized as the most sensitive cells to DON toxicity. These targets have been previously described to explain the modulation of the intestinal immune
Toxins 2015, 7 1953 response induced by DON and other trichothecenes [34]. Few studies have analyzed the action of NIV on intestinal morphology. Chronic ingestion of NIV induced gastrointestinal erosions in young pigs (2.5 or 5 mg/kg) [15], whereas C57BL/6 mice exposed to NIV subchronically or chronically by feeding did not show any alterations in the histological architecture of small intestine [35,36]. These differences could be related to the highest sensitivity of the pig, reported to be the most sensitive species to mycotoxins [6,37]. In the present study, the proliferation mucosal response at crypt level was significant for NIV. These results are in accordance with previous comparative studies of the two toxins, in vitro or in vivo, with other endpoints than the intestinal target. For example, NIV showed higher anorectic potency in mice [38].
3.4. DON and NIV Induced Apoptosis in Vivo on Loops
The intestinal effects observed after 4-h exposure to DON and NIV can be mediated by oxidative stress, inducing intestinal cell membrane alteration and apoptosis. DON-induced oxydative stress has been shown in splenic tissue in a rodent model [39], as well as lipid peroxidation in vitro in HepG2 cells exposed to levels similar to the present study [40]. The alterations caused by DON in jejunal explants have been correlated to MAPKs signaling pathway activation [22], and to up-regulation of pro-inflammatory cytokines [41].
3.5. Relevance of the Results for Risk Characterization
The effects of mycotoxins were assessed at realistic concentrations in the present study, considering the concentrations of mycotoxins to which the consumer can be exposed via food. The results are therefore of high relevance for risk characterization of DON and NIV exposure. DON concentrations of 0.16–2 μg/mL (0.5–7 μM) in the human gut can be considered as realistic [42]. The lower concentration corresponds to the mean estimated daily intake of French adult consumers on a chronic basis [43]. The higher concentration is the simulated levels that can be attained after the consumption of heavily contaminated food, and is occasionally encountered. The amount of NIV in cereal products varies considerably between different countries across the world (from 20–60 µg/kg in France, to 584–1780 µg/kg in China) [44]. Significant architectural and lesional alterations were observed from doses of 1 µM NIV in this study, which is consistent with the levels plausibly encountered in the gastrointestinal tract after the consumption of heavily contaminated food.
4. Materials and Methods
4.1. Animals
For explants sampling, six 4–5 week-old crossbred piglets were used, housed in the animal facility of the INRA ToxAlim Laboratory (Toulouse, France). For the loops experiment, three two-month-old Large White female pigs were used and housed in the animal facility at INRA Nouzilly. All animals were fasted for 6 h before explants sampling or loops surgery. The experimental procedures were conducted in accordance with European Guidelines for the Care and Use of Animals for Research Purposes and were approved by the INRA local ethical committees for animal experimentation
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(C2EA-86 for explants, and “Comité d’Ethique en Expérimentation Animale Val de Loire”, C2EA-06, for the loops experiments).
4.2. Toxins
DON was acquired from Sigma (St Quentin Fallavier, France) and NIV from Waco Pure Chemical Industries LTD (Osaka, Japan). Stock solutions of these mycotoxins were dissolved in dimethyl sulfoxide (DMSO Sigma, Saint-Quentin Fallavier, France) at the following concentrations: 15 mM DON and 10 mM NIV for explants, and at 30 mM DON and NIV for the loops experiments. These stock solutions were stored at −20 °C. Working dilutions were prepared in William’s medium E (WME-Sigma, Saint-Quentin Fallavier, France) for the explants, and in physiological saline solution for the loops. The concentrations range for the dose-response explants study was 0–10 µM, selected after preliminary explants cultures with 0.1 to 30 µM of each toxin.
4.3. Jejunum Explants Experiment (in Vitro)
4.3.1. Jejunal Explants
The procedure for the culture of explants was as previously described [23,24]. Explants were incubated for 4 h with WME at 37 °C under a CO2-controlled atmosphere with orbital shaking. Uncultured control tissue was placed in fixative immediately after dissection, as time 0 controls (T0, n = 12, two explants/pig). In view of the possible effects of DMSO on intestinal morphology, the final concentration of 0.1% DMSO corresponding to the highest DMSO concentration in the working dilutions was tested in 42 explants (with and without DMSO: 84 explants). Twelve explants were exposed to purified DON and NIV at each of the concentration 1, 3 and 10 µM, for 4 h, respectively (two explants/pig for each condition).
4.3.2. Histological Scoring
For histological analysis, the explants fixed in 10% formalin (VWR, Strasbourg, France) were embedded in paraffin (VWR) and sectioned at 3–5 µm thickness parallel to the villus axis and stained with hematoxylin (VWR) and eosin (CML, Nemours, France) (HE) using standard procedures. The resulting slides were analyzed independently by two observers, at 100× magnification. The histological changes were evaluated using a tissue scoring system [23] with minor modifications. The scoring system included both architectural and lesional criteria, as shown in Table 1. The maximum score was attributed to the T0 tissue, before incubation, for each criterion. The architectural score included the number of villi per explant and the fusion of villi. This latter was expressed as the 97.5th percentile of the percentage of fused villi (number of fused villi/non fused villi 100×). The score of 3 for villus fusion corresponded to a maximum of 11% fused villi. At least 25 villi needed to be counted per explant to obtain the score of 3. The lesional score included morphology of enterocytes (score 3 for columnar epithelium), the degrees of edema and apoptosis in the lamina propria (score 2 for slight flattening of villi), and the extent of discontinued epithelium qualified as apical denudation of villi. This endpoint was quantified by the 97.5th percentile of the percentage of the villi showing apical denudation (score 3 for T0 explants).
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For explants lesions, score 3 corresponded to a maximum of 10% apical denudation and scores of 2, 1, and 0, to 11%–40%, 41%–70%, and 71%–100%, respectively. For lesion of the lamina propria, localized edema was scored as 1, whereas multifocal edema and apoptosis were scored as 0. The total score was calculated by taking into account the degree of severity for the lesions (severity factor). For each lesion, the score (according to intensity or observed frequency) was multiplied by the severity factor of 2. The total score for each explant was then obtained from the sum of each criterion. Each score value was the result of 2 explants/pig/condition. The maximum score (22 points) indicated overall integrity of the intestine. The histological scoring system was applied to compare the microscopic changes observed after 4-h exposure of the explants to DON and NIV.
Table 1. Explants histological scoring: endpoints used and severity factor. Score component Criteria (severity factor) End-point Score Columnar epithelium 3 <50% cuboid epithelium 2 Enterocytes morphology (2) >50% cuboid epithelium 1 Flattened epithelium 0 0%–10% 3 Lesional part of the Score 11%–40% 2 Apical denudation of villi (2) 41%–70% 1 71%–100% 0 No lesions, slight flattening of villi 2 Lesions of lamina propria (2) Localized edema and apoptosis 1 Multifocal edema and apoptosis 0 0%–11% 3 12%–40% 2 Villi fusion (1) 41%–70% 1 71%–100% 0 Architectural part of the Score ≥25 3 16–24 2 Number of villi (1) 5–15 1 ≤4 0
4.4. Jejunum Loops Experiment (in Vivo)
4.4.1. Jejunal Loops Injection and Sampling
A 1-m long segment of intestine was surgically prepared in the jejunum, to constitute the loops as previously described [19,45]. This segment was then subdivided into consecutive segments, designated as “loops” (10 cm long, 6 loops), separated by “inter-loops”. Three treatments, control (Ctrl), DON and NIV at 10 µM concentration were used for each of the 3 pigs (1 to 2 loops/pig for each condition, n = 6 loops for the controls) by injecting 3 mL of each test condition into each loop. Four hours after surgery, the pigs were euthanized by barbiturate overdose (pentobarbital, Vetoquinol, Lure, France) and the created jejunum-loop segments were collected. These loops were washed twice with physiological serum prior to fixation. In addition, a non-loop segment was sampled and processed in parallel.
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4.4.2. Histological Processing
A routine histological processing sequence (from 10% buffered formalin to paraffin block) was used. Paraffin sections 4-µm thick were stained with HE to assess architectural changes and immunohistochemically labeled (IHC) to assess proliferation and apoptosis.
4.4.3. Immunohistochemistry
Two commercial antibodies were used as previously described [46,47]. Briefly, four-micrometer paraffin-embedded transverse sections from formalin-fixed jejunum specimens were dewaxed in toluene and rehydrated by an acetone bath then deionized water. Antigen retrieval was performed in 10 mM citrate buffer pH 6.0 for 30 min in a water bath at 95 °C. Cooled sections were then incubated in Dako peroxidase blocking solution (Dako S2023) to quench endogenous peroxidase activity. Non-specific binding was blocked by incubation in normal goat serum (dilution 1:10, Dako X0902) for 20 min at room temperature. The primary antibodies were anti-Ki-67antigen (Dako M7240, dilution 1:50) and anti-active caspase-3 (R&D system, AF835, dilution 1:300). Sections were incubated with primary antibodies for 50 min at room temperature (RT). Bound primary antibodies were detected with EnVision™ + Horse Radish Peroxydase (HRP) Systems (Dako, K4061) 30 min at RT. Peroxidase activity was revealed by 3,3′-diaminobenzidine tetrahydrochloride substrate (Dako K3468). Finally, sections were counterstained with Harris hematoxylin, dehydrated and coverslipped.
4.4.4. Architectural Changes
Eclipse E400 Nikon microscope, with DS-FI camera driven by NIS-D element software (Nikon) was used to capture images (100× magnification) and take the measurements for architectural evaluation of the digestive mucosa. A total of about 30 well-oriented villi and crypts per loop were selected on each section. A villus was measured from the tip to the shoulder (crypt-villus junction) and a crypt was measured from the shoulder to its base (Figure 1c). The crypt-depth to villus-height ratio was calculated to assess the intestinal architectural changes.
4.4.5. Proliferative and Apoptosis Indexes
Proliferative and apoptotic cells were counted after immunohistochemistry in several sites on the mucosa. A minimum of 20 well-oriented villi and crypt units were assessed on scanned marked slides (Panoramic 250 Flash II–3D Histech), and analyzed with Pannoramic Viewer software (v. 1.15.2, 3DHISTECH Ltd, Budapest, Hungary,). Proliferative cells were counted in the upper one-third of the villi, i.e. villus tip (positive cells/total cells: lamina propria plus epithelial cells), and in the bottom two-thirds of the crypts from the basis (proliferative index of crypt enterocytes) (Figure 1c). For apoptosis, the total cells counts, lamina propria counts and villus enterocytes counts were measured in villus tip (upper one-third). The enterocyte apoptotic index was calculated by dividing the positive enterocyte number by the total number of enterocytes (×100) in the villus tips while the proliferative index of crypt enterocytes was calculated by dividing the number of positive enterocytes by the total number of enterocytes (×100) in the crypt bases (Table 2).
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Table 2. Summary of the cell counts and indexes used for assessing proliferation and apoptosis in loops.
Counted Endpoint area Cells counts Indexes (Figure 2) Proliferation Total cells: lamina Villus tip propria cells + enterocytes Proliferative index of crypt enterocytes: Crypt bases Crypt enterocytes number of positive enterocytes/total number of enterocytes (×100) Apoptosis Enterocytes Lamina propria cells Enterocyte apoptotic index: number of positive Villus tip (mainly immune cells) enterocytes/total number of enterocytes (×100) Total cells: lamina propria + enterocytes Ratio
Proliferation/Apoptosis Total cells: lamina Total-cell proliferation Villus tip propria cells + to total cell apoptosis ratio enterocytes
4.5. Statistical Analysis
The two experiments were designed as randomized blocks. The explants and loops data are presented as means ± SEM, expressed as percentages of the control values. Plots of fits versus residuals followed by Bartlett’s test, and normal plots of the residuals by Anderson-Darling’s test were carried out to confirm the assumptions of homogeneity of variances and the normal distribution of residuals, respectively. If these assumptions did not hold, the data were normalized and homogenized by log10 or square root transformation prior to being analyzed. All tests were performed using the MINITAB package software (V.13.0, Minitab Inc., State College, PA, USA). The data were analyzed by applying the GLM option of ANOVA analysis, followed by pairwise comparisons, Tukey’s or Bonferroni’s tests. The datasets for the control loops and non-loops were analyzed with the Wilcoxon matched pairs test and paired t-test for apoptosis and proliferation counts, respectively [48].
5. Conclusions
To conclude, the present study shows that pig intestinal explants and loops provide concordant results and permit investigating the digestive effects of DON and NIV with a reduced number of animals (implementation of the 3Rs). Acute NIV exposure induced mucosal changes at a lower concentration than DON in vitro. In vivo, lamina propria cells showed a higher sensitivity than enterocytes to NIV-induced apoptosis. Our results demonstrate that NIV toxicity is not similar to DON on the digestive target, highlighting the need of a specific hazard characterization for NIV health risk assessment.
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Acknowledgments
This study was supported by the ANR DON & Co project. S Cheat was supported by doctoral fellowships from TECHNO I Scholar Program, Erasmus Mundus. J. Gerez was supported by Grant No. 593/08 from CAPES/COFECUB. The authors are grateful to AM Cossalter and the CIRE team for the animals care and handling; to O. Lafaix for helping in the loops experiments; to P. Pinton and S. Desto for helping in explants experiments; to C. Bleuart and I. Pardo for technical assistance for immunohistochemistry; to F. Lyazhri for statistical advice; and to D. Warwick for language editing. The loops surgery was conducted at INRA Centre de recherche Val de Loire (Plate-forme CIRE Chirurgie et Imagerie pour la Recherche et l’Enseignement, UMR Physiologie de la Reproduction et des Comportements (INRA 0085, CNRS 7247, université François-Rabelais de Tours, Institut français du cheval et de l’équitation), 37380 Nouzilly, France).
Author Contributions
M.K.C., I.P.O., and I.R.L. have contributed to the conception and the design of the study. S.C. and J.C. carried out the loops experiments. J.R.G. and I.A.K. carried out the explants experiments. S.C. and J.R.G. analyzed, interpreted the data, and drafted the manuscript under the direct supervision of M.K.C. M.K.C., I.P.O., I.R.L. and A.P.B. revised critically the article. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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3.2. Paper 2: Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes
86 Alternative in vivo pig loops model for toxicity study: Deoxynivalenol and nivalenol show synergism on jejunal enterocytes
Sophal Cheat 1,2,3,*, Philippe Pinton 1,2 , Anne-Marie Cossalter 1,2 , Juliette Cognie 4, Isabelle Raymond- Letron 5,6 , Isabelle P. Oswald 1,2 , Martine Kolf-Clauw 1,2,*
1 Université de Toulouse, Institut National Polytechnique-Ecole Nationale Vétérinaire (INP-ENVT), Unité Mixte de Recherche UMR 1331 Toxalim, Research Center in Food Toxicology, 23 chemin des Capelles, F-31300 Toulouse, France 2 INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, 180 chemin de Tournefeuille F-31027 Toulouse, France 3 Faculty of Animal Science and Veterinary Medicine, Royal University of Agriculture, P.O. box 2696, Phnom Penh, Cambodia 4 Plate-forme CIRE Chirurgie et Imagerie pour la Recherche et l’Enseignement UMR 085 PRC, INRA, 37380 Nouzilly, France 5 INP-ENVT, Université de Toulouse, F-31300 Toulouse, France 6 STROMALab UMR5273 UPS EFS INSERM U1031, 1 Avenue Jean Poulhes, 31403 Toulouse, France
* Authors to whom correspondence should be addressed; E-mails: [email protected] or [email protected] (S.C.); [email protected] (M.K.-C.); Tel.: +855-12555-572 (S.C.); +33-561-193-283 (M.K.-C.); Fax: +33-561-193-978 (M.K.-C.).
Abstract
Food and feed safety pose an issue due to mycotoxins, secondary fungal metabolites and usually found as mixtures in cereals products. The aim of this work was to analyze the effects of two main mycotoxins, alone or associated, deoxynivalenol (DON) and nivalenol (NIV), on the intestinal pig mucosa either after a single exposure in vivo , or after repeated exposure of animals. The animals received a natural contaminated feed, with DON (2.89 mg.kg -1 feed) or with DON+NIV (3.50 mg.kg -1 /0.72 mg.kg -1 feed) for 28 days. The loops model was developed to assess an acute single exposure of DON and NIV individually and in combination (1:1) at 1, 3 and 10 µM for 24h. Histological investigations, including morphometry, proliferation and apoptosis assessments were conducted. Both experiments were concordant for the total-cell proliferation decreased at the villus tips after DON or DON+NIV at 10 µM acutely after 24h, or repeatedly after 28 days, by 30-35% and 20-25%, respectively in loops and in animals. In loops model, apoptotic enterocytes at villus tips increased dose-dependently by either DON, NIV alone or in combination. The combination in loops at 10 µM showed higher effects on proliferation and apoptosis than DON alone. The interaction analysis showed synergism between DON and NIV for villus apoptotic enterocyte. It is concluded that proliferative enterocyte and total-cell proliferation at the villus tips were sensitive to DON and DON+NIV in both models. Intestinal loops, in the context of 3Rs, represent a model allowing to investigate the digestive effects of
87 mycotoxins and of food contaminants, and can contribute to improve our knowledge on plausible interactions of contaminants present simultaneously at the intestinal level.
Keywords : Apoptosis, Fusarium fungi, intestine mucosa, morphometry, surgical model
1. Introduction
The worldwide contamination of agricultural grain commodities by mycotoxins raise a high concern for food and feed safety (Streit et al 2013). Mycotoxins are low-molecular-weight secondary metabolites produced by toxigenic fungi (Bouhet and Oswald 2005). The Fusarium fungi are commonly found on cereals grown in the temperate regions of America, Europe and Asia. They produce mycotoxins, Fusarium toxins, including deoxynivalenol (DON) and nivalenol (NIV). F. graminearum and F. culmorum on wheat are both co-producers of DON and NIV (Logrieco et al 2002; Bottalico and Perrone 2002). These toxins cause a variety of toxic effects in both animals and human (Creppy 2002). DON may have adverse health effects after acute or chronic administration. After acute administration of high dose, DON produces mainly decrease in feed consumption (anorexia) and emesis (vomiting) of neurogenic origin (Gaigé et al 2013). The repeated ingestion of low dose of mycotoxins in pig alters the intestine (induces tissue lesions, modulate the immune cell count as well as the cytokine synthesis and decreases the expression of proteins involved in cell adhesion). Thus it may predispose animals to infections by enteric pathogens (Bracarense et al 2012; Pinton et al 2009). DON is capable to disrupt proliferation, to induce programmed cell death and to alter genes expression (Petska 2010). Pig is the most sensitive species to DON and to NIV toxicity (Pestka and Smolinski 2005; EC 2000). Because of its digestive physiology is very similar to that of human (Kararli 1995), pig can be regarded as the most relevant animal model for extrapolating to human (Rotter et al 1996). NIV health damage is regarded as a serious problem. NIV is considered to be one of the mycotoxins needing regulation (SCOOP EU 2003; EFSA 2013). However, the occurrence of NIV contamination is limited to some parts of Europe and Asia. Consequently, NIV has been poorly studied, and the risks have not been evaluated (EFSA 2013). In vitro , NIV inhibited proliferation of human lymphocytes (Thuvander et al 1999). In young male pigs fed with 2.5 or 5 mg purified NIV/kg feed for 3 weeks, there was no sign of altered feed intake or body weight and no vomiting or clinical problem (Hedman et al 1997b). The rate of absorption of NIV in pig was 11-43% during 7.5 hours after feeding with 0.05 mg NIV/kg bw twice daily (Hedman et al 1997a). Globally, mycotoxin co-occurrence is common (Schatzmayr and Streit 2013), particularly in finished feed: for example 58% of which 4548 samples contained two or more mycotoxins (Streit et
88 al 2013). DON and NIV usually co-occur in grains and grain products, and the DON concentration is generally higher than that of NIV (Yazar and Omurtag 2008; Schothorst and van Egmond 2004). DON and NIV were detected in 57% (n=11022) and 16% (n=4166), respectively, in food samples in the European Union (Schothorst and van Egmond 2004). The combination of NIV with DON, T- 2 toxin, or DAS resulted in an additive effect in vitro on human lymphocytes (Thuvander et al 1999). Interaction between DON and NIV were synergistic in vitro Caco-2 cells (Alassane-Kpembi et al 2013). Less additive effect was observed in ex vivo explant (data not published). Most researches have been targeted on gastrointestinal tract because it is the first organ exposed to food/feed contaminants or xenobiotics (Haschek et al 2010). The gastrointestinal tract plays multi-function roles in regulation, storage, propulsion, digestion, absorption, secretion, barrier activity and elimination (Pinton and Oswald 2014; Gelberg 2012; Haschek et al 2010). In the approach of “3Rs”-Replacement, Reduction and Refinement (Russell and Burch 1959), an alternative experimental model will be needed. Many biological models have been used in toxicity study such as in vitro (cell culture; Pinton et al 2009) and ex vivo (explants; Kolf-Clauw et al 2009) in parallel to conventional animal experiments (Hedman et al. 1997a & 1997b). The intestinal loops model has been developed previously in parasitology or in bacteriology studies (Gerdts et al 2001; Pernthaner et al 1996; Vandenbroucke et al 2011). Jejunal loops model is an in vivo model allowing to analyze the in situ effects of toxics on intestinal mucosa (Cheat et al 2015). In our previous study, the digestive effects of DON and NIV were investigated in explants and in loops after 4-h exposure, and we identified villus apoptosis and proliferation as sensitive endpoints (Cheat et al 2015). In the present study, we investigated these endpoints to compare the digestive effects of DON and NIV alone or associated, after a single 24-h exposure in loops, or after 28-day repeated exposure of animals.
2. Materials and methods
2.2. Purified toxins
DON was acquired from Sigma (St Quentin Fallavier, France) and NIV from Waco Pure Chemical Industries LTD (Osaka, Japan). Stock solutions of these mycotoxins were dissolved in dimethyl sulfoxide ( DMSO) at the 30 mM DON and NIV for the loops experiments. These stock solutions were stored at -20 °C. Working dilutions were prepared in physiological saline solution. The concentrations at 0 (Ctrl), 1, 3 and 10 µM were used for the dose-response of individual or combined mycotoxins.
89 2.2. Animals and toxins exposure
2.2.1. Loops For the loops experiment, two 2-month-old Large White female pigs weighting 20 and 25 kg were used and housed in the animal facility at INRA Nouzilly. The experimental procedures were conducted in accordance with European Guidelines for the Care and Use of Animals for Research Purposes and were approved by the Val de Loire local ethical committees for animal experimentation (C2EA-06). The animals were fasted for 6h before loops surgery. A 2-m long segment of jejunum was surgically prepared, to constitute the loops as previously described (Girard- Misguich et al 2011; Meurens 2009). This segment was then subdivided into consecutive segments, designated as “loops” (10 cm long, 12 loops), separated by “inter-loops”. Treatments, control (Ctrl), DON and NIV at 1 µM, 3 µM and 10 µM and DON+NIV (1:1) at 1 µM, 3 µM and10 µM concentration were used for each of the 2 pigs (1 loops/pig for each condition) by injecting 3 mL of each test condition into each loop. After surgery (24 h), the pigs were intravenously (IV) euthanized by barbiturate overdose and the jejunum loop were collected. These loops were washed twice with physiological serum prior to fixation in 10% formalin. In addition, jejunum normal segments (nL) were sampled and processed in parallel.
2.2.2. Animal experiment For the animal experiment, 24-crossbred castrated-male piglets (4-week old) with a mean weight of 11.2 (SD 1.2) kg were used. Pigs were acclimatized for 1 week in the animal facility of the Toxalim laboratory, INRA (Toulouse, France) before the experimental protocols. They were randomly allocated to 4 experimental batch pens with equal numbers. The experimental procedures were conducted in accordance with European Guidelines for the Care and Use of Animals for Research Purposes and were approved by the INRA local ethical committees for animal experimentation (Directive 63/2010/EEC). DON and NIV were obtained from a natural contaminated feed source. The levels of the other mycotoxins were below the detection limits or considered as negligible (Table A1.). A diet without toxins (Ctrl) or with natural single contamination (DON) at 2.89 mg DON/kg feed or co- contamination (DON+NIV) at 3.50 mg DON/kg with 0.72 mg NIV/kg feed were used to expose orally the animals for 28 days. Feed and water were provided ad libitum throughout the experimental period. The feed composition is given (Table A1.). Body weights were measured before starting the experiment (day 0). The body weights and feed intake were measured weekly and at the end of the experiment period (day 28). The blood samples were collected at days 0, 7 and at 28 for biochemistry analysis: total serum protein (TP),
90 albumin, fibrinogen, gamma-glutamyl transferase (GGT). At the end of the experiment, the pigs were fasted overnight prior to be subjected to electrical stunning and euthanized by exsanguination. The jejunum segments were immediately collected and processed as the “Swiss rolls” (Moolenbeek and Ruitenberg 1981), then fixed in 10% buffered formalin solution for histological procedure.
2.3. Histological assessment
2.3.1. Histological processing and architectural changes A routine histological processing sequence was used. Paraffin sections from the loops and from the animal experiment, 4-µm thick were stained with HE to assess architectural changes and immunohistochemically labeled (IHC) to assess proliferation and apoptosis. Eclipse E400 Nikon microscope, with DS-FI camera driven by NIS-D element software (Nikon) was used to capture images (x100 magnification) and take the examinations for architectural evaluation of the digestive mucosa. Approximately 30 well-oriented villi and crypts per loop were selected on each section to measure villus height and crypt depth. A villus was measured from the tip to the shoulder (crypt-villus junction) and a crypt was measured from the shoulder to its base, as described previously (Cheat et al 2015). The index was calculated as described previously (Cheat et al 2015). The crypt-depth to villus-height ratio was calculated to assess the intestinal architectural changes after the treatment (Haschek et al 2010).
2.3.2. Immunohistochemistry Briefly, four-micrometer paraffin-embedded transverse sections from formalin-fixed jejunum specimens were dewaxed in toluene and rehydrated by an acetone bath then deionized water. Antigen retrieval was performed in 10 mM citrate buffer pH 6.0 for 30 min in a water bath at 95°C. Cooled sections were then incubated in Dako peroxidase blocking solution (Dako S2023) to quench endogenous peroxidase activity. Non-specific binding was blocked by incubation in normal goat serum (dilution 1:10, Dako X0902) for 20 min at room temperature. Two commercial antibodies were used as previously described (Abot et al 2013; Laprie et al 1998). The primary antibodies were anti-Ki-67antigen (Dako M7240, dilution 1:50) and anti-active caspase-3 (R&D system, AF835, dilution 1:300). Sections were incubated with primary antibodies for 50 min at room temperature (RT). Bound primary antibodies were detected with EnVision™ + Horse Radish Peroxydase (HRP) Systems (Dako, K4061) 30 min at RT. Peroxidase activity was revealed by 3, 3’-diaminobenzidine tetrahydrochloride substrate (DAKO, K3468). Finally, sections were counterstained with Harris hematoxylin, dehydrated and coverslipped.
91 Proliferative and apoptotic cells were counted after immunohistochemistry in several sites on the mucosa. A minimum of 20 well-oriented villi and crypt units were assessed on scanned marked slides (Panoramic 250 Flash II – 3D Histech), and analyzed with Pannoramic Viewer software (v. 1.15.2) as previously described (Cheat et al 2015). The counts were performed at villus tip and crypt basis. Proliferative and apoptotic cells were counted in the upper one-third of the villi, i.e. villus tip (positive-total cells: lamina propria plus epithelial cells or enterocyte only). Proliferative enterocytes were also counted in the bottom two-thirds of the crypts from the basis. The proliferative index of crypt enterocytes was calculated by dividing the number of positive enterocytes by the total number of enterocytes (x100) in the crypt bases. The ratios were calculated by dividing the number of proliferative cells by the number of apoptotic cells, for enterocytes or total cell ( lamina propria and epithelial cells).
2.3.3. Interaction analysis of the mycotoxins Median-effect doses and combination index (CI) were determined by the Chou-Talalay median-effect equation (Chou and Talalay 1984) as previously described in Kolf-Clauw et al (2013). Briefly, the median-effect plot of Chou was applied to the individual toxins, and a median- enterocyte-apoptosis dose (Dm) was calculated for each toxin (CompuSyn Software 2007). For the mixture of DON and NIV, the Dm for the mixture was calculated. The CI was calculated for a 10, 20 and 30 % increase of cell apoptosis.
2.4. Statistical analysis
The Completely randomized and the randomized blocks designs were used in the animal experiment and in the loop experimental model, respectively. The result s are presented as means ± SD. Plots of fits versus residuals followed by Levene’s and Bartlett’s test, and normal plots of the residuals by Anderson-Darling’s test were carried out to confirm the assumptions of homogeneity of variances and the normal distribution of residuals, respectively. If these assumptions did not hold, the data were normalized and homogenized by log 10 or square root prior to analysis to them being analyzed. If none of the transformations resolve these problems, then Kruskal-Wallis test was used. All tests were performed using the MINITAB package software (V.13.0, Minitab Inc., State College, PA, USA). The data were analyzed by applying the GLM option of ANOVA. The datasets of control loops and normal segments were analyzed with Wilcoxon matched pairs test and paired t- test. When there was a significant difference at P-value ≤0.05, then the all pairwise comparisons of means were generated by Tukey’s or Behrens Fisher tests and Asymptotic Mann-Whitney tests (Bate and Clark 2014; Festing et al 2002).
92
3. Results
3.1. Loops model
The loop results were obtained from 2 pigs after 24 h in situ incubation.
3.1.1. Comparison of loops segments with normal segments The morphometry was affected by the surgery in the loop (Table 1.). After surgery, the villi of the control loops were shorter and the crypts were deeper compared to normal segments. The ratio of crypt-depth to villus-height was significantly higher in control loops than in normal segments. The number of proliferative total cells or enterocytes at villus tip, the crypt index of proliferative enterocyte, the number of apoptotic cells at villus tip, and the ratio total-cell proliferation to total-cell apoptosis at villus tip did not differ between the surgical loop condition and normal segments.
3.1.2. Effect of the single mycotoxins on proliferation and apoptosis in loops a. Effect of DON DON induced a bell-shaped curve for the total number of proliferative cells at the villus tip. The two lowest concentrations, 1 and 3 µM DON increased the total proliferation by about 20% compared to the control loops (Fig. 1A-I), whereas 10-µM DON strongly reduced by about 35% the villus total-cell proliferation compared to control loops. Villus enterocyte proliferation was not affected at 1 or 10 µM but only at 3 µM was observed a slight decrease compared to the control loops (Fig. 1B-I). Villus total cell apoptosis did not show clear evidence of DON related effect (data not shown), while the number of apoptotic enterocytes at the villus tip increased dose-dependently, with a 20% increase at the highest DON concentration (Fig. 1C-I). The total-cell proliferation to total-cell apoptosis ratio showed a bell-shaped curve. The ratio was about twofold significantly lower at 10-µM DON compared to control loops and about two-to- threefold higher at 1 and 3 µM compared to control loops (Fig. 1D-I). b. Effect of NIV A similar dose-response tendency to that of DON was observed for the total number of villus proliferative cells, with a 40% decrease of total-cell proliferation at 10-µM NIV compared to
93 control loops (Fig. 1A-II). The enterocyte proliferation was not affected at any concentrations (Fig. 1B-II). Enterocyte apoptosis at the villus tips significantly increase in loops at 3 µM and 10 µM (Fig. 1C-II). At these concentrations, 3 µM and 10 µM, the ratio of enterocyte proliferation to enterocyte apoptosis decreased by about 30% (Fig. 1F-II). The total-cell proliferation to total-cell apoptosis ratio showed a bell-shaped curve, as for DON, with a twofold decrease at 10 µM DON, compared to control loops (Fig. 1D-II).
3.1.3. Effect of the mixture DON+NIV (1:1) on proliferation and apoptosis in loops Similar dose-response curves were observed with the binary combination, compared to single mycotoxins. For total-cell proliferation at villus tips, a bell-shaped curve similar to that of DON was observed, with an about 30% increase with the lowest doses, and an about 35% decrease of the total-cells proliferation at 10-µM DON+NIV (Fig. 1A-III). A dose-dependent relationship was found for the increase in enterocyte proliferation at villus tips, with 33% and 15% increase of enterocyte proliferation compared to control loops, at 10 and 3 µM respectively (Fig. 1B-III). DON+NIV increased dose-dependently enterocyte apoptosis at the villus tips at different concentrations ( P<0.001). DON+NIV (10 µM) increased the enterocyte apoptosis by 68% compared with control loops (Fig. 1C-III). Villus enterocyte proliferation to villus enterocyte apoptosis ratio was significantly decreased for treated loops reaching 30% compared to control loops, at all concentrations of the mixture (Fig. 1F-III).
3.1.4. Comparative effect of DON+NIV (1:1) in loops at 10 µM showed higher effects on proliferation and apoptosis than DON alone (Table B2.) Enterocyte proliferation at the villus tips was significantly increased by 33% for DON+NIV treated loops compared to control loops (Fig. 2A). The effect of DON+NIV was also found at crypt levels (Fig. 2B), where DON+NIV exposed loops decreased crypt cell proliferation by 12 %. A significant increase was observed in the villus enterocyte apoptosis for DON and DON+NIV loops by 21% and 68% (Fig. 2C), respectively, compared with control loops ( P<0,001). DON+NIV loops increased total-cell apoptosis at the villus tips by about 10% compared with DON loops and controls.
3.1.5. Interaction analysis of DON+NIV (1:1) for villus enterocyte apoptosis The effect between DON and NIV was synergist for villus enterocyte apoptosis (Fig. 3) with CI<1 for an effect below 70% of the control values.
94
3.2. Animal experiment
3.2.1. Zootechnical and clinical pathology: effect of DON and DON+NIV In this animal experiment, DON and DON+NIV natural contamination at 2.89 mg DON/kg feed and 3.50 mg DON+0.72mg NIV/kg feed did not affect weight gain, feed intake (Table A2.), blood total protein, gamma-glutamyl transferase (GGT), albumin and fibrinogen (Table A3.).
3.2.2. Morphometry, proliferation and apoptosis: effect of DON and DON+NIV (Table A4.) Dietary exposure to co-contamination DON+NIV significantly increased crypt depth by about 5% compared to DON and to control animals (Fig. 4A). DON+NIV exposed animals showed a 10-20% decreased in villus enterocyte proliferation compared to control animals (Fig. 4B). DON+NIV and DON exposed animals showed 20-25% decrease in the number of total proliferating cells at villus tips (Fig. 4C).
4. Discussion
4.1. Models
A conventional experimental animal model (animal experiment) was used to confirm endpoints described with loops model. The animal experiment allows to expose the animals to natural contaminated feed repeatedly (28-day study). Loops model is the alternative model that enables to reduce the number of animals in toxicology study. The interest of the loops model is that it has tremendous advantages compared with the models using individual pigs because one pig can provide approximately 30 individual loops (Girard-Misguich et al 2011; Boyen et al 2009; Meurens 2009) and allows multiple doses assessment. The intestinal loop model has been used in several species to explore the responses of the intestinal epithelial cells i.e. using bacteria in pigs (Vandenbroucke et al 2011; Boyen et al 2009), using bacteria in pigs and bovines (Bolton et al 1999), using nematode parasites in sheep (Pernthaner et al 1996), studying mucosal immune responses in sheep (Gerdts et al 2001). In our study, 24-h in situ incubation was used according to our research objective. Besides, this model allows the in situ incubation to be maintained for longer durations for example two weeks (Girard-Misguich et al 2011) or four weeks (Gerdts et al 2001) without macroscopic lesions or alteration of blood flow. However, the limitations of the loop model may be the preservation of normal metabolism for morphological preservation and structural integrity on one side. In this model, the comparison
95 between non-loop segment (nL) and control loops (Ctrl) for the tissue architecture such as villus height, crypt depth as well as crypt-depth to villus-height ratio revealed a change made by surgery. It is the first time, at our knowledge, that these changes are reported and we also previously reported vascular changes after 4-h exposure (Cheat et al 2015). A marked atrophy of the villi at the vicinity of ulcerative lesions has been reported in villosity architecture of infected and not infected jejunal loops (Girard-Misguich et al 2011). On another side, the concentration reaching the targets cell might be modulated potential by loops-to-loops transfer, and it is not possible to have neither repeated exposure nor natural-occurred mycotoxins treatment via feeding.
4.2. Exposure level and endpoints
The selected concentration ranging from 1 to 10 µM DON was related to previous studies (Bracarense et al 2012; Pinton et al. 2012; Marzocco et al 2009), and to realistic concentrations for the consumer via food. Each mycotoxin showed significant effects at 10 µM. This latter concentration corresponds to an exposure to 3 mg DON/kg contaminated feed or food, assuming that DON diluted in one liter of gastrointestinal fluid is ingested in one meal and is 100% bioavailable (Sergent et al 2006). The jejunum was chosen to prepare the loops, because it was easier than with duodenum or ileum (Girard-Misguich et al 2011). In animal experiment, jejunum was chosen because of previous experiments in vivo and ex vivo on this segment (Pinton et al 2009, 2012; Kolf-Clauw et al 2013) and because of its anatomy and physiology (Haschek et al 2010). The discussions will be focused on the highest dose (10 µM) of each toxin. In animal experiment, the exposure of natural contaminated DON and DON+NIV at 2.89 mg DON/kg feed and 3.50 mg DON+0.72mg NIV/kg feed did not induce any change in the zootechnical and pathological endpoints studied. The results of clinical pathology were in parallel to those of Alizadeh et al. (2015). Effects on the weight gain and feed intake has been shown in previous studies from 2 mg/kg feed for four weeks (Gerez et al 2015; Bracarense et al 2012; Pinton et al 2012). It is reported that DON is able to decrease nutrient absorption (Ghareeb et al 2015). In growing pig exposed to 0.9 mg DON/kg feed for 10 days, weight gain was negatively affected but not feed intake (Alizadeh et al 2015). So, in the present study, jejunum mucosa was exposed to subclinical level of toxins. Proliferation and apoptosis were chosen as potential sensitive endpoints, used in parallel to the loops model, to investigate, in absence of zootechnical effects, the consequences of the toxins in situ on the jejunum mucosa. Proliferation and apoptosis at different sites of the villi or crypts were selected because there was no modification between non-loops and control loops segment for theses endpoints. Trichothecenes are well known to act on highly proliferative cells (Pestka 2010), and the intestinal
96 epithelium has been identified as a target for DON and other derivatives in vitro , ex vivo , and in vivo (Pinton and Oswald 2014; Kolf-Clauw et al 2013). So, these two endpoints, proliferation and apoptosis, are specially relevant to assess the in situ mucosa changes, specifically affected in vitro and in vivo by the trichothecenes (Gerez et al 2015; Bianco et al 2012; Bracarense et al 2012; Pinton et al 2012; Marzocco et al 2009).
4.3. Effect of DON, NIV and DON+NIV on morphometry
In animal model, crypt depth was increased after DON+NIV exposure but not after DON exposure. Jejunal crypts were shown deeper after 3 mg/kg DON feeding for 10 weeks (Dänicke et al 2012) and after 0.9 mg/kg DON feeding for 10 days (Alizadeh et al 2015). These results contrast with previous other results showing shorter crypt depth after 1.5 mg/kg DON feeding (Gerez et al 2015). No change in crypt depth was observed after repeated exposure to 3 and 6 mg DON/kg feed for 5 weeks and to 3.1 mg DON/kg feed for 37 days (Klunker et al 2013; Bracarense et al 2012). The change of crypt depth is shown when more enterocytes need to be generated to migrate progressively along the side of its villus, particularly toward the tip, caused by mechanical, toxic or xenobiotic damages i.e. the case of malabsorptive diarrheas. Thus, the crypts elongate compared to healthy crypts, and this morphologic change is suggested to be site-specific of toxicant involved (Haschek et al 2010). Each crypt produces 300-400 cells per day from a relatively few stem cells, which in turn divide several more times in the lower and middle portion of the crypts. The rate of cell proliferation in the crypts with the rate of mature cell loss at the villus tips is coordinated by negative feedback mechanisms (Haschek et al 2010).
4.4. Effect of DON, NIV and DON+NIV on proliferation, apoptosis and the ratio proliferation/apoptosis
The experimental loops and the experimental animal models provided concordance for the total-cell proliferation decrease at the villus tips after exposure to 10 µM DON or DON+NIV (1:1) acutely after 24h or repeatedly after 28 days. These results are in accordance with the downregulation of total mRNA of proliferative marker Ki-67 in jejunum of piglet fed purified 0.9 mg DON/kg diet (Alizadeh et al 2015). This confirms that proliferation is a sensitive endpoint for the responses to DON or DON+NIV. Proliferative enterocytes were affected by DON+NIV in animal experiment. At villus tips, proliferative enterocytes decreased after 28 days. These results are in agreement with those of Bracarense et al (2012). However, proliferative enterocytes were found increased in loops treated at 10 µM. In loops model, the decrease of villus total-cell
97 proliferation after 24-h exposed to 10-µM DON and 10-µM NIV is concordant with our recently findings with the same concentration but 4-h exposure (Cheat et al 2015). In this previous study after exposure to DON and NIV, enterocyte proliferation was not affected, showing that lamina propria cells were more affected by these individual mycotoxins. When these two mycotoxins are mixed (DON+NIV), both villus total-cell proliferation and villus enterocyte proliferation were affected. The 24-h response of enterocytes to the toxins might be dependent on the cell renewal cycle because these epithelial cells of the small intestine are replaced every 2-4 days in adult and takes longer in neonate (Haschek et al 2010). In loops, the results showed bell-shaped dose-response curves for proliferation and apoptosis of total cells at villus tips and for the ratio proliferation to apoptosis for individual or combined DON and NIV. These non-linear of dose-response curves have been described previously for several endpoints, for example cell proliferation in human lung fibroblasts and apoptosis in mouse lymphocytes (Calabrese and Baldwin 2001). These biological responses could be related to the well-known immunomodulation described with DON and the mechanism of protein inhibition of NIV (Severino et al 2006). DON is low-dose stimulation and high-dose inhibition of immune response corresponding to lymphocytes proliferation. DON is known as ribotoxic stress response, where DON binds to ribosomes and inhibits protein synthesis. NIV inhibits peptidyl transferase with subsequent inhibition of peptide bond formation by acting on the initial step of protein synthesis and elongation-termination step. Therefore, trichothecenes, particularly DON and NIV are toxic to tissues with a high cell proliferation rate (Rocha et al 2005). This suggests a direct stimulatory response of immune cells and a response that occurred only as overcompensation to initial injury. These responses are concordant with the previously described induction of mitogen activated protein kinases (MAPKs) or increased reactive oxygen species (ROS) production in T- cells and B-cells (Krishnaswamy et al 2010; Pestka 2007) The high impact on apoptotic enterocytes with 10-µM DON and 10-µM DON+NIV in loops might be due to the impaired antioxidant status of cell and increased production of ROS, particularly induced by DON. It has been observed previously in in vitro studies with intestinal epithelial cells (Pestka 2007). The effect of DON and NIV on proliferation and apoptosis in the intestine, also described in other species (Payros, personal communication), are confirmed in our in vivo study with DON and NIV. So, human intestine should be similarly affected. The apoptotic enterocytes observed in loops showed similarities with several other human intestinal diseases for example microvillus inclusion disease and coeliac disease (Groisman et al 2000; Moss et al 1996).
4.5. Effect of DON on gene expression in vivo
98 In the present study in loops, we did not observe any differences after 24 h in cytokine genes expression for IL-1α, IL-1β, IL-6, IL-8, IL-21, IL12-p40, TGF-β (data not shown). Our negative results on cytokines from acute 24-h exposure might be due to the time needed for the response observed after repeated exposure: for example IL-1β expression was increased in duodenum of piglets fed a DON diet (0.9 mg/kg feed) for 10 days, IL-1β and IL-6 expression were up-regulated in jejunum of piglets fed a DON diet (3 mg/kg feed) for 35 weeks, or IL-8 expression were up- regulated in ileum of piglets fed a DON diet (3.5 mg/kg feed) for 42 days (Alizadeh et al 2015; Lessard et al 2015; Bracarense et al 2012). However the present result, tight junction protein expression for claudin-4, one important component of tight junction protein, was not altered (data not shown). DON exposure, through the activation mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (protein kinase B) signaling pathways, is capable to modulate inflammatory response and to influence the expression of tight junction proteins (Mishra et al 2014; Pinton et al 2009). In the similar exposed time (24h) in vitro , claudin-4 was up-regulated in Caco-2 cells exposed to increasing concentrations (1.39, 4.17, and 12.5 µM) of DON (Akbari et al 2014), whereas DON inhibits the expression of claudin-4 in IPEC-1 (Pinton et al 2009). The first hypothesis for addressing this difference between the in vitro and our in vivo loop result is the fact that the samples taken in the in vivo loop contained the entire intestinal wall, not only the epithelial cell layer. Another hypothesis could be the contact time of DON in loops too limited compared to in vitro studies.
4.6. Interaction analysis
In vitro , exposure of intestinal and non-intestinal cells to the trichothecenes alone or in combination decreased proliferation (Alassane-Kpembi, et al 2013; Wan et al 2013; Pinton et al 2012; Petska 2010, Thuvander et al 1999). Concordantly, a decrease was observed in enterocyte proliferation to enterocyte apoptosis ratio in loops after 10-µM DON+NIV. Alassane-Kpembi, et al (2013) found that NIV and DON+NIV were more toxic than DON alone in Caco-2 cells. The present study shows a similar type of interaction between DON and NIV on a different target. The interaction analysis confirmed synergism between the two mycotoxins for villus enterocyte apoptosis. Our results are of high relevance, because they were obtained in models (animals or loops) more complex than the cell lines. A recent study on the pig intestinal cell line IEC-6 suggested also a more than additive interaction between DON and NIV based on their pro-oxidant effect (Del Regno et al 2015).
Conclusion
99
The current study showed that proliferative enterocyte and total-cell proliferation at the villus tips were sensitive to DON and DON+NIV in both models. The loops allowed testing the interaction between the two mycotoxins, DON and NIV. Taking together from the previous study (Cheat et al. 2015), our data indicate that intestinal loops model, in the context of 3Rs, represents a relevant and sensitive model to investigate the digestive effects of food contaminants. Limitations to unique exposure and to non-natural occurring mycotoxin treatments are its main disadvantages.
Acknowledgement
This study was supported by the ANR DON & Co project. S Cheat was supported by doctoral fellowships from TECHNO I Scholar Program, Erasmus Mundus. The authors are grateful to the CIRE team for the animals care and handling, to the E5-team for helping in animal experiment sampling, to C Bleuart and I Pardo for technical assistance for immunohistochemistry, and to F Lyazhri for statistical advice. The loops surgery was conducted at INRA Centre de recherche Val de Loire (Plate-forme CIRE Chirurgie et Imagerie pour la Recherche et l’Enseignement, UMR Physiologie de la Reproduction et des Comportements (INRA 0085, CNRS 7247, université François-Rabelais de Tours, Institut français du cheval et de l’équitation), 37380 Nouzilly, France).
References
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Table and figure legends
Table 1. Comparison of control loops and non-loops segments for surgical effect after 24h post-surgery on morphometry, proliferation-cell and apoptosis-cell counts of the jejunum.
Figure 1. Proliferation and apoptosis in the jejunum loops after exposed to DON (I), NIV (II) and DON+NIV (1:1) (III) at 1, 3 and 10 µM for 24h. Mean values ± SD expressed as % of the control group (Ctrl). (A-I, A-II, A-III) Total-cell proliferation at villus tip (upper one-third); (B-I, B-II, B-III) Villus enterocyte proliferation; (C-I, C-II, C-III) Villus enterocytes apoptosis; (D-I, D-II, D-III) Total-cell proliferation to total-cell apoptosis ratio at villus tip; (E-I, E-II, E-III) Villus total-cell apoptosis; and (F-I, F- II, F-III) Villus enterocyte proliferation to apoptosis ratio. Letter a, b and c are different at P≤0.05; Behrens Fisher tests and Asymptotic Mann-Whitney tests; n = 2 or 4 (Ctrl) loops, 20 villi or crypts/loop.
Figure 2. Combined effect of the mixture of DON and NIV (1:1) in loops at 10 µM showed higher effects on proliferation and apoptosis than DON alone. Mean values ± SD expressed as % of the control group (Ctrl). (A) Enterocyte proliferation at villus tip (upper one-third), P<0.001; (B) Enterocytes proliferation at crypt base (lower two-third), P=0.001; and (C) Villus enterocyte apoptosis, P<0.001. Letter a, b and c are different at P≤0.05; Tukey’s test or Behrens Fisher tests and Asymptotic Mann-Whitney tests; n = 2 or 4 (Ctrl) loops, 20 villi or crypts/loop.
Figure 3. Isobolograms illustrating the combined effect of the mixture of DON and NIV (1:1) for reaching 10% (Fa = 0.1, circle ), 20 % (Fa = 0.2, square ), or 30 % increase (Fa = 0.3, triangle ) of villus enterocyte apoptosis in jejunal loops model. The points on each axis are mean concentrations of dose– response curves of each toxin alone ( CompuSyn® software analysis).
Figure 4. Jejunal morphology, proliferation and apoptosis in in vivo animal after exposed to DON natural single- at 2.89 mg.kg -1 feed and co-contamination to DON+NIV at 3.50 mg.kg-1 feed. Mean values ± SD expressed as % of the control group (Ctrl). (A) Crypt depth, P=0.007; (B) Enterocyte proliferation at villus tip (upper one-third), P<0.001 and (C) Total cell proliferation at villus tip, P=0.009.
106
Table 1. Comparison of control loops (Ctrl) and non-loops segments (nL) for surgical effect after 24h post-surgery on morphometry, proliferation-cell and apoptosis-cell counts of the jejunum Parameters nL Ctrl P-value Morphometry Villus height (µm) 378.1 ±63.8 339.6 ±64.5 0.014 ** Crypt depth (µm) 336.2 ±40.9 364.6 ±50.7 0.007 ** Crypt-depth to villus-height ratio 0.90 ±0.20 1.11 ±0.28 <0.001 *** Proliferation Villus total cell 33.08 ±6.17 35.23 ±4.86 0.127 w Crypt index (x100) 66.6 ±9.8 64.8 ±10.4 0.453 w Apoptosis Villus total cell 3.55 ±0.90 3.54 ±0.97 0.976 Ratio Total-cell proliferation to total-cell apoptosis 9.84 ±4.67 10.70 ±7.93 0.216 - Data presented as mean ± standard deviation (SD);Paired t-test - w = Wilcoxon matched pairs test - ** : Significant level P ≤0.01; *** : Significant level P ≤0.001 - Morphometry: n= 2 or 4 (Ctrl) loops, 30 villi or crypt /loop; Values are mean numbers per villus or per crypt - Proliferation and apoptosis: n= 2 or 4 (Ctrl) loops, 20 villi or crypt/loop; Values are mean numbers per1/3 villus tip or per 2/3 crypt base
107 250 (A-I) Villus total cell proliferation after 24h 250 (A-II) Villus total cell proliferation after 250 (A-III) Villus total cell proliferation after 24h 24h 200 P<0,001 200 P<0,001 200 P<0,001 b b a 150 b 150 a 150 a a a a c 100 c 100 b 100 % compared compared % Ctrl to % compared to Ctrl to compared % 50 50 50 compared % Ctrl to
0 0 0
250 (B-I) Villus enterocyte proliferation after 250 (B-II) Villus enterocyte proliferation 250 (B-III) Villus enterocyte proliferation after 24 after 24 24 P=0,023 200 200 200 P<0,001 P=0,751 c 150 a a 150 150 ab b ab b a 100 100 100 % compared compared % Ctrl to 50 compared % Ctrl to 50 50 % compared compared % Ctrl to
0 0 0
(C-III) Villus enterocyte apoptosis after (C-II) Villus enterocyte apoptosis after 250 (C-I) Villus enterocyte apoptosis after 24h 250 250 24h 24h P=0,021 P<0,001 P<0,001 c 200 200 200 c bc b bc ab b ab 150 a a 150 a 150 a
100 100 100
% compared compared % Ctrl to 50 compared % Ctrl to 50 compared % Ctrl to 50
0 0 0
(D-I) Villus total cell proliferation to total (D-II) Villus total cell proliferation to (D-III) Villus total cell proliferation to 400 400 400 cell apoptosis ratio after 24h total cell apoptosis ratio after 24h total cell apoptosis ratio after 24h 350 350 350 b P<0,001 b P<0,001 P<0,001 300 b 300 300 b b 250 250 250 200 200 ab 200 150 a 150 a 150 a c 100 100 c 100 c % compared compared % Ctrl to compared % Ctrl to compared % Ctrl to 50 50 50 0 0 0
(E-I) Villus enterocyte proliferation to 250 250 (E-II) Villus enterocyte proliferation to 250 (E-III) Villus enterocyte proliferation to apoptosis ratio after 24h apoptosis ratio after 24h apoptosis ratio after 24h 200 200 P=0,107 P=0,002 200 P=0,001 a a ab 150 150 150 c bc b 100 100 b b 100 % compared compared % Ctrl to
% compared compared % Ctrl to 50 50
% compared compared % Ctrl to 50 0 0 Ctrl DON1 DON3 DON10 Ctrl NIV1 NIV3 NIV10 0 Ctrl DON1+NIV1 DON3+NIV3 DON10+NIV10 Figure 1. Proliferation and apoptosis in the jejunal loops after exposed to DON (I), NIV (II) and DON+NIV (1:1) (III) at 1, 3 and 10 µM for 24h. Mean values ± SD expressed as % of the control group (Ctrl). (A-I, A-II, A-III) Total-cell proliferation at villus tip (upper one-third); (B-I, B-II, B-III) Villus enterocyte proliferation; (C-I, C-II, C-III) Villus enterocytes apoptosis; (D-I, D-II, D-III) Total- cell proliferation to total-cell apoptosis ratio at villus tip; (E-I, E-II, E-III) Villus total-cell apoptosis; and (F-I, F-II, F-III) Villus enterocyte proliferation to apoptosis ratio. Letter a, b and c are different at P≤0.05; Behrens Fisher tests and Asymptotic Mann-Whitney tests; n = 2 or 4 (Ctrl) loops, 20 villi or crypts/loop.
108 250 (A) Villus enterocyte proliferation after 24h
200 P<0,001 b 150 a a
100
% compared Ctrl to % compared 50
0
250 (B) Crypt enterocyte proliferation after 24h
200 P=0,001
150 a a b 100
% compared Ctrl to % compared 50
0
250 (C) Villus enterocyte apoptosis after 24h c 200 P<0,001 b 150 a
100
% compared Ctrl to % compared 50
0 Ctrl DON DON+NIV
Figure 2. Combined effect of DON and NIV (1:1) in loops at 10 µM showed higher effects on proliferation and apoptosis than DON alone. Mean values ± SD expressed as % of the control group (Ctrl). (A) Enterocyte proliferation at villus tip (upper one-third), P<0.001; (B) Enterocytes proliferation at crypt base (lower two-third), P=0.001; and (C) Villus enterocyte apoptosis, P<0.001. Letter a, b and c are different at P≤0.05; Tukey’s test or Behrens Fisher tests and Asymptotic Mann-Whitney tests; n = 2 or 4 (Ctrl) loops, 20 villi or crypts/loop.
109
(µM) DON DON
2.5 NIV (µM)
Figure 3. Isobolograms illustrating the combined effect of the mixture of DON and NIV (1:1) for reaching 10% (Fa = 0.1, circle ), 20 % (Fa = 0.2, square ), or 30 % increase (Fa = 0.3, triangle ) of villus enterocyte apoptosis in jejunal loops model. The points on each axis are mean concentrations of dose- response curves of each toxin alone ( CompuSyn® )
110 200 (A) Crypt depth after 28 days 200 (C) Villus total cell proliferation after 28 days
P=0,007 P=0,009 150 150 a a b a b b 100 100
% compared Ctrl to % compared 50
% compared Ctrl to % compared 50
0 0
200 (B) Villus enterocyte proliferation after 28 days 200 (D) Crypt-depth to villus-height ratio after 28 days P<0,001 P<0,001 150 a 150 a b a b b 100 100
50 50 % compared Ctrl to % compared Ctrl to % compared
0 0 Ctrl DON DON+NIV Ctrl DON DON+NIV
Figure 4. Jejunal architecture, proliferation and apoptosis in in vivo animal after exposed to DON (2.89 mg.kg -1 feed) natural single- and co-contamination to DON+NIV (3.50 + 0.72 mg.kg -1 feed). Mean values ± SD expressed as % of the control group (Ctrl). (A) Crypt depth, P=0.007; (B) Enterocyte proliferation at villus tip (upper one-third), P<0.001; (C) Total cell proliferation at villus tips, P=0.009.; and (D) Crypt-depth to villus-height ratio, P<0.001.
111 3.3. General discussion
3.3.1. Benefits and limitations of the alternative models
Biological models can be used in toxicology studies such as in vitro , cell culture (Pinton et al. 2009); ex vivo, explants, (Kolf-Clauw et al. 2009) and in vivo , animal experiment (Claude 1957; Hedman et al. 1997) . Among those models, in vivo is costly, time consuming and limited by individual variations. In this work, we use conventional animal experiment ( in vivo ), but also alternative models either in vivo (loops) or ex vivo (explants). These models allow to reduce the number of animals used in agreement with the 3Rs recommendation of Russel and Burch (1959). However, the in vivo model provides a complete animal biological response to the treatments. Loop is an in vivo model the created loops being in situ maintained inside the pigs. This model has been used in bacterial infection studies on different animal species (Girard-Misguich et al. 2011; Vandenbroucke et al. 2011; Gerdts et al. 2001; Bolton et al. 1999; Pernthaner et al. 1996). To the best of our knowledge, to date, the use of this model is neglected in toxicologic studies. The discussions below are related to the concordant results obtained from the three types of model.
3.3.2. In animal experiment
Feed intake and weight gain have been found to be depressed in several studies after repeated exposure to DON (Xiao et al. 2013; Pinton et al. 2012; Danicke et al. 2012a; Danicke et al. 2012b). However in the present study, DON and DON+NIV, natural contamination at 2.89 mg DON/kg feed and 3.50 mg DON+0.72 mg NIV/kg feed did not impact weight gain nor feed intake, and of the clinical biochemistries investigated such as blood total protein, gamma-glutamyl transferase (GGT), albumin and fibrinogen did not change. The proportion of co-contamination used here in animal experiment were similar to that of DON and NIV in the analyzed samples from food collected in the European Union (Schothorst and van Egmond 2004). These toxins are of concern for human and animal health (EFSA 2013). A similar result for the unchanged-weight gain was reported in DON-exposed animals at 1.5 mg.kg -1 diet (Gerez et al. 2015). Our study showed a high toleration of the pigs to the levels of mycotoxins used while various studies reported that pig could be tolerate up to DON 0.9 mg.kg -1 feed without any adverse effects on feed intake and live weight gain (EFSA 2007).
The current study shows that villus height was increased after DON exposure but not after DON+NIV exposure in animal experiment. In general assumption, intestinal morphological
112 changes of small intestine, caused by injurious agents such as DON (Diesing et al. 2011), xenobiotics (Haschek et al. 2010) are reduction of the villus height because of villus contraction, which support the restoration of the barrier function through minimizing the surface area (Blikslager et al. 2007). By contrast, the present result is not the case when villi were higher than control animals after DON exposure. DON from 1.5-3 mg.kg -1 feed reduction of villus height has been previously reported in animal experiments (Gerez et al. 2015, Lucioli et al. 2013, Bracarense et al. 2012; Pinton et al. 2012). Our observation could also be incidental, as DON+NIV had no effect on villus height in the current study which is not consistent with observations of Gerez et al. (2015) using a tertiary combinations of DON+NIV+ZEA.
3.3.3. Proliferation
Trichothecenes have been reported to highly act on proliferative cells, and the intestinal epithelium has been identified as a target for DON and other derivatives in vitro , ex vivo , and in vivo (Kolf- Clauw et al. 2013; Gauthier et al. 2013; Bianco et al. 2012; Bracarense et al. 2011; Pinton et al. 2012). Although effects have been found for subchronical exposure, the current study, showed significant impact, starting from a 3 µM concentration on proliferative enterocytes with combined DON and NIV after 24h on loops.
At intestinal epithelium, enterocytes originate from the small intestinal crypts which surround the base of each villus. Biologically, these enterocytes progressively move along the side of the villus toward the tip, crypt-villus axis. As senescent enterocytes slough from the tip of villi, the absorptive epithelium is replaced from below by dividing epithelium cells. Each crypt produces 300-400 cells per day from a relatively few stem cells that produce committed daughter cells, which in turn divide several more times in the lower and middle portion of the crypts and are replaced every 2-4 days in adult, but replacement take longer in the neonate (Haschek et al. 2010; Booth and Potten 2000). However, this basic biological phenomenon for tissue homeostasis was effectively induced only when the combined DON and NIV were ingested repeatedly. The result of the impact of mycotoxins on proliferative enterocytes is in correlation to a concept that intestinal epithelial cells are important targets for the toxic effects of mycotoxins resulted only when they are exposed for subchronical period (Bouhet et al. 2004). Damage of intestinal epithelium cell can be induced and might have consequences for animal and human health exposed to xenobiotics or contaminated feed/food (Pinton et al. 2009).
113 Watson (1995) suggested that more studies are required to describe the fate of cells at the villus tip. Thus the current studies, at least in part, support this suggestion since the negative feedback mechanisms coordinate the rate of cell proliferation in the crypts with the rate of mature cell loss at the villus tip (Haschek et al. 2010), although it is not certain if the form of apoptosis is responsible for epithelial cell sloughing from the villus tips (Watson 1995). The result of total-cell count, assuming that most cells were lymphocytes, DON and DON+NIV inhibited proliferation at 10 µM in 24-h loops. In a dose-dependent manner, limited variation was sensitive between individuals or combined DON and NIV in 24-h loops and the inhibition was in accordance with Thuvander et al. (1999).
3.3.4. Apoptosis
In the small intestinal mucosa, apoptosis has been extensively studied (Groisman et al. 2000). However, to the best of our knowledge, it seems to be scarce using this biomarker in mycotoxicologic study, particularly for DON and NIV. Apoptosis is defined that cells undergoing apoptosis are morphologically characterized by condensation of nuclear chromatin into caps at the edge of the nucleus and detachment from their neighbors (Watson 1995). In particular, caspase- dependent apoptosis plays an executive role in the pathogenesis of a huge number of human diseases i.e. autoimmune diseases, multiple types of cancer, (Favaloro et al. 2012), and diseases of intestinal tract, for instance microvillous inclusion disease (Groisman et al. 2000; Watson 1995). Therefore, quantifying this biomarker will improve our knowledge of the health risk caused by mycotoxin.
In this study, apoptotic cells were counted at the villus tips compared to the crypts. Groisman et al. (2000) found that apoptotic cells were located in the upper third of villi in the normal small intestinal biopsies. Therefore, the upper one-third of villi where the current study assessed is corroborative the findings of Groisman et al. (2000) and the descriptions of Haschek et al. (2010) and cellular events of Watson (1995). At 24-h acute exposure in loops, individual 10 µM DON and starting from 3 µM NIV significantly increased apoptotic enterocyte at the villi, while the combination of this two mycotoxins affected even from 1 µM concentration. The lower doses, for example in NIV or in the combined DON and NIV, seemed to produce modulations with biological activity below the traditional toxicological threshold. Thus, it is assumed that follows a partern of hormesis, a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition (Calabrese 2002). It also well known that mycotoxins are immunomodulators for gastrointestinal tract (Oswald et al. 2005). A strong impact of the combined mycotoxins in the
114 present study reflects the results from previous reports. Thuvander et al. (1999) found that combinations of NIV with DON or T-2 toxin, DAS resulted in an additive effect. Caco-2 cells showed a different interaction effect between DON and NIV at in vitro cell level (Alassane-Kpembi et al. 2013). As results, the present combination of DON and NIV showed interaction effect on enterocytes for apoptosis in 24-h loops, and proliferation from both 24-h loops and animal experiment because the effects were greater when these two mycotoxins were combined.
The apoptotic enterocyte increase after 24-h exposed to 10 µM DON and DON+NIV, show similarity in microvillus inclusion disease (MID) and coeliac disease. MID is a specific disorder causing severe refractory diarrhea with diffuse villus atrophy without inflammatory changes in neonates. Coeliac disease is characterized by flat mucosa despite epithelial hyperproliferation in human (Groisman et al. 2000; Moss et al. 1996). The lesional changes such as villi atrophy, flattening of the epithelial cells found in explant after DON or NIV exposure were similar to those disease characteristics. In both cases, Groisman et al. (2000) and Moss et al. (1996) hypothesized that increased apoptosis is a major factor in the increased cell loss in MID and coeliac disease even if another forms of cell loss are exist. This is apparently shown in the present study. The absolute numbers of proliferating cells in the current study surpassed those of apoptotic cells at the villi. Thus, proliferative enterocyte to apoptotic enterocyte ratio at the villi was calculated, showing that DON+NIV had lower ratio in comparison with control loops and similar in comparison with DON treated loops. In loops model, many interleukin inflammatory genes which response to an inflammation were quantified by qRT-PCR, expressed no difference in 10-µM DON after 4-h and 24-h exposure. These results, at least in part, could support the similar characteristics of DON and MID affect intestinal health.
Either in 4-h loops or in 24-h loops a biological response for proliferation and apoptosis occurred immediately after exposure to DON or NIV. It was clearly shown that enterocyte apoptosis was not affected after 4-h exposure to DON or NIV. However, both proliferation and apoptosis of the total- cell counts were affected, with NIV impacted greater than DON after 4-h exposure. So, our studies show that lamina propria cells, mainly lymphocytes are more sensitive than enterocytes. The strong impact of NIV is confirmed with Wan et al. (2013) that NIV had the potency greater than DON in cell viability. Although, in the current study, these biological responses were not seen after 24-h incubation, suggesting that compensating responses might loss at this time because significant effects were found from individual, except for dose-dependent NIV and combined mycotoxins on the same counts.
115 A balance between cell proliferation and cell apoptosis is thought to preserve the normal architecture in the small intestine. It is reported that proliferative cells are found in the base of the crypts, and apoptotic cells are seen most frequently toward the tips of the villi (Watson 1995; Hall et al. 1994). In our study, one hand showed in 4-h loops that apoptotic enterocytes at the villi were not different after DON or NIV exposures, although crypt index proliferation was increased by NIV. On the other hand, 24-h loops showed that apoptotic enterocytes at the villi were significantly increased in DON or DON+NIV treated loops, even though crypt proliferation index was not affected at 10-µM exposure. These might be the insufficient time (after 4 h) shown for engulfment of apoptotic bodies by adjacent cells that would permit reutilization of the cellular materials.
3.4. General conclusion
In explant, the mucosa changes were induced by NIV at lower concentrations than DON. The total- cell count, mainly lymphoid cells, showed higher sensitivity than enterocytes to NIV-induced apoptosis after 4-h exposure in loops model. In loops model, DON+NIV showed high impact on proliferative enterocytes at both villus tip and crypt base after 24-h exposure. It also impacted enterocyte apoptosis and total-cell count at villus tip. In animal experiment, DON and DON+NIV decreased total-cell count proliferation at the villus tip while only DON+NIV affected enterocyte proliferation and significantly elongated the crypt depth. DON or NIV exposure has characteristics, at least in part, related to human diseases, microvillus inclusion disease and celiac disease leading to impaired nutrient absorption.
NIV alone or in combination with DON induced enterocyte proliferation and enterocyte apoptosis at the villi of pig small intestine following acute or repeated exposure. The interaction was shown on enterocytes for apoptosis in 24-h loops, and for proliferation in both 24-h loops and animal experiment. Thus, enterocytes were found to be a biomarker for the toxin effects for the two models, loops and animal experiment. In addition, loops model allowed in situ testing possible for dose-response and analyzing the interaction of the mycotoxins, despite the fact that it is not convenient to model repeated exposures. Owing to the lack of sufficient toxicological data for most of the masked mycotoxins and emerging mycotoxins, the loops model may contribute to risk assessments of these toxins.
3.5. Perspectives
116 The blood tests for the mycotoxin concentrations will indicate the real internal exposure of the animals, and these can verify the absorption, distribution, metabolism and excretion patterns in toxicology. The immunomodulation impact from individual or combined of DON and NIV at lower concentrations i.e. 1 µM or 3 µM, should be considered.
More efforts will be given to differentiate enterocytes and their migration rate along the villus axis. Furthermore, to identify the cells that mainly involved in the mucosa level in response to immune response i.e. neutrophils, eosinophils, monocytes, lymphocytes, etc. Then, proliferation and apoptosis scenarios, particularly in lamina propria should be considered.
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139 Appendixes
A. Animal experiment
Table A1 . Composition of the experimental diets (%, g.kg -1 or kcal.kg -1 based on a DM content of 88%); ARVALISADAESO-ITP 2002 Ctrl DON DON+NIV DON+ Composition (%) Uncontaminated Wheat Galopin 46.95 - 49.08 - Contaminated Wheat 17885 ++ 13.98 36.45 Contaminated maize 10-19077 - - 25.00 - Contaminated Wheat 17828 - 47.00 - 24.00 Uncontaminated Corn 18469 25.00 10.00 - 11.00 Soybean meal 48 21.00 22.00 18.80 21.50 Lysine HCL 0.57 0.55 0.63 0.56 L-Threonine 0.25 0.25 0.25 0.25 DL-Methionine 0.18 0.18 0.18 0.20 L-Tryptophan 0.05 0.04 0.06 0.04 Vitamins and minerals premix 4.00 4.00 4.00 4.00 Soybean oil 2.00 2.00 2.00 2.00 Nutrients (%DM) Dry matter 872.6 882.4 874.0 877.2 Total nitrogen 171.4 172.5 173.9 171.1 Crude fiber 23.7 24.2 23.3 23.9 Starch (Ewers methods) 442.0 435.3 446.4 434.9 Crude Fat 36.2 32.9 36.6 33.1 Minerals 59.4 60.7 58.4 60.3 Calcium 12.8 12.7 12.7 12.8 Phosphor 6.8 7.0 6.8 7.0 Digestible Energy 3437 3471 3456 3450 Net energy 2502 2515 2517 2503 Net energy (g4) 2494 2507 2507 2494 Total nitrogen/Net energy (g4) 69 69 69 69 LysineT 12.5 12.5 12.4 12.5 LysineD 11.5 11.5 11.5 11.5 LysineD/Net energy (g4) (g/1000 kcal) 4.6 4.6 4.6 4.6 MethinoninD/LysinD (%) 37 36 36 38 Methionine + CysteineD/LysineD 61 60 60 62 ThreonineD/LysineD 67 67 66 66 TryptophanD/LysineD 20 20 20 19 TryptophanD /Neutral amino acid 6 6 6 6 Mycotoxins (mg.kg -1) DON 0.03 2.89 3.5 4.63 NIV <0.01 0.07 0.72 0.05 ZEA <0.01 0.01 0.08 0.045 15-acétyl DON <0.01 0.01 0.19 0.015 3-acétyl DON <0.01 <0.01 <0.01 <0.01
140 Table A2. Effect of DON and DON+NIV after 28-day repeated exposure with DON natural single- at 2.89 mg.kg -1 feed and co-contamination to DON+NIV at 3.50 mg DON+ 0.72 mg NIV.kg -1 feed on weight gain and feed intake Parameters Ctrl DON DON+NIV P-value Weight gain (kg/pig) Average daily gain 0.51 ±0.12 0.57 ±0.10 0.49 ±0.06 0.358 Day 0 11.02 ±1.43 10.87 ±1.29 11.22 ±1.09 0.893 Day 7 13.78 ±2.16 13.20 ±1.4 13.20 ±1.55 0.800 Day 14 16.93 ±2.79 17.30 ±2.39 16.38 ±1.49 0.787 Day 21 20.95 ±3.32 21.90 ±3.03 20.40 ±1.64 0.644 Day 28 25.27 ±4.71 26.53 ±3.81 24.75 ±2.16 0.699 Feed intake (kg/pig) Average weekly intake 6.15 ±1.18 6.43 ±1.88 5.72 ±1.52 0.815 Day 7 4.63 4.12 3.65 N/a Day 14 5.80 5.70 5.60 N/a Day 21 7.05 7.72 6.47 N/a Day 28 7.13 8.17 7.17 N/a - Data presented as mean ± standard deviation (SD), ANOVA - N/a: not assessable - Weight gain or feed intake, n= 6 pigs
Table A3. Effect of DON and DON+NIV after 28-day repeated exposure with DON natural single- at 2.89 mg.kg -1 feed and co-contamination to DON+NIV at 3.50 mg DON+ 0.72 mg NIV.kg -1 feed on blood biochemistry Parameters Ctrl DON DON+NIV P-value Total protein (g/L) Day 0 54.33 ±5.43 48.33 ±1.70 50.82 ±2.05 0.060 kw Day 28 61.10 ±3.47 63.28 ±4.61 61.00 ±3.08 0.512 % changed 13.03 a ±8.54 30.92b ±8.40 20.16 ab ±6.90 0,005 Albumin (g/L) Day 0 29.35 ±3.57 27.53 ±2.71 29.53 ±1.90 0.417 Day 28 34.87 ±2.60 36.27 ±2.68 34.50 ±1.78 0.422 % changed 20.28 ±17.95 32.17 ±8.67 17.37 ±11.19 0.155 Fibrinogen (g/L) Day 0 17.28 ±4.37 19.15 ±3.57 20.26 ±1.84 0.408 Day 7 20.24 ±4.10 23.07 ±2.33 22.80 ±2.62 0.293 Day 28 23.52 ±3.81 20.62 ±1.25 22.48 ±3.03 0.246 % changed 38.24 ±11.44 11.52 ±26.83 22.80 ±2.62 0.060 Gamma-glutamyl transferase (U/L) Day 0 82.17 a ±32.59 52.83 ab ±9.54 47.67 b ±14.05 0.020 Day 28 60.17 ±17.88 69.17 ±21.99 60.50 ±18.87 0.674 % changed -20.28a ±27.78 29.59 b ±24.37 31.27b ±43.26 0.025 - Data presented as mean ± standard deviation (SD), ANOVA, n=6 pigs - kw : P-value from Kruskal-Wallis test - a, b mean values with different superscripts within the same row are different at P ≤0.05; Tukey’s test
141 Table A4. Effect of DON and DON+NIV after 28-day repeated exposure with DON natural single- at 2.89 mg.kg -1 feed and co-contamination to DON+NIV at 3.50 mg DON+ 0.72 mg NIV.kg -1 feed on morphometry, proliferation-cell and apoptosis-cell counts of the jejunum Parameters Ctrl DON DON+NIV P-value Morphometry Villus height (µm) 419.4 a ±95.1 481.3 b ±89.9 425.2 a ±103.2 <0.001 kw Crypt depth (µm) 340.4 a ±45.4 337.1 a ±57.6 354.8 b ±60.3 0.007 kw Crypt-depth to villus-height ratio 0.877 a ±0.25 0.742 b ±0.23 0.889 a ±0.25 <0.001 kw Proliferation Villus total cell 77.80 a ±21.31 57.83 b ±14.70 62.38 b ±16.78 0.009 Villus enterocytes 50.21 a ±17.82 45.06 a ±13.36 39.91 b ±10.71 <0.001 kw Lamina propria (cell) 27.59 a ±8.68 12.77 b ±3.52 22.47 a ±6.04 0.002 kw Apoptosis Peyer’s Patches (cell) 35.17 a ±9.83 38.93 b ±7.71 32.03 a ±12.82 0.009 kw - Data presented as mean ± standard deviation (SD), ANOVA - kw = P-values from Kruskal-Wallis test - a, b mean values with different superscripts within the same row are different at P ≤0.05; Tukey’s test or Behrens Fisher tests and Asymptotic Mann-Whitney tests - Morphometry: n= 6 pigs, 30 villi or crypt /loop; Values are mean numbers per villus or per crypt - Proliferation and apoptosis: n= 6 pigs, 20 villi or crypt/loop; Values are mean numbers per1/3 villus tip or per 2/3 crypt base
142 B. Loops model
Table B1. Gene expressions of jejunal loops treated with 10-µM DON for 4-h in situ incubation. Genes Ctrl DON P-value IL-1α 1 ±0.872 0.702 ±0.559 0.221 IL-1β 1 ±0.661 0.584 ±0.257 0.106 w IL-6 1 ±0.471 0.583 ±0.499 0.276 IL-8 1 ±0.763 0.838 ±0.599 0.595 IL-10 1 ±0.486 1.070 ±0.613 0.632 IL12-p40 1 ±0.914 0.548 ±0.283 0.590w IL-21 1 ±0.845 0.980 ±0.603 0.923 TLR-1 1 ±0.507 1.034 ±0.263 0.784 TLR-2 1 ±1.710 0.650 ±1.220 0.254 TLR-5 1 ±0.523 0.719 ±0.185 0.267 TLR-9 1 ±0.736 0.924 ±0.569 0.606 CCL-20 1 ±2.110 0.140 ±0.106 0.787 w NF-κB 1 ±0.551 0.752 ±0.231 0.178 TNF-α 1 ±0.739 0.782 ±0.281 0.556 TGF-β 1 ±0.656 1.100 ±0.834 0.845 IFN-γ 1 ±0.336 1.183 ±0.416 0.205 ALP 1 ±0.771 0.701 ±0.182 0.281 w EDN-2 1 ±0.820 0.859 ±0.630 0.646 Lysozyme 1 ±1.420 0.444 ±0.541 0.229 PCNA 1 ±0.566 0.744 ±0.197 0.324 Claudin-1 1 ±0.474 0.838 ±0.366 0.551 Claudin-2 1 ±1.010 0.582 ±0.341 0.262 Claudin-3 1 ±0.997 0.990 ±0.988 0.979 Claudin-4 1 ±0.852 0.613 ±0.300 0.214 Occludin 1 ±0.250 0.808 ±0.338 0.376 E-cadherin 1 ±0.823 0.635 ±0.238 0.787 w ZO-1 1 ±0.267 0.777 ±0.199 0.188 - Data presented as mean ± standard deviation (SD); n= 5 (pigs); Paired t-test - w = Wilcoxon matched pairs test
143 Table B2. DON+NIV (1:1) after 24h exposure at 10µM in loops showed higher effects on proliferation-cell and apoptosis-cell counts but not morphometry of the jejunal loops Parameters Ctrl DON DON+NIV P-value Morphometry Villus height (µm) 339.6 ±64.5 325.5 ±55.1 341.0 ±63.5 0.175 Crypt depth (µm) 364.6 ±50.7 375.2 ±38.3 363.6 ±42.7 0.269 Crypt-depth to villus-height ratio 1.11 ±0.28 1.14 ±0.21 1.17 ±0.24 0.196 Proliferation Villus total cell 35.23 a ±4.86 22.60 b ±4.33 22.40 b ±4.55 <0.001 Villus enterocytes 18.39 a ±4.47 19.15 a ±4.70 24.43 b ±4.91 <0.001 Crypt enterocytes 50.54 a ±4.47 50.58 a ±4.70 44.53 b ±4.91 0.001 Apoptosis Villus enterocyte 2.46 a ±0.98 2.98 b ±0.80 4.13 c ±1.30 <0.001 kw Lamina propria (cell) 2.38 ±0.93 2.25 ±1.16 2.03 ±0.95 0.290 kw Villus total cell 3.54 a ±0.97 3.56 ab ±0.94 3.96 b ±0.92 0.039 kw Ratio Enterocyte proliferation to apoptosis 9.23 a ±5.71 7.08 ab ±3.02 6.58 b ±2.80 0.021 kw Total-cell proliferation to total-cell apoptosis 10.70 a ±3.54 6.75 b ±2.23 5.97 b ±1.92 <0.001 - Data presented as mean ± standard deviation (SD), ANOVA - kw = P-values from Kruskal-Wallis test - a, b, c mean values with different superscripts within the same row are different at P ≤0.05; Tukey’s test or Behrens Fisher tests and Asymptotic Mann-Whitney tests - Morphometry: n= 2 or 4 (Ctrl) loops, 30 villi or crypt /loop; Values are mean numbers per villus or per crypt - Proliferation and apoptosis: n= 2 or 4 (Ctrl) loops, 20 villi or crypt/loop; Values are mean numbers per1/3 villus tip or per 2/3 crypt base
Table B3. Gene expressions of jejunal loops treated with 10-µM DON for 24-h in situ incubation. Genes Ctrl DON P-value IL-1α 1 ±0.786 0.999 ±0.243 0.998 IL-1β 1 ±1.210 0.173 ±0.118 0.541 IL-6 1 ±1.160 0.275 ±0.017 1.000 w IL-8 1 ±0.666 0.393 ±0.187 0.498 IL-21 1 ±0.807 0.994 ±0.936 0.961 IL12-p40 1 ±0.205 0.703 ±0.460 0.642 TGF-β 1 ±0.317 0.605 ±0.004 0.371 w Claudin-4 1 ±0.233 0.642 ±0.126 0.132 - Data presented as mean ± standard deviation (SD); n= 2 (pigs); Paired t-test - w = Wilcoxon matched pairs test
144 Title: Individual and combined effects of the trichothecenes deoxynivalenol and nivalenol ex vivo and in vivo on pig intestinal mucosa
Abstract
Deoxynivalenol (DON) and nivalenol (NIV), major fusariotoxins and worldwide cereal contaminants, raise concerns for intestinal health. The impact of DON and NIV on pig intestinal mucosa was investigated after acute exposure on jejunal explants after 4 hours (0 to 10 µM), on jejunal loops after 4 hours and 24 hours (0 to 10 µM), and after 28-day natural contamination feeding of animals. On explants, dose-dependent increases in the histological changes were induced. The decrease in the overall proliferative villus cells was concordant between animal experiment and loops, reaching after 4 hours in loops 13% and 30%, and after 24 hours 35 and 40 % for DON and NIV respectively, at 10 µM. In loops, villus apoptosis increased after DON and NIV at 10 µM. After 24 hours, apoptotic enterocytes increased dose-dependently by DON, NIV, or the combination DON+NIV (1:1). The interaction analysis showed synergism between DON and NIV for villus enterocyte apoptosis.
Keywords: mycotoxins; intestinal health; loops; enterocytes; cell proliferation; apoptosis; deoxynivalenol; nivalenol
AUTEUR : Sophal CHEAT
TITRE : Etudes ex vivo et in vivo des effets des trichothécènes déoxynivalenol et nivalénol, seuls ou combinés, sur la muqueuse digestive du porc
DIRECTEUR DE THESE : Pr. Martine KOLF-CLAUW
LIEU ET DATE DE SOUTENANCE : Ecole Nationale Vétérinaire de Toulouse, 08 juillet 2015
RESUME : Déoxynivalenol (DON) et nivalénol (NIV), fusariotoxines majeures des céréréales peuvent endommager l’intestin. Les effets de DON et NIV sur la muqueuse intestinale ont été étudiés chez le porc, après exposition aigüe d’explants jéjunaux après 4 heures (0 à 10 µM), d’anses jéjunales après 4 et 24 heures (0 à 10 µM), et après exposition d’animaux pendant 28 jours, à des aliments naturellement contaminés. Ex vivo sur les explants, des modifications histologiques dose-dépendantes ont été induites. In vivo , la diminution du nombre de cellules villositaires en prolifération a été concordante pour les anses jejunales et chez les animaux, atteignant, pour DON et NIV respectivement, 13 et 30% après 4h, et après 24 heures 35% et 40%, à 10 µM. Dans les anses, l’apoptose a été induite au niveau des villosités à 10µM de DON et de NIV. Après 24 heures, le nombre d’entérocytes apoptotiques a augmenté de façon dose dépendante avec DON, NIV, et DON+NIV (1:1), et l’analyse d’interaction a montré une synergie pour ce paramètre.
MOTS -CLES: Mycotoxines, santé digestive, anses, entérocytes, prolifération cellulaire, apoptosis, deoxynivalénol, nivalénol DISCIPLINE ADMINISTRATIVE : Pathologie, Toxicologie, Génétique et Nutrition
INTITULE ET ADRESSE DU LABORATOIRE : Equipe de BioToMyc: Biosynthèse & Toxicité des Mycotoxines UMR INRA-INPT-UPS 1331 Toxalim Ecole Nationale Vétérinaire 23 chemin des Capelles, BP 87614 31076 Toulouse Cedex 03, France