Optimal Ovodefensins

Lead Research Organisation: University of Edinburgh
Department Name: Royal (Dick) School of Veterinary Scienc

Abstract

Concern about the development of resistance to antibiotics has reached the public consciousness with predictions that we are heading for a dark age where our ability to fight infection is severely compromised. Although the indiscriminate medical prescribing of antibiotics is part of the problem, their extensive use in agriculture has very much been in the spotlight. The use of antibiotics to promote growth and prevent infections in farmed animals is widespread in much of the world. Although this practice is banned in Europe, antibiotics are still widely used to treat diseases, especially of the gastrointestinal tract, and total use in animals has risen as the scale of production has increased.

The need for responsible use of antibiotics and stewardship of this precious resource is likely to lead to significant restrictions on agricultural use. This presents tough challenges for the poultry industry, where maintaining good gut health is critical to the efficient production of poultry meat. Poultry supply around one third of the world's animal protein and although 60 billion birds are reared worldwide each year, demand is fast accelerating as our population grows. A need therefore exists for alternatives to antibiotics to improve gut health and energy retention to sustain the efficiency of animal production, not least as we compete with animals for dietary resources such as grain.

We have characterised a novel family of antimicrobial peptides from eggs which possess potent activity against a number of bacteria. These small peptides, which we called ovodefensins, have been synthesised inexpensively on a large scale by our industrial collaborators. When tested in animals and artificial gut models they have potent favourable effects on bacterial communities in the gut. For example, in a critical region of the lower gut there is an increase in favourable organisms such as Lactobacilli and less undigested food is present, which indicates that the gut is working well. Indeed growth of the chickens was improved by 6% over just 3 weeks by in-feed administration of an ovodefensin, which is comparable to the effect of growth-promoting antibiotics.

We propose to understand how the ovodefensin peptides exert their effect by examining the gut tissue and contents from chickens that have been fed the peptides. We will look for changes in gut structure and gene expression, as well as differences in the metabolites and microbes in the gut contents that may be associated with the beneficial effects of ovodefensins on gut health and growth. We also want to understand how treatment in the early days of a chick's life influences the colonisation of the gut by bacteria and how this influences maturation and function of the gut in later life.

In terms of the ovodefensin peptides themselves we have found a wide range of these molecules across the egg-laying birds and reptiles. Evolution, driven presumably by the bacteria the eggs are exposed to, has resulted in diverse ovodefensins with different amino acids sequences but also in number of amino acids between key amino acids that determine the shape of the molecule. We want to understand how the key features of the molecules dictate the ability to kill bacteria and promote growth to inform the design of ovodefensins with improved activity. We will take a structured approach using gallin, a potent ovodefensin, to alter key amino acids that effect the properties of the molecule that are thought to be important for its activity against bacteria. Ultimately we aim to use the knowledge gained to design optimised ovodefensins which will have improved activity against bacteria and show even more potent activity to improve gut health in chickens and reduce the need to use antibiotics. The project is founded on research funded by BBSRC and AB Vista and benefits from substantial contributions from our industrial partners, reflecting the value to industry of the proposed research.

Technical Summary

Poultry are vital to global food security. For decades, in-feed antibiotics have been used as growth-promoters to increase productivity. However, this has driven the evolution of resistance, threatening the efficacy of human and veterinary medicines. Use of antibiotics in prophylaxis and as growth-promoters is prohibited in the EU but therapeutic use has increased to near pre-ban levels so alternatives are urgently needed. We recently characterised a novel antimicrobial peptide from egg white (the ovodefensin gallin) synthesised in a recombinant fungal system. It improves poultry growth in a manner associated with altered intestinal microbiota and metabolite profiles. We wish to optimise the activity of ovodefensins by altering residues and motifs predicted from the 3D-NMR structure of gallin. In nature there is variability in the amino acid sequence and spacing between the conserved 6 cysteine motif that make this family particularly interesting. We will probe the importance of cysteine bonds, spacing and hydrophobicity in the 1st/2nd cysteine region, spacing and an ovodefensin-specific fifth beta-sheet in the 4th/5th cysteine region and ovodefensin charge, which can range from +3 to +6. Trichoderma-expressed ovodefensins will be tested in poultry with substantial cash and in-kind contributions from AB Vista, both to optimise dose, timing and duration of administration, to compare gallin with mutated gallin and naturally-occurring ovodefensins, and to compare an optimised ovodefensin with currently used antibiotics. From these trials, correlates of bird performance and gut health will be identified using transcriptomics and histological measurements, as well as microbiome and metabolite profiling. The project has the potential to enhance avian health and productivity at a time of fast accelerating demand. AB Vista are ideal partners, having already driven the widespread adoption of enzymes as dietary additives and identified correlates of gut health.

Planned Impact

The principal benefit from the proposed research will be an understanding of how the feeding of small doses of naturally-occurring and modified ovodefensins produces beneficial effects on the performance of poultry. By linking ovodefensin structure to function and identifying correlates of in vivo activity we will be able to design and evaluate optimal ovodefensins with greater antimicrobial and growth-promoting activity to improve avian welfare and productivity. The scope to achieve this impact is reflected in the substantial cash and in-kind contributions of AB Vista, which has a proven record of producing commercially successful dietary additives founded on basic science.

The removal of prophylactic antibiotics from animal feed has resulted in problems of enteric disease in poultry and other monogastic species. The cost of such restrictions in poultry (even with large parts of the world still using growth-promoters) is estimated to be $2 billion owing to reduced feed conversion efficiency, lower yields and increased morbidity and mortality due to infectious disease. The discovery of compounds which can be used in place of antibiotic growth-promoters has the potential to deliver major improvements in the efficiency of production, welfare and environmental impact of animal agriculture. If the project is successful in its aim of being part of the solution to reducing the prophylactic or therapeutic use of antibiotics in animals and transmissible resistance it will contribute substantially to societal and economic prosperity in the medium- to long-term.

Whilst our principal aim is improving monogastric gut health and energy retention from the diet there is a strong imperative to develop novel antimicrobials for a wider set of applications. This will be aided by our characterisation of a wider range of host-defence peptides and their properties, novel methods to produce large quantities, reduced costs of production and realistic tests of efficacy. Some of these challenges have been met in regard to ovodefensins, in particular the inexpensive high-yield production of gallin and taeniopygin-2 in recombinant Trichoderma and evidence of growth-promotion following in-feed administration in a commercial setting. Because the ovodefensins are able to kill bacteria that are not growing there may be applications in surface sanitisation of food and surfaces where antibiotics that target growth-dependent processes are often ineffective.

The value of the impacts above would be felt worldwide and contribute to the aim of ensuring a sustainable and efficient supply of quality food for the world. AB Vista is a UK company (part of AB Agri in turn part of AB Foods) with a global reach and this provides a basis for knowledge-based wealth creation that will benefit the UK. The production of a usable product for use in animals does not involve the regulatory hurdles of human medicine; however that is not to say there are not regulatory challenges. These can take longer to overcome than the process of engineering and producing recombinant products, some of which we already have in production. It is reasonable to expect around 5 years to complete the procedures required to bring novel dietary additives to the market because of this, however AB Vista has a proven record of doing so with enzymes to improve digestion of non-starch polysaccharides and thus energy retention. Our partnership is already working well and no delays are anticipated as the named researcher has already worked with AB Vista and Alimetrics to characterise gallin and other members of the ovodefensin family.

Publications

10 25 50
 
Description A number of preparations of ovodefensin like molecules have been trialled as feed additives in broiler diets. There have been indications of improved performance with indications of correlated improvements in the microbiota and its metabolic products. Assesment of the repeatability of these observations are continuing to be made and the most successful compounds are being used in more challenging situations for the chickens gut health.

There was a set back in the synthesis of molecules to test the effectiveness of different modifications to the ovodefensins. A new supplier has been used and we have good molecules to test and that aspect of the project is back on target. This has led to some clear indications of how the structure of ovodefensins can be altered to improve antimicrobial activity in particular the role of the cysteine bonds.
The function of genes and cellular phenotypes in animal systems
We were unable to synthesise the peptide when alanine was used to replace cysteine at C3-C6. All others cysteine substitutions could be synthesised. Interestingly the peptide with the most potent antibacterial activity was where the C1-C5 cysteines had been replaced. A low dose of 10µM gave more than 50% reduction against S. Aureus test microorganism. When all cysteines were replaced so no disulphide bonds would be formed significant antibacterial activity versus S. Aureus remained, similar to the unaltered structure with ~ 50% reduction at 100µM. Disrupting the C2-C4 disulphide bond lead to lower antibacterial activity even at higher doses (100µM to 200µM). These results suggest that the C3-C6 bond is an important for the structure of the ovodefensin since the peptide was unable to be folded after synthesis, whilst the removal of the C2-C4 bond gave limited antibacterial activity suggesting this may also be important for the ovodefensin to function properly. The removal of C1-C5 lead to a significant increase in antibacterial activity indicating this disruption could increase the antimicrobial potency. Future research would be needed assess modified ovodefensin in a feed trial to see if it provided any benefit to growth and intestinal health.
Exploitation Route Our partners ABvista have completed trials to improve the formulation and production of the molecules which work best. This is based on the results of the trials already completed and informed by the structure function test results. Colleagues in France are using some of the peptides we synthesised to understand the effects of specific changes on the beta turns in this novel group of peptides using X ray crystallography.
The evidence on the structure and in particular the cysteine bonds should aid understanding of activity effects.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description The data produced from animal trials of peptide fed to animals has led to further industry funding of animal trials. Specifically, trials have been designed to assess the effect of ovodefensin supplementation in a necrotic enteritis challenge trial. If successful this may lead to the registration of a peptide feed additive to improve the welfare of rapidly growing birds. Studies on the structure of the peptide suggest some options to improve the activity against micro-organisms. The samples generated during this grant have also been used to support a Nuffield school placement.
First Year Of Impact 2017
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description OPEN day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact To introduce our work on the sustainability of poultry and egg production to a wider audience. We had a number of activities which introduced the role of the cuticle in preventing bacterial penetration of the egg. For many people this was a revelation. It allowed an opening to discussions on modern farming and to challenge many misconceptions. There was also a lot of hands on opportunities. The event was so successful that we have developed it into a grant for building kits for using at farm visits.
Year(s) Of Engagement Activity 2016
URL http://www.bbsrc.ac.uk/news/people-skills-training/2016/160107-pr-farm-science-at-open-farm-sunday/
 
Description Presentation at World Poultry Congress (Beijing) Ovodefensin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Largest Poultry science conference >4000 people. 50% industry Interest engendered from industry
Year(s) Of Engagement Activity 2016