Comparing effects of natural betaine and betaine hydrochloride on gut physiology in broiler chickens.

Betaine is a well-known component of poultry diets with various effects on nutritional physiology. For example, increased water retention due to the osmolytic effect of betaine increases the volume of the cell, thereby accelerating the anabolic activity, integrity of cell membrane, and overall performance of the bird. Betaine is a multifunctional component (trimethyl derivative) acting as the most efficient methyl group donor and as an organic osmolyte, which can directly influence the gastrointestinal tract integrity, functionality, and health. So far, nothing is known about the effect of betaine on the intestinal barrier in chickens. In addition, little is known about comparing natural betaine with its synthetic form. Therefore, an animal study was conducted to ascertain the effects of betaine supplementation (natural and synthetic) on performance and intestinal physiological responses of broilers. One hundred and five 1-day-old broiler chicks were randomly assigned into 3 groups with 35 birds each: control, natural betaine (1 kg active natural (n)-betaine/ton of feed) and synthetic (syn)-betaine-HCL (1 kg active betaine /ton of feed). Histological assessment showed lower jejunal crypt depth and villi height/crypt depth ratio in syn-betaine-HCL group compared with natural n-betaine fed birds. Furthermore, it was found that syn-betaine-HCL negatively affects the integrity of the intestine by increasing the intestinal paracellular permeability in both jejunum and cecum as evidenced by a higher mannitol flux. Additionally, syn-betaine-HCl significantly upregulated the IFN-γ mRNA expression at certain time points, which could promote intestinal permeability, as it plays an important role in intestinal barrier dysfunction. Body weight (BW) and body weight gain (BWG) did not differ (P > 0.05) between the control birds and birds supplemented with syn-betaine-HCL. However, the BW and BWG were significantly (P < 0.05) improved by the dietary inclusion of n-betaine compared with other treatments. Altogether, the dietary inclusion of n-betaine had a positive effect on performance and did not negatively affect gut paracellular permeability. Furthermore, our results show that syn-betaine-HCl induces changes in the intestine, indicating an alteration of the intestinal histology and permeability. Thus, natural or synthetic betaine has different effects, which needs to be considered when using them as a feed supplement.

Paracellular intestinal permeability of chickens induced by DON and/or C. jejuni is associated with alterations in tight junction mRNA expression.

Toxins, antigens, and harmful pathogens continuously challenge the intestinal mucosa. Therefore, regulation of the intestinal barrier is crucial for the maintenance of mucosal homeostasis and gut health. Intercellular complexes, namely, tight junctions (TJs), regulate paracellular permeability. TJs are mainly composed of claudins (CLDN), occludin (OCLN), tight junction associated MARVEL-domain proteins (TAMPS), the scaffolding zonula occludens (ZO) proteins and junction-adhesion molecules (JAMs). Different studies have shown that a Campylobacter infection can lead to a phenomenon so-called "leaky gut", including the translocation of luminal bacteria to the underlying tissue and internal organs. Based on the effects of C. jejuni on the chicken gut, we hypothesize that impacts on TJ proteins play a crucial role in the destructive effects of the intestinal barrier. Likewise, the mycotoxin deoxynivalenol (DON) can also alter gut permeability in chickens. Albeit DON and C. jejuni are widely distributed, no data are available on their effect on the tight junctions' barrier in the broiler intestine and consequences for permeability. Therefore, the aim of this study was to analyze the interaction between DON and C. jejuni on the gut barrier by linking permeability with gene expression of TJ proteins and to determine the relationships between the measurements. Following oral infection of birds with C. jejuni NCTC 12744 at 14 days of age, we demonstrate that the co-exposure with DON has considerable consequences on gut permeability as well as on gut TJ mRNA expression. Co-exposure of DON and C. jejuni enhanced the negative effect on paracellular permeability of the intestine, which was also noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain time points in both jejunum and caecum. Furthermore, the increased paracellular permeability was associated with significant changes in TJ mRNA expression in the small and large intestine. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni resulted in a decreased barrier function via up-regulation of pore-forming tight junctions (CLDN7 and CLDN10), as well as the cytosolic TJ protein occludin (OCLN) that can shift to various paracellular locations and are therefore able to alter the epithelial permeability. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni affects the paracellular permeability of the gut by altering the tight junction proteins. Furthermore, analysing of correlations between TJs revealed that the mRNA expression levels of most tight junctions were correlated with each other in both jejunum and caecum. Finally, the findings indicate that the molecular composition of tight junctions can be used as a marker for gut health and integrity.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

The Mycotoxin Deoxynivalenol (DON) Promotes Campylobacter jejuni Multiplication in the Intestine of Broiler Chickens With Consequences on Bacterial Translocation and Gut Integrity.

Deoxynivalenol (DON) is one of the major health concern in poultry production as it targets epithelial cells of the gastrointestinal tract and contributes to the loss of the epithelial barrier function. It is well-documented that DON severely compromises various important intestinal functions in coincidence with aggravated clinical symptoms in livestock. In addition, a prolonged persistence of intestinal pathogens (e.g., Salmonella, Clostridium) in the gut has also been reported in pigs and chickens, respectively. Similar to DON, recent studies demonstrated that an experimental Campylobacter infection has severe consequences on gut health. Through experimental infection, it was found that Campylobacter (C.) jejuni negatively affects the integrity of the intestine and promotes the translocation of bacteria from the gut to inner organs. So far, no data are available investigating the simultaneous exposure of DON and C. jejuni in broilers albeit both are widely distributed. Thus, the aim of the present study was to explore the interaction between DON and C. jejuni which is of a significant public and animal health concern as it may affect the prevalence and the ability to control this pathogen. Following oral infection of birds at 14 days of age with C. jejuni NCTC 12744, we show that the co-exposure to DON and C. jejuni has a considerable consequence on C. jejuni loads in chicken gut as well as on gut permeability of the birds. A reduced growth performance was found for DON and/or C. jejuni exposed birds. Furthermore, it was found that the co-exposure of DON and C. jejuni aggravated the negative effect on paracellular permeability of the intestine already noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain times or intestinal segments. Furthermore, the increased paracellular permeability promotes the translocation of C. jejuni and E. coli to inner organs, namely liver and spleen. Interestingly, C. jejuni loads in the intestine were higher in DON-fed groups indicating a supportive growth effect of the mycotoxin. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni has not only considerable consequences on gut integrity but also on bacterial balance. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni could have a significant impact on gut health and bacteria translocation leading to an increased risk for public health.

Campylobacter jejuni increases the paracellular permeability of broiler chickens in a dose-dependent manner.

In recent years, several studies emphasize the deleterious effects of Campylobacter jejuni on the chicken intestine. In this context, it was shown that C. jejuni, contrary to the general belief, has a negative influence on the gut barrier in chickens. More precisely, we demonstrated that C. jejuni affects gut physiology characterized by changes in ion transport and transepithelial ion conductance, but the underlying mechanism is yet to be investigated. In the actual study, to determine epithelial paracellular permeability, the mucosal to serosal flux of 14C-mannitol in the small and large intestine was measured applying Ussing chamber. A total of seventy-five 1-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to 3 different groups (n = 25 with 5 replicates/group) and infected at 14 d of age with a high (108 colony forming units [CFU]) or a low (104 CFU) dose of C. jejuni and a third group kept as noninfected control. Infection with the low dose of C. jejuni resulted in delayed cecal colonization but equalized at 21 d postinfection, independent of the dose. Invasion of liver and spleen with C. jejuni was only noticed in birds infected with 108 (CFU). Body weight (BW) and body weight gain of all birds infected with C. jejuni were lower than in the control group and varied with the dose of infection, confirming a negative correlation between the infection dose and birds BW. Mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in all C. jejuni infected birds compared with control birds. Likewise, significant differences in mannitol flux of both jejunum and cecum were detected depending on the infection dose of C. jejuni. The correlation analyses revealed a positive relationship between Campylobacter dose and mannitol flux of both jejunum and cecum. Altogether, the actual results emphasize that the adverse effect of C. jejuni on gut permeability arises in a dose-dependent manner.

Feeding of deoxynivalenol increases the intestinal paracellular permeability of broiler chickens.

In recent years, the deleterious effects attributed to mycotoxins, in particular on the intestine, faced increased attention and it was shown that deoxynivalenol (DON) causes adverse effects on gut health. In this context, it has been repeatedly reported that DON can alter the intestinal morphology, disrupt the intestinal barrier and reduce nutrient absorption. The underlying mechanism of a compromised intestinal barrier caused by DON in chickens has yet to be illustrated. Although, DON is rapidly absorbed from the upper parts of the small intestine, the effects on the large intestine cannot be excluded. Additionally, a damaging effect of DON on the gut epithelium might decrease the resistance of the gut against infectious agents. Consequently, the objectives of the present studies were: (1) to investigate the impact of DON on the epithelial paracellular permeability by demonstrating the mucosal to serosal flux of 14C-mannitol in the small and large intestine applying Ussing chambers and (2) to delineate the effects of DON on the colonization and translocation of Escherichia coli. Both parameters are well suited as potential indicators for gut barrier failure. For this, a total of 75 one-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to three different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Body weight (BW) and BW gain of birds in the group fed with 10 mg/kg DON were significantly lower than in group with 5 mg/kg DON and the control group. Moreover, the mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in DON-fed groups compared to control birds. Consistent with this, DON enhanced the translocation of E. coli with a higher number of bacteria encountered in the spleen and liver. Altogether, the actual results verified that DON can alter the intestinal paracellular permeability in broiler chickens and facilitates the translocation of enteric microorganisms such as E. coli to extra-intestinal organs. Considering that moderate levels of DON are present in feed, the consumption of DON-contaminated feed can induce an intestinal breakdown with negative consequences on broiler health.

Re-thinking the chicken-Campylobacter jejuni interaction: a review.

Chickens are recognized as an imperative source of thermophilic Campylobacter spp., carrying this microorganism in high numbers in their intestinal tract. For a long time, Campylobacter jejuni has been considered as a commensal microorganism which colonizes its primary host rather than infecting it, in the absence of any obvious clinical signs. However, recent studies question this and argue for a deeper understanding of the host-bacteria interaction. Following oral uptake, it was demonstrated that C. jejuni interacts intimately with the gut epithelium and influences cellular functions of the host, with consequences on nutrient absorption. The immune reaction of the host which was revealed in some studies confirmed the infectious nature of C. jejuni. In agreement with this, an increased expression of pro-inflammatory cytokine genes was noticed. The ability to induce intestinal damage and to modulate the barrier function of the intestinal epithelia has further consequences on gut integrity, as it facilitates the paracellular passage of C. jejuni into the underlying tissues and it supports the translocation of luminal bacteria such as Escherichia coli to internal organs. This is associated with an alteration of the gut microbiota as infected birds have a significantly lower abundance of E. coli in different parts of the intestine. Some studies found that the gut microbiota influences the infection and translocation of C. jejuni in chickens in various ways. The effects of C. jejuni on the intestinal function of chickens already indicate a possible interference with bird performance and welfare, which was confirmed in some experimental studies. Furthermore, it could be demonstrated that a Campylobacter infection has an influence on the movement pattern of broiler flocks, supporting experimental studies. The intense interaction of C. jejuni with the chicken supports its role as an infectious agent instead of simply colonizing the gut. Most of the findings about the impact of Campylobacter on chickens are derived from studies using different Campylobacter isolates, a specific type of bird and varying experimental design. However, experimental studies demonstrate an influence of the aforementioned parameters on the outcome of a certain trial, arguing for improved standardization. This review summarizes the actual knowledge of the host-pathogen interaction of C. jejuni in chickens, emphasizing that there are still major gaps despite recently gained knowledge. Resolving the cascade from oral uptake to dissemination in the organism is crucial to fully elucidating the interaction of C. jejuni with the chicken host and to assess the clinical and economic implications with possible consequences on preventive interventions.

Enteric Pathogens and Their Toxin-Induced Disruption of the Intestinal Barrier through Alteration of Tight Junctions in Chickens.

Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing bird's health. The intestinal epithelial barrier serves as the first line of defense between the host and the luminal environment. It consists of a continuous monolayer of intestinal epithelial cells connected by intercellular junctional complexes which shrink the space between adjacent cells. Consequently, free passing of solutes and water via the paracellular pathway is prevented. Tight junctions (TJs) are multi-protein complexes which are crucial for the integrity and function of the epithelial barrier as they not only link cells but also form channels allowing permeation between cells, resulting in epithelial surfaces of different tightness. Tight junction's molecular composition, ultrastructure, and function are regulated differently with regard to physiological and pathological stimuli. Both in vivo and in vitro studies suggest that reduced tight junction integrity greatly results in a condition commonly known as "leaky gut". A loss of barrier integrity allows the translocation of luminal antigens (microbes, toxins) via the mucosa to access the whole body which are normally excluded and subsequently destroys the gut mucosal homeostasis, coinciding with an increased susceptibility to systemic infection, chronic inflammation and malabsorption. There is considerable evidence that the intestinal barrier dysfunction is an important factor contributing to the pathogenicity of some enteric bacteria. It has been shown that some enteric pathogens can induce permeability defects in gut epithelia by altering tight junction proteins, mediated by their toxins. Resolving the strategies that microorganisms use to hijack the functions of tight junctions is important for our understanding of microbial pathogenesis, because some pathogens can utilize tight junction proteins as receptors for attachment and subsequent internalization, while others modify or destroy the tight junction proteins by different pathways and thereby provide a gateway to the underlying tissue. This review aims to deliver an overview of the tight junction structures and function, and its role in enteric bacterial pathogenesis with a special focus on chickens. A main conclusion will be that the molecular mechanisms used by enteric pathogens to disrupt epithelial barrier function in chickens needs a much better understanding, explicitly highlighted for Campylobacter jejuni, Salmonella enterica and Clostridium perfringens. This is a requirement in order to assist in discovering new strategies to avoid damages of the intestinal barrier or to minimize consequences from infections.

Age-Related Differences in the Luminal and Mucosa-Associated Gut Microbiome of Broiler Chickens and Shifts Associated with Campylobacter jejuni Infection.

Despite the importance of gut microbiota for broiler performance and health little is known about the composition of this ecosystem, its development and response towards bacterial infections. Therefore, the current study was conducted to address the composition and structure of the microbial community in broiler chickens in a longitudinal study from day 1 to day 28 of age in the gut content and on the mucosa. Additionally, the consequences of a Campylobacter (C.) jejuni infection on the microbial community were assessed. The composition of the gut microbiota was analyzed with 16S rRNA gene targeted Illumina MiSeq sequencing. Sequencing of 130 samples yielded 51,825,306 quality-controlled sequences, which clustered into 8285 operational taxonomic units (OTUs; 0.03 distance level) representing 24 phyla. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Tenericutes were the main components of the gut microbiota, with Proteobacteria and Firmicutes being the most abundant phyla (between 95.0 and 99.7% of all sequences) at all gut sites. Microbial communities changed in an age-dependent manner. Whereas, young birds had more Proteobacteria, Firmicutes, and Tenericutes dominated in older birds (>14 days old). In addition, 28 day old birds had more diverse bacterial communities than young birds. Furthermore, numerous significant differences in microbial profiles between the mucosa and luminal content of the small and large intestine were detected, with some species being strongly associated with the mucosa whereas others remained within the luminal content of the gut. Following oral infection of 14 day old broiler chickens with 1 × 108 CFU of C. jejuni NCTC 12744, it was found that C. jejuni heavily colonized throughout the small and large intestine. Moreover, C. jejuni colonization was associated with an alteration of the gut microbiota with infected birds having a significantly lower abundance of Escherichia (E.) coli at different gut sites. On the contrary, the level of Clostridium spp. was higher in infected birds compared with birds from the negative controls. In conclusion, the obtained results demonstrate how the bacterial microbiome composition changed within the early life of broiler chickens in the gut lumen and on the mucosal surface. Furthermore, our findings confirmed that the Campylobacter carrier state in chicken is characterized by multiple changes in the intestinal ecology within the host.

Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during Campylobacter jejuni colonization.

Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca(2+)]i signaling in the intestines of broiler chickens. For this, we compared: control birds (n = 60) and C. jejuni-infected birds (n = 60; infected orally with 1 × 10(8) CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca(2+) indicator Fluo-4 and two-photon microscopy, we revealed that [Ca(2+)]i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca(2+)]i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca(2+)]i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca(2+) signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health.

Impacts of the feed contaminant deoxynivalenol on the intestine of monogastric animals: poultry and swine.

Deoxynivalenol (DON) is one of the most prevalent cereal contaminants with major public health concerns owing to its high toxigenic potentials. Once ingested, DON first and foremost targets epithelial cells of the gastrointestinal tract, whose proper functioning, as the first line of defence, is of paramount importance for the host's health. Emerging evidences, summarized in this article, suggest that DON produces its toxicity primarily via activation of the mitogen-activated protein kinases (MAPKs) signalling pathway and alteration in the expression of genes responsible for key physiological and immunological functions of the intestinal tissue of chickens and pigs. The activation of MAPKs signalling cascade results in disruption of the gut barrier function and an increase in the permeability by reducing expression of the tight junction proteins. Exposure to DON also down-regulates the expression of multiple transporter systems in the enterocytes with subsequent impairment of the absorption of key nutrients. Other major intestinal cytotoxic effects of DON described herein are modulation of mucosal immune responses, leading to immunosupression or stimulation of local immune cells and cytokine release, and also facilitation of the persistence of intestinal pathogens in the gut. Both of the last events potentiate enteric infections and local inflammation in pigs and poultry, rendering enterocytes and the host more vulnerable to luminal toxic compounds. This review highlights the cytotoxic risks associated with the intake of even low levels of DON and also identifies gaps of knowledge that need to be addressed by future research.

Campylobacter infection in chickens modulates the intestinal epithelial barrier function.

Asymptomatic carriage of Campylobacter jejuni is highly prevalent in chicken flocks. Thus, we investigated whether chronic Campylobacter carriage affects chicken intestinal functions despite the absence of clinical symptoms. An experiment was carried out in which commercial chickens were orally infected with C. jejuni (1 × 10(8) CFU/bird) at 14 days of life. Changes in ion transport and barrier function were assessed by short-circuit current (I(sc)) and transepithelial ion conductance (G(t)) in Ussing chambers. G(t) increased in cecum and colon of Campylobacter-infected chicken 7 d post-infection (DPI), whereas G t initially decreased in the jejunum at 7 DPI and increased thereafter at 14 DPI. The net charge transfer across the epithelium was reduced or tended to be reduced in all segments, as evidenced by a decreased I sc. Furthermore, the infection induced intestinal histomorphological changes, most prominently including a decrease in villus height, crypt depth and villus surface area in the jejunum at 7 DPI. Furthermore, body mass gain was decreased by Campylobacter carriage. This study demonstrates, for the first time, changes in the intestinal barrier function in Campylobacter-infected chickens and these changes were associated with a decrease in growth performance in otherwise healthy-appearing birds.

The feed contaminant deoxynivalenol affects the intestinal barrier permeability through inhibition of protein synthesis.

Deoxynivalenol (DON) has critical health effects if the contaminated grains consumed by humans or animals. DON can have negative effects on the active transport of glucose and amino acids in the small intestine of chickens. As the underlying mechanisms are not fully elucidated, the present study was performed to delineate more precisely the effects of cycloheximide (protein synthesis inhibitor, CHX) and DON on the intestinal absorption of nutrients. This was to confirm whether DON effects on nutrient absorption are due to an inhibition of protein synthesis. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Addition of D-glucose or L-glutamine to the luminal side of the isolated mucosa of the jejunum increased (P < 0.001) the Isc compared with basal conditions in the control tissues. However, the Isc was not increased by the glucose or glutamine addition after pre-incubation of tissues with DON or CHX. Furthermore, both DON and CHX reduced Gt, indicating that the intestinal barrier is compromised and consequently induced a greater impairment of the barrier function. The remarkable similarity between the activity of CHX and DON on nutrient uptake is consistent with their common ability to inhibit protein synthesis. It can be concluded that the decreases in transport activity by CHX was evident in this study using the chicken as experimental model. Similarly, DON has negative effects on the active transport of some nutrients, and these can be explained by its influence on protein synthesis.

Campylobacter jejuni influences the expression of nutrient transporter genes in the intestine of chickens.

The gastrointestinal tract represents the first barrier against pathogens. However, the interaction of Campylobacter with intestinal epithelial cells and its effects on the intestinal function of chickens are poorly studied. Therefore, the goal of the present study was to characterize the effects of C. jejuni oral infection on the mRNA expression of nutrient transporters in the intestine. Newly hatched specific pathogen-free (SPF) chickens were orally infected with C. jejuni (NCTC 12744; 1 × 10(8)CFU/bird) at 14 days of age. Quantitative RT-PCR analyses at 14 days-post infection (dpi) revealed that the relative gene expression of the sodium/glucose cotransporter (SGLT-1) and the peptide transporter (PepT-1) was down-regulated (P<0.05) in all investigated segments (duodenum, jejunum and cecum) of Campylobacter-infected birds, while the facilitated glucose transporter (GLUT-2) was down-regulated (P<0.05) in jejunal and cecal tissues only. Furthermore, down-regulation (P<0.05) of the cationic amino acid transporter (CAT-2) and the excitatory amino acid transporter (EAAT-3) was seen in the jejunum, and down-regulation (P<0.05) of the l-type amino acid transporter (y(+)LAT-2) was noticed in the duodenum of infected birds. The decreased expression of intestinal nutrient transporters coincided with a decrease (P<0.05) in body weight and body weight gain during a 2-week post infection period. For the first time, it can be concluded that nutrient transporter expression is compromised in the small and large intestine of Campylobacter-infected birds with negative consequences on growth performance. Furthermore, the down-regulation of mRNA expression of glucose and amino acid transporters may result in accumulation of nutrients in the intestinal lumen, which may favor C. jejuni replication and colonization.

In vitro exposure to Escherichia coli decreases ion conductance in the jejunal epithelium of broiler chickens.

Escherichia coli (E. coli) infections are very widespread in poultry. However, little is known about the interaction between the intestinal epithelium and E. coli in chickens. Therefore, the effects of avian non-pathogenic and avian pathogenic Escherichia coli (APEC) on the intestinal function of broiler chickens were investigated by measuring the electrogenic ion transport across the isolated jejunal mucosa. In addition, the intestinal epithelial responses to cholera toxin, histamine and carbamoylcholine (carbachol) were evaluated following an E. coli exposure. Jejunal tissues from 5-week-old broilers were exposed to 6×10(8) CFU/mL of either avian non-pathogenic E. coli IMT11322 (Ont:H16) or avian pathogenic E. coli IMT4529 (O24:H4) in Ussing chambers and electrophysiological variables were monitored for 1 h. After incubation with E. coli for 1 h, either cholera toxin (1 mg/L), histamine (100 µM) or carbachol (100 µM) were added to the incubation medium. Both strains of avian E. coli (non-pathogenic and pathogenic) reduced epithelial ion conductance (Gt) and short-circuit current (Isc). The decrease in ion conductance after exposure to avian pathogenic E. coli was, at least, partly reversed by the histamine or carbachol treatment. Serosal histamine application produced no significant changes in the Isc in any tissues. Only the uninfected control tissues responded significantly to carbachol with an increase of Isc, while the response to carbachol was blunted to non-significant values in infected tissues. Together, these data may explain why chickens rarely respond to intestinal infections with overt secretory diarrhea. Instead, the immediate response to intestinal E. coli infections appears to be a tightening of the epithelial barrier.

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.

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.

Effects of feed contaminant deoxynivalenol on plasma cytokines and mRNA expression of immune genes in the intestine of broiler chickens.

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1ß, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κß1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1ß, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1ß, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κß1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.

Dietary inulin alters the intestinal absorptive and barrier function of piglet intestine after weaning.

An experiment was conducted to study the effects of dietary inulin supplementation on the electrophysiological properties of small intestine of suckling and weaned piglets as indicators for glucose absorption and barrier function. Ten sows were divided into two groups, receiving either a control diet, or a diet with 3% inulin. The diets were fed from 3 weeks ante partum to 6 weeks post partum. In the first 2 weeks of life, piglets received only sow's milk. Irrespective to sex and without castration of males, four piglets (one piglet of each litter) from each group were selected and sacrificed on day 10 of age. The gastrointestinal tract of each piglet was removed and segments were immediately taken from the mid-jejunum and mounted in Ussing chambers. Furthermore, at weaning (6 weeks old) 8 piglets were randomly selected irrespective to sex and males were un-castrated (4 animals from sows received control diet and 4 animals from sows received 3% inulin supplemented diet) and fed for 2 weeks either control weaning diet or inulin supplemented diet. Thereafter segments of the mid-jejunum were used to investigate the effect of inulin on the gut electrophysiology of weaned piglets. The increase in short-circuit current (Isc) after the addition of glucose is an indicator of higher glucose absorption and the higher tissue conductance (Gt) of the epithelium suggested a higher intestinal permeability to paracellular Na(+). In suckling piglets, the addition of d-glucose on the luminal side of the isolated jejunal mucosa increased (P<0.001) the Isc in the inulin-supplemented and control groups compared to basal values. Electrogenic glucose transport (ΔIsc) was similar in suckling piglets from sows fed inulin or control diet, suggesting that feeding of inulin to the mother sows had no effect on glucose absorption across the jejunal mucosa of suckling piglets. However, the dietary inulin supplementation after weaning increased the ΔIsc (P<0.001) compared with the controls, suggesting that the inulin supplementation increased the electrogenic transport of glucose across the jejunal mucosa of weaned piglets indicating higher glucose absorption. Furthermore, the Gt was higher in the inulin-supplemented weaned piglets than in control piglets, which could be due to the increased paracellular permeability to Na(+). In conclusion, dietary inulin increased the glucose transport and altered the intestinal barrier by increasing the intestinal permeability in the jejunal mucosa of post-weaned piglets. Furthermore, the results indicated that inulin has a positive effect on glucose absorption in the piglet small intestine after weaning and subsequently the dietary inulin offers a promising approach to avoid post-weaning gastrointestinal tract disorders in pigs.

The impact of the Fusarium mycotoxin deoxynivalenol on the health and performance of broiler chickens.

The aim of the present experiment was to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on morphometric indices of jejunum and to follow the passage of deoxynivalenol (DON) through subsequent segments of the digestive tract of broilers. A total of 45 1-d-old broiler chickens (Ross 308 males) were randomly allotted to three dietary treatments (15 birds/treatment): (1) control diet; (2) diet contaminated with 1 mg DON/kg feed; (3) diet contaminated with 5 mg DON/kg feed for five weeks. None of the zootechnical traits (body weight, body weight gain, feed intake, and feed conversion) responded to increased DON levels in the diet. However, DON at both dietary levels (1 mg and 5 mg DON/kg feed) significantly altered the small intestinal morphology. In the jejunum, the villi were significantly (P < 0.01) shorter in both DON treated groups compared with the controls. Furthermore, the dietary inclusion of DON decreased (P < 0.05) the villus surface area in both DON treated groups. The absolute or relative organ weights (liver, heart, proventriculus, gizzard, small intestine, spleen, pancreas, colon, cecum, bursa of Fabricius and thymus) were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls. DON and de-epoxy-DON (DOM-1) were analyzed in serum, bile, liver, feces and digesta from consecutive segments of the digestive tract (gizzard, cecum, and rectum). Concentrations of DON and its metabolite DOM-1 in serum, bile, and liver were lower than the detection limits of the applied liquid chromatography coupled with mass spectrometry (LC-MS/MS) method. Only about 10 to 12% and 6% of the ingested DON was recovered in gizzard and feces, irrespective of the dietary DON-concentration. However, the DON recovery in the cecum as percentage of DON-intake varied between 18 to 22% and was not influenced by dietary DON-concentration. Interestingly, in the present trial, DOM-1 did not appear in the large intestine and in feces. The results indicate that deepoxydation in the present study hardly occurred in the distal segments of the digestive tract, assuming that the complete de-epoxydation occurs in the proximal small intestine where the majority of the parent toxin is absorbed. In conclusion, diets with DON contamination below levels that induce a negative impact on performance could alter small intestinal morphology in broilers. Additionally, the results confirm that the majority of the ingested DON quickly disappears through the gastrointestinal tract.