Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues.

Whether the different NR-AMPs could ever be utilized as drugs not only against prokaryotic (bacteria) pathogens but eukaryotic (fungal pathogens, and parasitic protists) depends on the side effects. To get experimental experience about the option of applying EPB-produced antimicrobials to pathogens, and parasites of veterinary significance, we present here the results of an in vitro, and an accompanying in vivo study on chicken. In the in vitro study, we tested the cytotoxic potential of the cell-free conditioned culture media (CFCM) of three entomopathogenic bacterium species, - X. budapestensis, DSM16342 (EMA); X. szentirmaii DSM16338 (EMC); Photorhabdus luminescens ssp. akhurstii TT01 - on chicken tissue culture cells, namely, on the Leghorn Male Hepatoma (LMH), [92] cells, (a permanent confluent hepato-carcinoma cell line). Each CFCM proved rather cytotoxic in this test. In the in vivo study, we fed freshly hatched male broiler chickens for 42 days with XENOFOOD [39] which contained autoclaved cultures of EMA, and EMC). These bacteria were grown on standard chicken (starter and grower) [HM3] [u4] feed, and the whole culture was used as a “food supplement”. [HM5]. It had been known that these EPB species cannot grow that is, not viable) atbody temperature (above 33 C).

Background: Although there is a great emphasis on meat safety in the poultry industry, chicken meat is a frequent source of the zoonotic Campylobacter jejuni infections. The possibility of carcass contamination is correlated with the number of C. jejuni bacteria in the intestinal tract of the birds, therefore, controlling bacterial colonization is of special importance already at the flock level. Application of short chain fatty acids could be an alternative strategy meeting the needs of the animals and the requirements of food safety regulations, as well. Objectives: In this study, we aimed to assess the antibacterial efficacy of butyrate on various C. jejuni strains at two pH values (6.0 and 7.4) in vitro, together with enrofloxacin and ampicillin sensitivity. Materials and Methods: Eight C. jejuni strains (7*10(5) CFU/ml) were incubated in Bolton broth buffer with different concentrations of sodium butyrate for 48 hours. Minimal inhibitory and bactericidal concentrations (MIC and MBC) of butyrate were determined by colony counting on Campylobacter selective agar plates. Conventional agar-diffusion test was used for the evaluation of antibiotic sensitivity. Results and Discussion: Butyrate exerted its inhibitory effect only at 100 mmol/1 concentration at pH 7.4, while its effectivity remarkably increased on the lower pH: MIC was detected to be 5 mmol/1 and MBC was measured strain -dependently as 5 or 7.5 mmol/1. All tested strains were sensitive to enrofloxacin, only one showed ampicillin-resistance, and all strains with the exception of one showed similar sensitivity to butyrate. Decreased butyrate susceptibility of this single field isolate was associated with ampicillin resistance. Based on our results, it can be stated that butyrate applied in the required concentration and at lower pH acts effectively against most C. jejuni strains in vitro. Hence, it might be a useful tool to reduce enteral C. jejuni colonization. However, several additional factors might influence the antibacterial efficacy of butyrate under in vivo conditions, which should be further analysed.

As heat stress is a major emerging issue in poultry farming, investigations on the molecular mechanisms of the heat-triggered cellular response in chickens are of special importance. In the present study, 32-day-old Ross 308 broiler chickens were subjected to 37 °C environmental temperature combined with 50% relative humidity for 4 or 8 h respectively. Following sampling, redox parameters such as malondialdehyde (MDA), reduced glutathione (GSH), protein carbonyl levels as well as glutathione peroxidase activity were assessed in liver, spleen, and kidney homogenates. The concentrations of small heat shock proteins (sHSP-s) HSP27, αA- and αB-crystallins were also investigated. Among these organs, the liver was found the most susceptible to heat-provoked oxidative stress, indicated by enhanced lipid peroxidation and rapid activation of protective pathways, including the definite increase of glutathione peroxidase activity and the excessive utilization of αA- and αB-crystallin proteins. Heat-associated decline of protein carbonylation and GSH content was observed in the liver in correlation with the increased involvement of αA- and αB-crystallins in cellular defense, resulting supposedly in an overcompensation mechanism. These data highlight the hepatic sensitivity to acute heat shock, potential adaptation mechanisms, and the specific role of sHSP-s in the restoration of physiologic cell function.

A healthy microbiota present in the small intestine contributes significantly to small intestinal function, including digestion, nutrient absorption and health. The current study investigated the effects of a prebiotic, a probiotic and a symbiotic supplementation on ileal microbiota composition of broilers at 7 days of age. In a total of 574 male Ross 308 day-old chickens were divided into four groups using six replicate pens and 24 chickens per pen. A maise-soybean based control diet (C), a control diet supplemented with probiotics (Broilact; Br), a control diet supplemented with symbiotic (inulin, yeast, Bacillus subtilis; Sy) and a wheat based diet supplemented with wheat bran (W) were formulated. On day 7 of life, two chickens per pen were slaughtered and ileal chymus samples were collected. For microbiota analysis 16S rRNA (V3-V4 region) gene targeted Illumina MiSeq sequencing was used. Feeding all diets supplemented increased the diversity to varying degrees compared to the control (C) diet (p=0.006). As a conclusion, all supplementation substantially influenced ileal microbiota of broiler chickens at an early age. All these results could offer some information for the future study on the relationship between early intestinal microbiota and the compounds of the feed.

Trichothecene mycotoxins such as T-2 toxin cause severe problems for agriculture, as well as for veterinary medicine. As liver is one of the key organs in metabolism, the main aim of our study was to investigate the immunomodulatory and cytotoxic effects of T-2 toxin, using primary hepatocyte mono-culture and hepatocyte—nonparenchymal cell (predominantly Kupffer cell) co-culture models of chicken. Cultures were exposed to 10 (T10 group), 100 (T100 group) and 1000 (T1000 group) nmol/L T-2 toxin treatment for 8 or 24 h. Alterations of cellular metabolic activity, the production of reactive oxygen species (extracellular H2O2), heat shock protein 70 (HSP70), and the concentration of different inflammatory cytokines such as interleukin (IL-)6 and IL-8 were investigated. Metabolic activity was intensely decreased by T-2 toxin administration in all of the cell culture models, in every applied concentration and incubation time. Concentrations of HSP70 and IL-8 were significantly increased in hepatocyte mono-cultures exposed to higher T-2 toxin levels (both in T100 and T1000 groups for HSP70 and in T1000 group for IL-8, respectively) compared to controls after 24 h incubation. Similarly, IL-6 levels were also significantly elevated in the T100 and T1000 groups in both of mono- and co-cultures, but only after 8 h of incubation time. In spite of the general harmful effects of T-2 toxin treatment, no significant differences were observed on reactive oxygen species production. Furthermore, the two cell culture models showed different levels of H2O2, HSP70, and IL-8 concentrations independently of T-2 toxin supplementation. In conclusion, the established primary cell cultures derived from chicken proved to be proper models to study the specific molecular effects caused by T-2 toxin. Metabolic activity and immune status of the different examined cell cultures were intensively affected; however, no changes were found in H2O2 levels.

Feed additives that can improve intestinal health and maintain a diverse and resilient intestinal microbiota of poultry are of great importance. Thus, the current study investigated the effects of a single strain butyric acid-producing Clostridium (C. butyricum) with (symbiotic) or without wheat bran supplementation on cecal microbiota composition and gut health characteristics of broiler chickens. In total, 384 male Ross 308 day-old chickens were divided into four dietary treatment groups and fed ad libitum until day 37 of life. Cecal samples were taken for Illumina sequencing and pH and short-chain fatty acid analyses, as well as for histological analysis at the end of the experimental period. Neither of the supplemented diets improved chicken growth performance. Caecum was dominated by the members of Bacteroidetes phyla followed by Firmicutes in each dietary group. At the genus level, Bacteroides, Oscillospira, Akkermansia, Faecalibacterium, Ruminococcus and Streptococcus genera exceeded 1% relative abundance. Dietary treatment influenced the relative abundance of the Akkermansia genus, which had a lower relative abundance in the C. butyricum group than in the other groups and in the symbiotic group compared to the wheat bran supplemented group. Dietary treatment also altered cecal crypt depth and had a trend to modify the cecal fermentation profile. Additive effects of wheat bran and C. butyricum supplementation were not detected. Our results suggest that Akkermansia muciniphila colonization in chicken can be influenced by diet composition.

Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte-nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte-nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte-nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

The effects of a single strain lactic acid producing bacteria (LAB) (Lactobacillus farciminis 5x109 CFU/kg) and a single strain butyric acid producing bacteria (BAB) (Clostridium butyricum 2.5x109 CFU/kg) with or without wheat bran supplementation (WB), were investigated on the production traits and on several gut characteristics of broiler chickens. In total, 576 male Ross 308 day-old chickens were divided into 24 floor pens and fed a corn-soybean based control diet (C) and five other probiotic or wheat bran supplemented diets (LAB, BAB, LAB+WB, BAB+WB, C+WB) in 4 replicates. The wheat bran content of the starter, grower and finisher diets were 3, 6 and 6%, respectively. During the 37 day long fattening period, growth rate, feed intake were recorded and feed conversion was calculated. At the end of the trial, 8 chickens per treatment were slaughtered and the following parameters investigated: trypsin, lipase and amylase activity of the jejunal chyme, ileal histomorphology and Lactobacillus load. None of the treatments resulted significant differences in the production traits (P>0.1). BAB supplementation tended to decrease digestive enzyme activity. Feeding WB in all combination increased crypt depth (P=0.002), ileal muscle layer thickness (P=0.001) and decreased the villi: crypt ratio (P=0.037) in the ileum.

In this literature review the authors present the favourable pharmacological effects of milk thistle (Silybum marianum). The medicinal herb has been used since ancient times, primarily because it has liver-protective effects, promotes liver functions and facilitates the regeneration of hepatocytes. The most important active ingredient of milk thistle is silymarin, which is mainly concentrated in the seed shell. It composed of flavonolignans and flavonoids (70-80%) and a mixture of polyphenol-like molecules (20-30%) that are not precisely defined chemically. The success of applications at the cellular level is due to the toxin-binding, antioxidant, protein synthesis enhancing, anti-fibrotic, anti-carcinogenic, antiviral and anti-inflammatory effects of the plant's active ingredients. In addition, it can be well integrated into the treatment of insulin resistance and diabetes, can help milk production, and has cardio- and neuroprotective properties. Its beneficial antimicrobial effects in the gut can contribute to the healthy intestinal flora. Most of the biological effects have been proven in vitro. The absorption of active ingredients of the milk thistle is quite low (20-50%). Furthermore, after absorption, the hepatic enzymatic biotransformation of silymarin is intense. Thus, the study of in vivo effects is more difficult and much less biological efficacy can be detected compared to in vitro investigations. Diets containing the active ingredients of milk thistle can be used well in the treatment of companion animals. Also, diets of exotic birds, parrots and pigeons may contain milk thistle extract. In racing horses, herb-based cure is often used to prevent stress-induced negative health effects. In the case of ruminants, pig, dog, cat and poultry species, the milk thistle seed, the oil and various by-products produced after oil-pressing can be mixed in the diet.

From the results of metagenomic research studies, we are just beginning to understand the positive and negative effects of the microbiome on the host. Metagenomics is the study of genetic materials from environmental or hostassociated microbiota to identify the microbial diversity and its functions. These new sequencing techniques aim at analysing not only the human microbiome, but also that of the most commercial animal species, including poultry. Highthroughput sequencing technologies have facilitated studies of the microbiome complex by allowing more comprehensive identification of microbes than traditional culture methods. Poultry digestive tract has significant bacterial activity, which can be seen as a unique complex microbial ecosystem. Microbial community in gastrointestinal tract (GIT) plays an important role in overall health and function of host, be it in human or animals. Newly developed sequencing platforms such as next-generation sequencing (NGS) have allowed the important researches into the diversity and functions of gut microbiota of various livestock animals. Beginning in the chicken crop, starch breakdown, and lactate fermentation are mediated by various Lactobacillus spp. at cell densities up to 10(9) /g. Lactobacilli also dominate the proventriculus, and the gizzard. In the gizzard, where the majority of mechanical and chemical breakdown of feed is performed, the low pH of gastric juices containing hydrochloric acid and pepsin decrease the total number of cells below 10(8) /g. The small intestine harbours large (10(8)-10(9) /g) bacterial populations dominated by Lactobacillus, Enterococcus, and various Clostridiaceae. The most abundant groups in the chicken caeca were found to be Clostridiaceae, Bacteroidaceae, Lactobacillus and Proteobacteria. In our article, the latest research results on the gastrointestinal microbiome of the domestic chicken are reviewed.

The contribution of the intestinal tract to differences in residual feed intake (RFI) has been inconclusively studied in chickens so far. It is also not clear if RFI-related differences in intestinal function are similar in chickens raised in different environments. The objective was to investigate differences in nutrient retention, visceral organ size, intestinal morphology, jejunal permeability and expression of genes related to barrier function, and innate immune response in chickens of diverging RFI raised at 2 locations (L1: Austria; L2: UK). The experimental protocol was similar, and the same dietary formulation was fed at the 2 locations. Individual BW and feed intake (FI) of chickens (Cobb 500FF) were recorded from d 7 of life. At 5 wk of life, chickens (L1, n = 157; L2 = 192) were ranked according to their RFI, and low, medium, and high RFI chickens were selected (n = 9/RFI group, sex, and location). RFI values were similar between locations within the same RFI group and increased by 446 and 464 g from low to high RFI in females and males, respectively. Location, but not RFI rank, affected growth, nutrient retention, size of the intestine, and jejunal disaccharidase activity. Chickens from L2 had lower total body weight gain and mucosal enzyme activity but higher nutrient retention and longer intestines than chickens at L1. Parameters determined only at L1 showed increased crypt depth in the duodenum and jejunum and enhanced paracellular permeability in low vs. high RFI females. Jejunal expression of IL1B was lower in low vs. high RFI females at L2, whereas that of TLR4 at L1 and MCT1 at both locations was higher in low vs. high RFI males. Correlation analysis between intestinal parameters and feed efficiency metrics indicated that feed conversion ratio was more correlated to intestinal size and function than was RFI. In conclusion, the rearing environment greatly affected intestinal size and function, thereby contributing to the variation in chicken RFI observed across locations.

Aims: To elucidate the anticoccidial potential of antimicrobial peptides from Xenorhabdus budapestensis on both causative pathogens (prokaryotic Clostridium perfringens and eukaryotic Eimeria tenella) . Objectives: (1) To establish if the antimicrobial compounds of the cell-free culture media (CFCM) of the entomopathogenic symbiotic bacterium species, X. budapestensis DSM 16342 (EMA) and X. szentirmaii DSM 16338 (EMC) were active against 13 independent pathogenic isolates of Clostridium perfringens in vitro ; (2) To create a sterile, autoclaved, bio-preparation called “XENOFOOD”, for future in vivo feeding studies, aimed at determining the efficacy, and side-effects, of EMA and EMC on C. perfringens in chickens. Study Design: Clostridium perfringens samples (LH-1-LH24) were collected from chickens and turkeys, and were deposited in the frozen stock collection of Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary, where the in vitro assays were carried out on 13 of these isolates. Place and Duration of Study: Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary between September 2013 and February 2014. Methodology: Adaptation of our previously published in vitro bioassays for aerobic tests for the anaerobic bacteria Clostridium perfringens . When preparing “XENOFOOD” we benefitted from our experimental data about the heat tolerance and endurance to proteolytic enzymatic digestion of the studied antimicrobial peptides. Results: The studied antimicrobial peptides were heat-stable, trypsin and pepsin resistant. All but one of 13 C. perfringens isolates was sensitive to EMA-CFCM. XENOFOOD (made here) is not toxic for chicken, (unpublished). Conclusion: Since these cell-free cultures killed E. tenella cells, but were toxic to permanent chicken liver (LMH) cells, we need to run in vivo feeding tests to determine the gastrointestinal (ileac), anti- Clostridium and anti- Eimeria biological effects of the these heat, - and proteolysis tolerant antimicrobial peptides.