Predisposing effect of the mycotoxins deoxynivalenol and fumonisins on necrotic enteritis in broiler chickens

Mycotoxins are secondary fungal metabolites, which frequently contaminate feed and food raw materials. The Fusarium mycotoxins deoxynivalenol (DON) and fumonisins (FBs) are the most frequently detected mycotoxins on a worldwide basis. The majority of feed samples however comply with European Union (EU) maximum guidance levels. The EU maximum guidance level for poultry feed is set at 5 mg DON/kg feed and 20 mg fumonisin B1 (FB1) + fumonisin B2 (FB2)/kg feed. Although these low to moderate contamination levels do not result in obvious clinical symptoms of intoxication, they may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host pathogen interactions and thus a different outcome of eventual infections. Necrotic enteritis (NE) is one of the economically most important enteric diseases in poultry production. NE in broiler chickens may arise when changes in the gut microbial homeostasis allow colonization of the virulent Clostridium perfringens strains expressing the NetB toxin gene. The presence of predisposing factors such as coccidiosis and fishmeal is required in the pathogenesis of NE, allowing the proliferation of C. perfringens by either providing nutrients and/or favorable ecological niches. The general aim of this thesis was to examine whether DON and FBs, at concentrations in the feed approaching EU maximum guidance levels, predispose for NE in broiler chickens; and to gain insights in the mechanisms responsible for this interaction. Chapter 1 describes an experimental C. perfringens infection study in order to evaluate the predisposing effect of DON exposure to the development of NE in broiler chickens. After C. perfringens challenge, DON significantly increased the percentage of birds developing subclinical NE compared to the control group (47±3.0% vs. 20±2.6%). A negative impact of DON was demonstrated on intestinal morphology and the intrinsic component of the intestinal barrier, which is composed of the epithelial cell layer and the tight junctions that interconnect these epithelial cells. Consequently, this intestinal damage led to an increased luminal protein content by leakage of plasma proteins or altered absorption of dietary proteins, which stimulate clostridial proliferation and subsequently the development of NE. In Chapter 2 the impact of feeding a FBs contaminated diet on the intestinal morphology and microbiota composition was studied. The uptake of FBs contaminated feed also induced a negative impact on the intrinsic component of the intestinal barrier, and on the intestinal microbiota as well. A reduced abundance of the immunomodulating bacteria Candidatus Savagella and Lactobacillus spp. such as L. johnsonii, and an increased number of C. perfringens were observed in the ileum of birds fed a FBs contaminated diet. Subsequently, an experimental C. perfringens infection study was performed investigating the predisposing effect of FBs exposure to the development of NE in broiler chickens. After C. perfringens challenge, a significant higher percentage of birds developed subclinical NE in the group fed a FBs contaminated diet compared to the control group (45±2.2% vs. 30±5.5%). In Chapter 3, the impact of DON and FBs on the intestinal barrier was further examined for selected functional and structural extrinsic components of the intestinal barrier, to gain insights in the associated consequences for intestinal colonization and nutrient availability for C. perfringens. In the first part (Chapter 3.1), a negative impact of feeding broiler chickens a DON and/or FBs contaminated diet on intestinal mucus layer integrity was observed and induction of oxidative stress in intestinal epithelial cells. The last study (Chapter 3.2) investigated whether DON-induced intestinal damage influences the intestinal absorption of FBs, which may lead to an altered systemic exposure and increased toxic effects of the latter mycotoxin in broiler chickens. Although, no differences in toxicokinetic parameters of FB1 could be demonstrated between chickens fed a DON contaminated diet compared to birds fed a control diet. This doctoral thesis demonstrates for the first time that feeding a DON or FBs contaminated diet is a predisposing factor for the development of C. perfringens induced NE in broiler chickens. This coincides with negative effects on selected components of the intrinsic and extrinsic intestinal barrier of the chicken host, i.e. villus height, tight junctions, mucus, oxidative stress, and microbiota homeostasis. Consequently, exposure to DON and/or FBs at concentrations approaching the EU maximum guidance level in feed provides both nutrients and a favorable ecological niche for clostridial proliferation. Therefore, this doctoral thesis demonstrates that in addition to the best-known predisposing factors, DON and/or FBs contaminated feed should be included as major risk factor for the development of C. perfringens induced NE in broiler chickens

Chronic dietary intake of enniatin B in broiler chickens has low impact on intestinal morphometry and hepatic histology, and shows limited transfer to liver tissue

The Fusarium mycotoxin enniatin B (ENN B) is a so-called emerging mycotoxin frequently contaminating poultry feed. To investigate the impact of chronic ENN B exposure on animal health, broiler chickens were fed either a diet naturally contaminated with ENN B (2352 mu g/kg) or a control diet (135 mu g/kg) for 2, 7, 14, or 21 days. ENN B concentrations were determined in plasma and liver using a validated ultra-high performance liquid chromatographytandem mass spectrometry UHPLC-MS/MS method. Liver was evaluated histologically, and the villus length and crypt depth of the duodenum, jejunum, and ileum were measured. Histopathology of the livers did not reveal major abnormalities. Feeding an ENN B-contaminated diet could possibly inhibit the proliferation of enterocytes in the duodenal crypts, but did not affect villus length, crypt depth, or villus length-crypt depth ratio of the jejunum and ileum. ENN B levels in plasma and liver were significantly higher in the ENN B-fed group and ranged between <25-264 pg/mL and <0.05-0.85 ng/g, respectively. ENN B carry-over rates from feed to liver tissue were 0.005-0.014% and 0.034-0.109% in the ENN B and control group, respectively. Carry-over rates were low and indicated a limited contribution of poultry tissue-derived products to the total dietary ENN B intake for humans. The above results support the opinion of the European Food Safety Authority stating that adverse health effects from ENN B in broiler chickens are unlikely

Emerging Fusarium and Alternaria mycotoxins : occurrence, toxicity and toxicokinetics

Emerging Fusarium and Alternaria mycotoxins gain more and more interest due to their frequent contamination of food and feed, although in vivo toxicity and toxicokinetic data are limited. Whereas the Fusarium mycotoxins beauvericin, moniliformin and enniatins particularly contaminate grain and grain-based products, Alternaria mycotoxins are also detected in fruits, vegetables and wines. Although contamination levels are usually low (mu g/kg range), higher contamination levels of enniatins and tenuazonic acid may occasionally occur. In vitro studies suggest genotoxic effects of enniatins A, A1 and B1, beauvericin, moniliformin, alternariol, alternariol monomethyl ether, altertoxins and stemphyltoxin-III. Furthermore, in vitro studies suggest immunomodulating effects of most emerging toxins and a reproductive health hazard of alternariol, beauvericin and enniatin B. More in vivo toxicity data on the individual and combined effects of these contaminants on reproductive and immune system in both humans and animals is needed to update the risk evaluation by the European Food Safety Authority. Taking into account new occurrence data for tenuazonic acid, the complete oral bioavailability, the low total body clearance in pigs and broiler chickens and the limited toxicity data, a health risk cannot be completely excluded. Besides, some less known Alternaria toxins, especially the genotoxic altertoxins and stemphyltoxin III, should be incorporated in risk evaluation as well

Pathofysiologie van lipopolysaccharide geïnduceerde inflammatoire respons bij vogels

Inflammation is a protective response to infection and/or tissue damage and it induces migration of immune cells and mediators of immune response from the circulation to the infected and/or damaged tissue. This response will remove the initial noxe (e.g. lipopolysaccharide or LPS) and tissue healing will be stimulated. LPS is part of the outer membrane of gram-negative bacteria and causes an inflammatory response in birds due to its proinflammatory properties. As a result to this inflammatory response, birds develop a change in body temperature, increased production of proinflammatory cytokines and acute phase proteins, show leukocytosis and sickness behavior. The magnitude of these symptoms in birds depends on the bird species and differs from the symptoms in mammals. The characteristics and pathophysiology of an inflammatory response are frequently studied using LPS inflammation models. These models can further be applied for pharmacodynamic studies to assess the clinical effect of different anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs or NSAIDs. In this paper, an overview of the LPS-induced inflammatory response in birds is given

Mycotoxin occurrence in maize silage : a neglected risk for bovine gut health?

Forages are important components of dairy cattle rations but might harbor a plethora of mycotoxins. Ruminants are considered to be less susceptible to the adverse health effects of mycotoxins, mainly because the ruminal microflora degrades certain mycotoxins. Yet, impairment of the ruminal degradation capacity or high ruminal stability of toxins can entail that the intestinal epithelium is exposed to significant mycotoxin amounts. The aims of our study were to assess (i) the mycotoxin occurrence in maize silage and (ii) the cytotoxicity of relevant mycotoxins on bovine intestinal cells. In total, 158 maize silage samples were collected from European dairy cattle farms. LC-MS/MS-based analysis of 61 mycotoxins revealed the presence of emerging mycotoxins (e.g., emodin, culmorin, enniatin B1, enniatin B, and beauvericin) in more than 70% of samples. Among the regulated mycotoxins, deoxynivalenol and zearalenone were most frequently detected (67.7%). Overall, 87% of maize silages contained more than five mycotoxins. Using an in vitro model with calf small intestinal epithelial cells B, the cytotoxicity of deoxynivalenol, nivalenol, fumonisin B1 and enniatin B was evaluated (0-200 mu M). Absolute IC50 values varied in dependence of employed assay and were 1.2-3.6 mu M, 0.8-1.0 mu M, 8.6-18.3 mu M, and 4.0-6.7 mu M for deoxynivalenol, nivalenol, fumonisin B1, and enniatin B, respectively. Results highlight the potential relevance of mycotoxins for bovine gut health, a previously neglected target in ruminants