Intestinal changes and immune responses during Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.

Effects of cLFchimera peptide on intestinal morphology, integrity, microbiota, and immune cells in broiler chickens challenged with necrotic enteritis.

Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.

Performance of an Escherichia coli phytase expressed in Lactococcus lactis on nutrient retention, bone traits and intestinal morphology in broiler chickens.

The availability of plant phosphorus in the gut chicken can be improved by increasing phosphorus retention using phytase enzyme or a probiotic with phytase activity as an alternative. In this study, the efficacy of a recombinant probiotic, Lactococcus lactis (L. lactis), with a potential of phytase production was evaluated in broiler chickens. To this aim, 360 one-day-old male broiler Cobb 500 were divided into six treatments with six replicates and reared to 42 days of age. The experimental treatments included positive control diet containing adequate phosphorus (PC), negative control diet containing reduced available phosphorus (NC), negative control diet involving recombinant L. lactis (RLL), negative control diet containing both recombinant L. lactis and Lactobacillus salivarius (RLL + LBS), negative control diet including non-recombinant L. lactis (LL) and negative control diet containing Hostazym® . Growth performance, total tract apparent disappearance of phytate-P and nutrient retention, mineral content of the tibia and histomorphology of jejunum were evaluated at the age of 35 days. Based on the results, the phosphorus (P) deficiency in the diet reduced body weight (BW), average daily gain (ADG), length and strength of tibia and increased feed conversion ratio (FCR) compared to PC group. However, the supplementation of Hostazym® or RLL probiotic into the feed improved BW, ADG, FCR, disappearance of Phytate-P and retention of P, length and strength of the tibia in a level similar to PC treatment. Phosphorus content of tibia in the chickens fed P-deficient diets containing RLL was similar to that of the tibia in the control group. Excreta phytate and total P excretion of the chickens decreased when diets contained Hostazym® , RLL and RLL + LBS. In addition, the diet containing RLL + LBS probiotic increased villi height compared with other treatments (p < .05). Further, recombinant L. lactis could represent phytase activity in the gut environment of the chickens and could be an alternative to the commercial phytase.

Evaluation of whey protein sources on performance, liver antioxidants and immune responses of broiler chickens challenged with ethanol.

To evaluate the effects of whey protein (WP) sources on performance, liver antioxidants and immune responses of broiler chickens, 300 one-d-old male Ross 308 chickens were randomly allocated into six treatments with five replications of 10 birds each. This study was conducted on the basis of a completely randomized design in a 3 × 2 factorial arrangement with dietary supplemental WP sources (no WP; 2% WP and 2% whey protein concentrate (WPC)) and ethanol (zero and 6%) into drinking water. Birds were challenged with ethanol 6% from 10 to 24 days of age. Growth performance, immune responses, intestinal morphology and antioxidant enzymes were assessed in both challenged and non-challenged groups. There was a significant interaction between ethanol and WP sources for growth performance, so that weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were significantly improved in broiler chickens challenged with ethanol and fed with WP (p < .05). Intestinal morphology, humoral responses and heterophil to lymphocyte ratio (H/L) were not affected by WP sources. Cellular immunity responses were significantly lower in control group and challenged birds fed with WP compared with other groups after 48 hr of phytohemagglutinin-P (PHA-P) injection. Ethanol challenge decreased liver antioxidant enzyme activities and increased levels of malondialdehyde (MDA) and alanine aminotransferase (ALT) activities. Dietary inclusion of WP and WPC increased levels of liver antioxidant enzymes and decreased MDA and ALT activities. It can be recommended to use WP for improving the growth performance and liver antioxidant enzymes in broiler chickens challenged with ethanol.

Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers challenged with E. coli.

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics.

Production of Phytase Enzyme by a Bioengineered Probiotic for Degrading of Phytate Phosphorus in the Digestive Tract of Poultry.

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25 kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4 U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19 U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.

Effect of purine nucleosides on growth performance, gut morphology, digestive enzymes, serum profile and immune response in broiler chickens.

1. This study was conducted to evaluate the effects of purine nucleosides on performance, gut morphology, intestinal enzymes and immunity functions in broiler chickens from 0 to 21 d of age. 2. A total of 360 1-d-old male chickens (Cobb 500) were randomly assigned to 4 treatments with 6 replications. Experimental diets consisted of a control without any additives and diets containing 0.1% pure adenosine, 0.1% pure guanosine and 0.1% equal aliquots of pure adenosine and guanosine. Two birds per cage (12 birds per treatment) were killed on d 11 and 21 in order to obtain serum samples for lipid profile, jejunal samples for morphology and mucosal immunity, digestive enzymes for epithelial maturation, and bursa and spleen samples for relative weight of immune organs to live body weight. 3. Birds receiving adenosine in their diets showed a significant increase in body weight and average daily gain and a significantly lower feed conversion ratio compared to the control birds. Villus height and width in jejunal samples also increased significantly in birds supplemented with adenosine. Although maltase was not affected by the experimental diets, adenosine increased alkaline phosphatase and aminopeptidase. Adenosine and its combination with guanosine boosted mucosal immunity as a result of increased IgA production. While there was no significant difference among treatments regarding the relative weight of the spleen, adenosine increased the relative weight of the bursa of Fabricius. Present results also showed that adding guanosine to broiler diets had no significant effects on growth, gut morphology, enzymes activity and immunological indices. 4. In conclusion, the improvement in growth performance, gut morphology and immunity in birds receiving adenosine demonstrated that pure adenosine could be a beneficial feed additive for the poultry industry, while guanosine showed no significant improvement.