Insights on the Host Stress,Fear and Growth Responses to the Deoxynivalenol Feed Contaminant in Broiler Chickens

Mycotoxins pose an important danger to human and animal health. Poultry feeds are frequently contaminated with deoxynivalenol (DON) mycotoxin. It is thus of great importance to evaluate the effects of DON on the welfare related parameters in poultry industry. In the present study, the effects of contamination of broiler diet with 10 mg DON/kg feed on plasma corticosterone and heterophil to lymphocyte (H/L) ratio as indicators of stress, tonic immobility duration as an index for fear response and growth performance of broiler chickens were studied. In addition, the effect of a microbial feed additive either alone or in combination with DON contamination on these different aspects was also evaluated. The results showed that DON feeding significantly affected the welfare related parameters of broiler chickens. The feeding of DON contaminated diet resulted in an elevation of plasma corticosterone, higher H/L ratio and increased the fear levels as indicated by longer duration of tonic immobility reaction. Furthermore, DON reduced the body weight and body weight gain during the starter phase definitely at the second and third week. However, during grower phase, feeding of DON decreased the body weight at the fourth week and reduced the body gain at the fifth week. Addition of the microbial feed additive, a commercial antidote for DON mycotoxin, was able to overcome DON effects on stress index (H/L ratio), fearfulness and growth parameters of broilers. In conclusion, we showed for the first time that the DON feeding increased the underlying fearfulness and physiological stress responses of broilers and resulted in a reduction in the welfare status as indicated by higher plasma corticosterone, higher H/L ratio and higher fearfulness. Additionally, feeding the microbial feed additive was effective in reducing the adverse effects of DON on the bird''s welfare and can improve the performance of broiler chickens.     

Deoxynivalenol affects the composition of the basement membrane proteins and influences en route the migration of CD16+ cells into the intestinal epithelium

The numerous pores in the basement membrane (BM) of the intestinal villi are essential for the communication of enterocytes with cells in the lamina propria, an important mechanism for the induction of intestinal immune responses. The intestinal epithelial barrier is affected by the mycotoxin deoxynivalenol (DON) from both the apical (luminal) and basolateral (serosal) side. The pig is the most susceptible species to the anorectic and immune-modulating effects of DON, which is most prevalent in crops. We analysed in pigs the effect of DON-contaminated feed on the composition and perforation of the BM and the presence of CD16⁺ cells or their dendrites in the epithelium. In addition to in vivo experiments, in vitro studies were carried out. Using microarray analyses, the effects of DON on IPEC-J2 cells were studied with the focus on the BM. Our in vivo results showed in the control pigs: (1) a significant increased pore number (p?≤?0.001) in the jejunum in comparison to ileum, (2) no difference in the pore size, and (3) comparable frequency of intraepithelial CD16⁺ cells/dendrites in the jejunum and ileum. There was a marked trend that DON feeding increases: (1) the pore number in jejunum, and (2) the number of CD16⁺ cells/dendrites in the epithelium (Tukey–Kramer; p?=?0.055 and p?=?0.067, respectively). The in vivo results were extended with microarray analyses of epithelial cell (IPEC-J2 cells). The down-regulation of genes like syndecan, fibulin 6 and BM-40 was observed. These proteins are important factors in the BM composition and in formation of pores. Our results provide evidence that already low basolateral concentrations of DON (50 ng/mL) influence the production of the BM protein laminin by epithelial cells. Thus, DON affects the composition of the BM.     

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application

Background and Aims

Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.

Methods

A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.

Results

Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.

Conclusions

Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.     

Azolla leaf meal at 5% of the diet improves growth performance,intestinal morphology and p70S6K1 activation,and affects cecal microbiota in broiler chicken

With growing concern about including unconventional dietary protein sources in poultry diets to substitute the protein sources that are essential for human consumption such as soybean meal, Azolla leaf meal (ALM) has grown in popularity. In our prior experiment, ALM was used at inclusion rates of 5 and 10%. Five per cent inclusion of ALM increased broiler chicken growth performance, the concentration of cecal propionic acid, and activation of skeletal muscle p70S6 Kinase1 (p70S6K1) without having detrimental effects on the meat quality. Those results prompted us to further evaluate the effect of the same inclusion rates of ALM on phase feeding and intestine and liver health of the broiler chicks. The current study hypothesis is that dietary ALM positively affects phase feeding, intestinal morphology and p70S6K1 activation, cecal microbial gene expression, and improves the liver energy status. For this, we enrolled 135 one-day-old broiler chicks and collected growth performance data (starter, grower, and finisher stages) and samples of the gastrointestinal tract to analyse the morphology of the villi, immune-related organs, mucin, and abundance of intestinal p70S6K1. Cecal bacterial species were analysed using qPCR and liver samples were collected to analyse adenosine monophosphate (AMP) and ATP content and selected oxidative stress biomarkers. ALM increased BW and feed intake during the starter and grower phases but did not affect the feed conversion ratio. Liver oxidative stress and AMP: ATP ratio increased in chickens fed on a diet containing 10% ALM (AZ10; P < 0.05). Jejunum villi length and abundance of duodenal neutral mucin increased but villi of the ileum decreased in chickens fed on a diet containing 5% ALM (AZ5), while lymphoid follicle areas of the cecal tonsils decreased with both doses of ALM. Activation of p70S6K1 increased with AZ10 in the duodenum and AZ5 in the jejunum. In the gut, the family of Enterobacteriaceae decreased with both ALM doses. In conclusion, our results indicate an overall positive effect of dietary inclusion of ALM in the broiler chicken diet via its positive effect on intestinal morphology and function; however, a negative effect on the liver was observed with 10% ALM.     

不同品种、日龄和饲养方式对肉鸡肠道菌群的影响

目的研究品种、日龄和饲养方式对肉鸡肠道菌群的影响。方法选择北京油鸡和AA肉鸡各120只,分笼养和地面平养两种饲养方式,每种饲养方式60只鸡(5个重复,每个重复12只),于7、14、21和42日龄无菌收集十二指肠、回肠和盲肠内容物,分析各肠段肠道菌群变化。结果 (1)从十二指肠、回肠到盲肠微生物种类越来越丰富,相同肠段不同处理间随日龄的增加微生物种类也不断增加,十二指肠、回肠主要菌是植物乳酸杆菌和双歧杆菌,盲肠主要菌是拟杆菌和梭菌,且AA肉鸡拟杆菌数量大于北京油鸡;(2)品种对十二指肠和回肠条带数影响不显著,但北京油鸡大于AA肉鸡,日龄对回肠条带数的影响显著。品种与日龄的互作对盲肠的影响显著,品种、日龄和饲养方式三个处理的互作对回肠条带数的影响显著。(3)十二指肠各处理间肠道菌群的相似性在42日龄达到稳定,回肠在21日龄稳定,盲肠从7日龄到42日龄各处理间肠道菌群相似性变化不大。结论不同肠段的微生物区系有明显差异,品种、日龄和饲养方式对肉鸡肠道微生物区系有不同影响,但微生物区系会随着肉鸡日龄的增加趋于稳定。     

The effect of dietary supplementation of nitric oxide donor and inhibitor on nNOS expression in and motility of the small intestine of broilers

Abstract

We investigated the effect of dietary supplementation of sodium nitroprusside (SNP), a nitric oxide (NO) donor, and N-nitro-L-arginine methyl ester (L-NAME), a NO inhibitor, on neuronal nitric oxide synthase (nNOS) expression in and motility of small intestinum in broilers. A total of 560, one-day-old Ross 308 hybrid mixed sex broiler chicks were divided randomly into one control and seven treatment groups for a 42 day feeding trial including starter phase (0–21 days) and grower phase (22–42 days). The control group was fed a basal diet and the experimental groups were the fed basal diet supplemented with 25, 50, 100 and 200 mg/kg SNP and 25, 50 or 100 mg/kg L-NAME. Ten chickens from each group were sacrificed to collect samples on days 21 and 42. The expression patterns of nNOS immunoreactivity in nerve fibers were determined by immunohistochemistry. In the contractility studies, longitudinal isolated strips of duodenum, jejunum and ileum were treated with 10^?5 M L-arginine and 10^?4 M SNP. Immunohistochemistry revealed that nNOS expression was not detectable in the duodenum or ileum of either the control or experimental groups. On the other hand, nNOS immunoreactivity in the jejunum control group showed a strong reaction on day 21, but the reaction was weak on day 42. nNOS expression clearly was suppressed on day 21 by the diet supplemented with L-NAME, while the diet supplemented with SNP stimulated nNOS expression on day 21. Contractility experiments revealed that spontaneous contractility of isolated strips of duodenum, jejunum and ileum showed no significant difference among groups. Spontaneous contractions of all strips were inhibited by L-arginine and SNP in all groups. The percentage inhibition rate of spontaneous contractions of jejunum application on days 21 and 42 after L-arginine decreased in the group supplemented with 100 mg/kg L-NAME. The percentage inhibition rate on day 21 after SNP application decreased in both groups that received 50 and 100 mg/kg L-NAME. We demonstrated the expression pattern of nNOS in nerve fibers in jejunum of broiler chickens. Contractility studies revealed that the NOS-NO pathway may play a role in smooth muscle contraction of small intestine of chickens. Feeding strategies that supplement NO donor and NO inhibitor can be of physiological importance to small intestine motility owing to alteration of nNOS expression in the jejunum.     

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat

Abstract

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.     

Deoxynivalenol impair skin barrier function through the down regulation of filaggrin and claudin 1/8 in HaCaT keratinocyte

Deoxynivalenol (DON), a typical mycotoxin, is a substance that is biosynthesized mainly by the Fusarium species. It is usually found in wheat and other grains grown in the field. When it enters the human body, it causes severe diarrhea, abdominal pain, vomiting, and even death. In addition, DON is known to induce inflammation of the small and large intestine, and is also associated with the occurrence of cancer. However, until recently, the effects of DON on the human skin were unknown. To investigate how DON affects HaCaT, human immortalized keratinocytes, we used CCK-8 assay and a quantitative real-time RT-PCR method to detect changes in the expression of tight junctions and skin cell regulatory proteins. The CCK-8 assay was performed to determine the growth inhibitory concentration of keratinocytes by DON. DON affected the cell survival rate from 1 μM in a concentration dependent manner, with the minimum set as 1 μM and the maximum as 4 μM for all experiments. DON inhibited the mRNA expression of filaggrin by up to 71% and SERPINA1 up to 75%. The expression of AQP3 was reduced by up to 93% compared to the untreated control group. This may cause problems in the pH control function of the skin and weaken the function of moisturizing. In addition, in the presence of DON, the gene expression of claudin 1 and claudin 8, which are important proteins in the regulation of intercellular skin barrier, decreased by up to 47 and 80%, respectively. Snail/ Slug, suppressors of the claudin gene expression, each increased up to 625 and 974%, respectively. Also, the MMP9 gene increased by up to 515% in a concentrationdependent manner, perhaps causing a weakness of the barrier function of the skin. These results suggest that DON may causing the development of atopic skin by impairing the skin barrier and pH control of skin, as well as intestinal inflammation diseases. Therefore, particular attention should be paid to DON contamination during the development of cosmetic ingredients using grains.     

Effect of cereal type and plant extract addition on the growth performance,intestinal morphology,caecal microflora,and gut barriers gene expression of broiler chickens

Feeding broiler chickens on diets based on cereal grains of high non-starch polysaccharides content such as wheat and barley can negatively impact their performance and gut health. Plant extracts can be used as a potential tool to alleviate these negative effects. The present study assessed the effects of dietary cereal type and the inclusion of a plant extract blend (PEB) on the growth performance, intestinal histomorphology, caecal microflora, and gene expression of selected biomarkers for gut integrity in broiler chickens in a 42-d experiment. Ross-308 male broilers were assigned into different dietary treatments and fed on two cereal types (corn- vs. wheat/barley-based) with/without added graded concentrations of a PEB (0, 250, 500, 1000, and 2000 mg/kg diet). There were no significant differences in the growth performance parameters, intestinal histomorphology, and caecal microflora due to the impact of dietary cereal type. However, lactobacilli count in the caecal microflora was increased in the group fed on a corn-based diet. The PEB supplementation especially at a level of 500 to 1000 mg/kg diet significantly increased the average BW and decreased the feed conversion ratio. It also increased the villi length of duodenum, jejunum, and ileum, decreased the duodenal crypt depth, and increased the villi length to crypt depth ratio in the duodenum, jejunum and ileum. Supplementation of the PEB decreased the total bacterial and coliform count and increased the lactobacilli count in a linear pattern. Gene expression of Occludin and Junction Adhesion Molecule was significantly increased in the PEB supplemented diets, whereby no influence was observed on mucin expression. In conclusion, supplementation of a PEB at levels of 500–1000 mg/kg can be used as a tool to improve broiler performance and gut health.     

Iron absorption by small intestine of chickens

Iron (Fe) absorption by three segments (duodenum, jejunum, and ileum) of the small intestine of chickens was studied by a perfusion technique in vivo in closed circuit using⁵⁹Fe Cl₃ and was related to the histological characteristics of each segment. The serosal transfers of Fe for the duodenum and jejunum were the same (14%/cm), but significantly different (p<0.05) from those of the ileum (9%/cm), which may be explained by the morphological and histological properties of the gut of chickens. However, the presence of Fe in blood and in liver was significantly lower after perfusion of the jejunum and ileum than after perfusion of the duodenum. It is concluded that chickens show an early adaptation of small intestine to Fe absorption in response to the considerable loss of Fe suffered during the laying process.     

Single and Combined Effects of Deoxynivalenol Mycotoxin and a Microbial Feed Additive on Lymphocyte DNA Damage and Oxidative Stress in Broiler Chickens

The immune and intestinal epithelial cells are particularly sensitive to the toxic effects of deoxynivalenol (DON). The aim of this experiment was to study the effects of DON and/or a microbial feed additive on the DNA damage of blood lymphocytes and on the level of thiobarbituric acid reactive substance (TBARS) as an indicator of lipid peroxidation and oxidative stress in broilers. A total of forty 1-d-old broiler chicks were randomly assigned to 1 of 4 dietary treatments (10 birds per group) for 5 wk. The dietary treatments were 1) basal diet; 2) basal diet contaminated with 10 mg DON/kg feed; 3) basal diet contaminated with 10 mg DON/kg feed and supplemented with 2.5 kg/ton of feed of Mycofix Select; 4) basal diet supplemented with Mycofix Select (2.5 kg/ton of feed). At the end of the feeding trial, blood were collected for measuring the level of lymphocyte DNA damage of blood and the TBARS level was measured in plasma, heart, kidney, duodenum and jejunum. The dietary exposure of DON caused a significant increase (P = 0.001) of DNA damage in blood lymphocytes (31.99±0.89%) as indicated in the tail of comet assay. Interestingly addition of Mycofix Select to DON contaminated diet decreased (P = 0.001) the DNA damage (19.82±1.75%) induced by DON. In order to clarify the involvement of lipid peroxidation in the DNA damage of DON, TBARS levels was measured. A significant increase (P = 0.001) in the level of TBARS (23±2 nmol/mg) was observed in the jejunal tissue suggesting that the lipid peroxidation might be involved in the DNA damage. The results indicate that DON is cytotoxic and genotoxic to the chicken intestinal and immune cells and the feed additive have potential ability to prevent DNA damage induced by DON.     

Effect of early antibiotic intervention on specific bacterial communities and immune parameters in the small intestine of growing pigs fed different protein level diets

Antibiotics are designed to affect gut microbiota and subsequently gut homeostasis. However, limited information exists about short- and long-term effects of early antibiotic intervention (EAI) on gut homeostasis (especially for the small intestine) of pigs following antibiotic withdrawal. We investigated the impact of EAI on specific bacterial communities, microbial metabolites and mucosal immune parameters in the small intestine of later-growth-stage pigs fed with diets differing in CP levels. Eighteen litters of piglets were fed creep feed with or without antibiotics from day 7 to day 42. At day 42, pigs within each group were offered a normal- or low-CP diet. Five pigs per group were slaughtered at days 77 and 120. At day 77, EAI increased Enterobacteriaceae counts in the jejunum and ileum and decreased Bifidobacterium counts in the jejunum and ileum (P < 0.05). Moreover, tryptamine, putrescine, secretory immunoglobulin (Ig) A and IgG concentrations in the ileum and interleukin-10 (IL-10) mRNA and protein levels in the jejunum and ileum were decreased in pigs with EAI (P < 0.05). At day 120, EAI only suppressed Clostridium cluster XIVa counts in the jejunum and ileum (P < 0.05). These results suggest that EAI has a short-term effect on specific bacterial communities, amino acid decarboxylation and mucosal immune parameters in the small intestine (particularly in the ileum). At days 77 and 120, feeding a low-CP diet affected Bifidobacterium, Clostridium cluster IV, Clostridium cluster XIVa and Enterobacteriaceae counts in the jejunum or ileum (P < 0.05). Moreover, feeding a low-CP diet increased the concentrations of Igs in the jejunum and decreased pro-inflammatory cytokines levels in the jejunum and ileum (P < 0.05). At day 120, feeding a low-CP diet increased short-chain fatty acid concentrations, reduced ammonia and spermidine concentrations and up-regulated genes related to barrier function in the jejunum and ileum (P < 0.05). These results suggest that feeding a low-CP diet changes specific bacterial communities and intestinal metabolite concentrations and modifies mucosal immune parameters. These findings contribute to our understanding on the duration of the impact of EAI on gut homeostasis and may provide basis data for nutritional modification in young pigs after antibiotic treatment.     

Acidomucin goblet cell expansion induced by parenteral nutrition in the small intestine of piglets

Total parenteral nutrition (TPN) impairs small intestine development and is associated with barrier failure, inflammation, and acidomucin goblet cell expansion in neonatal piglets. We examined the relationship between intestinal goblet cell expansion and molecular and cellular indices of inflammation in neonatal piglets receiving TPN, 80% parenteral + 20% enteral nutrition (PEN), or 100% enteral nutrition (control) for 3 or 7 days. Epithelial permeability, T cell numbers, TNF-alpha and IFN-gamma mRNA expression, and epithelial proliferation and apoptosis were compared with goblet cell numbers over time. Epithelial permeability was similar to control in the TPN and PEN jejunum at day 3 but increased in the TPN jejunum by day 7. By day 3, intestinal T cell numbers were increased in TPN but not in PEN piglets. However, goblet cell expansion was established by day 3 in both the TPN and PEN ileum. Neither TNF-alpha nor IFN-gamma mRNA expression in the TPN and PEN ileum correlated with goblet cell expansion. Thus goblet cell expansion occurred independently of overt inflammation but in association with parenteral feeding. These data support the hypothesis that goblet cell expansion represents an initial defense triggered by reduced epithelial renewal to prevent intestinal barrier failure.     

A Redescription of the Life Cycle of Eimeria mitis Tyzzer, 1929

Ten-day-old broiler chickens were inoculated with oocysts of a characterized strain of Eimeria mitis , and tissues were fixed at 4, 8, or 24-h intervals after inoculation for histopathological examination. Tissue collections were initiated at the time of inoculation and extended up to 168 h postinoculation. The preferred site of development of E. mitis was found to be the ileum although more limited development of the parasite also took in the jejunum, cecal pouches, cloaca, and bursa of Fabricius. No distinctive and consistent intestinal lesions were macroscopically evident even in heavily parasitized chickens. The prepatent period was approximately 92 h postinoculation. The histopathological features of the E. mitis infections were characterized using conventional bright-field microscopy as well as both scanning and transmission electron microscopy. No extra-intestinal development of the parasite was observed.     

A redescription of the life cycle of Eimeria mitis Tyzzer, 1929

Ten-day-old broiler chickens were inoculated with oocysts of a characterized strain of Eimeria mitis, and tissues were fixed at 4, 8, or 24-h intervals after inoculation for histopathological examination. Tissue collections were initiated at the time of inoculation and extended up to 168 h postinoculation. The preferred site of development of E. mitis was found to be the ileum although more limited development of the parasite also took in the jejunum, cecal pouches, cloaca, and bursa of Fabricius. No distinctive and consistent intestinal lesions were macroscopically evident even in heavily parasitized chickens. The prepatent period was approximately 92 h postinoculation. The histopathological features of the E. mitis infections were characterized using conventional bright-field microscopy as well as both scanning and transmission electron microscopy. No extra-intestinal development of the parasite was observed.     

Barrier properties and tight junction protein expression along the longitudinal axis of rat intestine

The tight junction (TJ) protein family of claudins is a major determinant of barrier properties in a wide variety of epithelia. Aim of the study was to compare epithelial barrier properties with the presence of TJ proteins in exactly defined intestinal segments. Transepithelial resistance (R(t)) of duodenum, jejunum, ileum and colon tissue preparations was measured in Ussing chambers. In parallel, expression of TJ proteins was analyzed by Western blots. Colon was characterized by higher R(t) than more proximal segments. However, among the small intestinal segments, R(t) was highest in duodenum and lowest in ileum. Along the intestine different claudins were detected by Western blotting with different signal intensities. Colon showed strongest signals for sealing claudins in accordance with R(t), whereas predominant expression of permeability-mediating claudins was observed in small intestinal segments. Along the intestine, claudins show a marked segment-specific expression which is in accordance with respective barrier properties.