Identification and development of novel antimicrobial peptides as alternatives to antibiotic growth promoters in poultry

Poultry are vital to food security, with 60 billion chickens reared worldwide per annum and demand fast accelerating. For many years antibiotic growth promoters have been used to promote energy retention from the diet and control intestinal bacterial growth. Antibiotic use for prophylaxis or growth-promotion in farmed animals is prohibited under EU Directives due to human health concerns, but a pressing need exists to maintain the efficiency of animal production by finding alternatives. Antimicrobial peptides (AMPs), part of the innate immune system exist naturally in most species and could provide a vast array of potential therapeutics. Microbial resistance to AMPs is unlikely due to their relatively unspecific mode of action, their ability to target multiple sites within a cell and diverse immune-modulatory activities. The avian egg provides antimicrobial protection through many mechanisms including AMPs which are incorporated into the egg white by the hen. The ovodefensin family and ‘transiently expressed in neural precursors’ (TENP) have been identified as potential novel antimicrobials in egg white and therefore formed the basis of the peptide portfolio of this study. TENP was first identified as having a role in neurological development but has since been shown to be an important egg component constituting ~0.1-0.5% of the total protein. TENP is conserved across avian species being found in chicken, turkey, duck and zebra finch. Its homology with the bacterial permeability-increasing family of innate immune genes suggests it may contribute to antimicrobial function in the egg. This study confirmed that expression of TENP is confined to the albumen forming region of the oviduct in adult hens and is under gonadal steroid control, typical of an oviduct and egg specific gene. The ovodefensin family are β defensin related antimicrobial peptides thought to be restricted to the albumen producing region of the avian oviduct. This study identified twenty five novel ovodefensin members through genome analysis, expanding the ovodefensin family to include reptiles for the first time. Phylogenetic analysis showed a unique example of the evolution of a cysteine spacing motif alongside traditional sequence evolution. The expression of eight ovodefensins was shown to be oviduct specific supporting the hypothesis that ovodefensins evolved to protect the egg. Antimicrobial activity for three ovodefensins from chicken and duck was investigated against gram negative organisms E. coli and Salmonella including pathogenic strains as well as a gram positive organism, S. aureus, for the first time. The spectrum of activity varied greatly between peptides suggesting a link between structure and function. Inclusion of recombinant ovodefensin peptides in the feed of chickens showed beneficial effects on the gut microbiome, metabolite profile and most crucially an increase in mean body weight. This demonstrates the potential of antimicrobial peptides as alternatives to antibiotic growth promoters in poultry

Cuticle deposition improves the biosecurity of eggs through the laying cycle and can be measured on hatching eggs without compromising embryonic development

The cuticle is part of the egg's natural defense and it can be improved by genetic selection. Prior to adoption of this measurement in breeding programs, questions that need to be addressed are whether improved cuticle deposition will result in a reduced risk of eggs becoming contaminated and whether selection for this trait will have any unintended consequences on the incubation process. Bacterial penetration experiments were carried out using eggs from a pedigree line of broiler breeders (BB) and Rhode Island Red (RIR) layers. Within the natural variation in cuticle deposition in each line, a good cuticle was shown to reduce an egg's susceptibility to penetration by Escherichia coli (BB, P = 0.023) and Salmonella typhimurium (RIR, P < 0.001). Deglycosylation of cuticle proteins had little effect on their antimicrobial activity. The effect of bird age on cuticle deposition was also examined. Shell color decreased with age as anticipated; however, we found no evidence that cuticle deposition decreases with age, at least up to 50 wk. A thicker cuticle could affect the water vapor conductance (WPC) of hatching eggs. The WPC of eggs was, therefore, measured on eggs selected from the top and tail of the cuticle distribution, this time in a Lohmann Selected Leghorn (LSL) pedigree line. Broiler breeder eggs were also tested. No evidence of a relationship between cuticle deposition and WPC was found for LSL or BB eggs. Cuticle deposition measurements require eggs to be stained. Here, we show that this has no adverse effect on embryo development at d 12 of incubation. Thus, we conclude that cuticle deposition is important in preventing bacterial penetration of eggs in genetically divergent breeds of chicken and that the measurement can be practically incorporated into breeding programs. This will contribute to improving the biosecurity of eggs by reducing vertical and horizontal transmission of potentially zoonotic and pathogenic organisms from parent to offspring

Ovodefensins, an Oviduct Specific Antimicrobial Gene Family Have Evolved in Birds and Reptiles to Protect the Egg by Both Sequence and Intra Six Cysteine Sequence Motif Spacing

Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg

Review on docosahexaenoic acid in poultry and swine nutrition: Consequence of enriched animal products on performance and health characteristics

Omega-3 polyunsaturated fatty acids (n-3 PUFA) are linked to a variety of health benefits against human disorders and disease. However, the typical western diet is generally low in n-3 PUFA and high in n-6 PUFA, suggesting that the recommended intake of these essential fatty acids is seldom achieved. Therefore, dietary enrichment of animal meat and eggs with n-3 PUFA could help increase consumption of these fatty acids. Fish oils and microalgae (MA) are rich sources of long chain n-3 PUFA, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Feeding these marine products has been shown to increase DHA content of tissues and yolk, however, this may also lead to an increased requirement for anti-oxidants to prevent oxidative deterioration and associated negative sensory attributes. Nonetheless, increased DHA has been linked to promising results in animal growth, fertility, immunity and bone strength in both pigs and poultry. These findings suggest that feeding DHA-rich ingredients to mono-gastric can enrich human diets as well as providing additional benefits to the animal. Keywords: DHA, Microalgae, Performance, Anti-oxidan

Dietary betaine reduces plasma homocysteine concentrations and improves bone strength in laying hens

Abstract 1. In this study, we tested the hypothesis that betaine’s methyl-donor properties would reduce homocysteine concentrations, which was recognised in a previous genetics study to be linked to bone quality, and improve bone quality. This was combined with phytase treatment, as phosphorus is critical for bone mineralisation. 2. Using a 2 X 2 arrangement, a total of 1920 Lohmann LSL-lite chickens in 24 replicates of 20 chickens were fed one of 4 diets containing dietary betaine (0 or 1000 mg/kg) and phytase (300 or 1000 FTU/kg) from one day old until end-of-lay. Blood and bone samples were collected at 45 and 70 weeks of age. 3. Hens that were fed betaine had lower plasma homocysteine level (P < 0.05), higher tibia breaking strength (P < 0.05), and higher tibia density (P < 0.05). 4. Egg production and egg quality were excellent throughout the study and were not affected by the dietary treatments. 5. In summary, the addition of dietary betaine was successful at reducing plasma homocysteine concentrations and improving bone strength in laying hens which could be used as an intervention to alleviate welfare concerns

Comparative biology and expression of TENP, an egg protein related to the bacterial permeability-increasing family of proteins

The ‘transiently expressed in neural precursors’ (TENP) gene product is a member of the bacterial/permeability-increasing (BPI) family of antimicrobial proteins but was first identified as having a role in an early neurological event occurring in post-mitotic cells. However, recent characterisation of the egg white proteome has shown that TENP is an important egg component constituting ~ 0.1–0.5% of the total protein and suggesting it is expressed in the adult oviduct. In this study we confirmed quantitatively that the expression of TENP is largely confined to the tubular glands of the magnum of the oviduct, where egg white synthesis occurs, with around 10,000 times more expression than in the embryo where TENP was first identified. TENP expression is significantly increased with the administration of oestrogen or progesterone (P < 0.001) and is reduced in regressed oviducts (P < 0.001) demonstrating gonadal steroid control, typical of an oviduct and egg specific gene. A putative translational start site for TENP has been characterised and the evidence indicates that it is expressed as one predominant transcript. In comparison with the published sequence, insertion and deletion events have been identified causing a partial frame-shift that results in an altered amino acid sequence to that previously documented. TENP is conserved across divergent avian species being found in chicken, turkey, duck and zebra finch and its expression profile confirmed in both chicken and duck. Similarity searches have shown homology with the BPI-like family of innate immune genes, particularly with palate, lung and nasal epithelial clone (PLUNC) members of this family. We therefore believe that at least in adults the role of TENP is as a major component of egg, particularly the white and it is probable that it contributes to its antimicrobial function

163 Xylanase and fermentable xylo-oligosaccharides improve performance in grower-finisher pigs fed a corn-soybean meal based diet

Abstract Cecal inoculum collected from broilers fed a diet supplemented with xylanase showed increased gas production in an in vitro fermentation study with arabinoxylan-based substrates, indicating that intestinal microbiota can adapt to increase the capability to ferment arabinoxylans when pre-exposed to xylo-oligosaccharides (XOS) produced by xylanase. As the most abundant non-starch polysaccharides in a corn-soybean meal-based diet is arabinoxylan, this study tested the hypothesis that a blend of a xylanase and XOS will improve growth performance of pigs compared with those fed a diet supplemented with xylanase alone. One hundred twenty male crossbred (Landrace x Large white x Duroc) pigs (77 days average age; 30 kg average body weight -BW) were allocated to one of three treatments: CTR (control diet), XYL (CTR diet supplemented with 100 g/t of xylanase [Econase XT, AB Vista]) and XYL+XOS [CTR diet supplemented with 100 g/t of xylanase and XOS (Signis, AB Vista)]. Each treatment had ten replicates, with 4 animals each. The control diet was formulated with corn, soybean meal, DDGS and cassava meal as major ingredients. Pelleted diets were fed over 3 phases: grower (30–60 kg), finisher 1 (60–80 kg) and finisher 2 (80–100 kg). Average daily gain (ADG) and daily feed intake (ADFI) were measured from 30 to 100 kg and feed efficiency calculated (FCR). No treatment effects were observed on ADFI. Pigs offered the XYL+XOS had increased ADG (CTR = 1.058 vs XYL+XOS= 1.086 g/pig/d; P = 0.02) compared with those supplemented with xylanase alone (CTR = 1.058 vs XYL = 1.067 g/pig/d; P = 0.40). Feed efficiency was improved with XYL (2.65; P = 0.042) and XYL+XOS (2.60; P &lt; 0.001) compared with CTR (2.71). These results suggest that xylanase in combination with XOS further improves daily gain and FCR compared with a xylanase alone. Interestingly the differences between XYL and XYL+XOS become more apparent in older animals, when the microbiome matures and possibly digests fiber more effectively. <jats:p /

A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens

ABSTRACTNontyphoidal Salmonella spp. are a leading cause of human gastrointestinal infections and are commonly transmitted via the consumption of contaminated meat. To limit the spread of Salmonella and other food-borne pathogens in the food chain, bacteriophage (phage) therapy could be used during rearing or pre-harvest stages of animal production. This study was conducted to determine if a phage cocktail delivered in feed is capable of reducing Salmonella colonization in experimentally challenged chickens and to determine the optimal phage dose. A total of 672 broilers were divided into six treatment groups T1 (no phage diet and unchallenged); T2 (phage diet 106 PFU/day); T3 (challenged group); T4 (phage diet 105 PFU/day and challenged); T5 (phage diet 106 PFU/day and challenged); and T6 (phage diet 107 PFU/day and challenged). The liquid phage cocktail was added to mash diet with ad libitum access available throughout the study. By day 42 (the concluding day of the study), no Salmonella was detected in faecal samples collected from group T4. Salmonella was isolated from a small number of pens in groups T5 (3/16) and T6 (2/16) at ∼4 × 102 CFU/g. In comparison, Salmonella was isolated from 7/16 pens in T3 at ∼3 × 104 CFU/g. Phage treatment at all three doses had a positive impact on growth performance in challenged birds with increased weight gains in comparison to challenged birds with no phage diet. We showed delivering phages via feed was effective at reducing Salmonella colonization in chickens and our study highlights phages offer a promising tool to target bacterial infections in poultry

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%–92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry.</p

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%–92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry.</p