Using Antimicrobial Proteins to Control Necrotic Enteritis in Meat ChickensRIRDC Publication No. 09/024

RIRDC

Innovation for rural Australia

Using Antimicrobial Proteins to Control Necrotic Enteritis in Meat Chickens

by Scott A. Sheedy and Robert J. Moore

March 2009 RIRDC Publication No 09/024 RIRDC Project No CSA-32A

2009 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 1 74151 828 8 ISSN 1440-6845 Using Antimicrobial Proteins to Control Necrotic Enteritis in Meat Chickens Publication No. 09/024 Project No. CSA-32A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details Dr Robert J Moore CSIRO Livestock Private Bag 24 Geelong VIC 3220 Phone: 03 5227 5760 Fax: 03 5227 5555 Email: rob.moore@csiro.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web: 02 6271 4100 02 6271 4199 rirdc@rirdc.gov.au. http://www.rirdc.gov.au

Printing by Union Offset Printing, Canberra Electronically published by RIRDC in March 2009

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ForewordIn-feed antibiotics are widely used in the broiler chicken industry, largely to maintain flock health. These products play an important role in the poultry growers ability to economically produce a healthy and nutritious product. In the last decade or so there have been increasing concerns expressed about the potential impact of infeed antibiotic use on the development and spread of antibiotic resistant bacteria of importance in a human health context. Because of these concerns there is an international trend towards a reduction in the use of in-feed antibiotics in the intensive animal production industries. The withdrawal of these products is likely to lead to a requirement for alternative methods for the maintenance of animal health and productivity. In the poultry industry, necrotic enteritis, caused by Clostridium perfringens, has been identified as a specific threat that could increase in importance following a reduction in the use of in-feed antibiotics. This report describes the development and study of one possible alternative strategy for controlling this potentially devastating disease. The report describes studies aimed at harnessing antimicrobial proteins to control disease. Some natural antimicrobial proteins produced by plants and animals are classified in a category of molecules known as defensins, while others produced by bacteria are called bacteriocins. A number of specific bacteriocins capable of killing the pathogen, C. perfringens, have been expressed in live vector bacteria and these constructs have been delivered to chickens to colonize the gut and set up a protective barrier to C. perfringens colonization and proliferation. To assess the prophylactic potential of these antimicrobials, the chickens were challenged in an experimentally induced necrotic enteritis model. Across a series of trials protection from disease was sometimes seen, however this could not be consistently achieved. The balance of conditions required to consistently produce protection are not understood. If the critical tipping point determining the success of these treatments can be understood and controlled, then with further development and testing the products described in this report may provide a means of controlling bacterial pathogens in broiler chickens and hence may offer producers an alternative way of maintaining flock health. This project was funded from industry revenue which is matched by funds provided by the Government. This report, an addition to RIRDCs diverse range of over 1800 research publications, forms part of our Chicken Meat R&D program, which aims to support increased sustainability and profitability in the chicken meat industry by focusing research and development on those areas that will enable the industry to become more efficient and globally competitive and that will assist in the development of good industry and product images. Most of our publications are available for viewing, downloading or purchasing online through our website: www.rirdc.gov.au.

Peter OBrien Managing Director Rural Industries Research and Development Corporation

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AcknowledgmentsThe authors wish to thank Dr Mark Ford, Dr Volker Haring, Anthony Keyburn, Kathy Sproat, Chrystal Wood and the staff of the CSIRO Livestock Industries animal facilities for their involvement and contributions to the project. We also thank Prof. Julian Rood and his team at Monash University for valuable input towards the project. We would also like to thank RIRDC for supporting the work, and various travel and presentations associated with it.

AbbreviationsAmp100 CCEC EntP GFP Nal35 NE P126 PedPA1 qRT-PCR Rif200 Resistance to ampicillin at 100 g/mL CSIRO Chicken E. coli isolate Enterocin P Green Fluorescent Protein Resistance to naladixic acid at 35 g/mL Necrotic enteritis Piscicolin 126 Pediocin PA-1 Quantitative real time polymerase chain reaction Resistance to rifampicin at 200 g/mL

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ContentsForeword ...............................................................................................................................................iii Acknowledgments................................................................................................................................. iv Abbreviations........................................................................................................................................ iv Executive Summary............................................................................................................................. vii 1. Introduction ....................................................................................................................................... 1 1.1 Background to the project ........................................................................................................... 1 1.2 Concept to be tested .................................................................................................................... 1 1.3 In-feed antibiotics ....................................................................................................................... 1 1.4 Necrotic enteritis ......................................................................................................................... 2 1.5 Clostridium perfringens .............................................................................................................. 2 1.6 Antimicrobial peptides................................................................................................................ 3 1.7 Live delivery ............................................................................................................................... 3 2. Objectives ........................................................................................................................................... 5 3. Methodology....................................................................................................................................... 6 3.1 Identification of antimicrobial genes to express and deliver....................................................... 6 3.2 Strategy for expression of antimicrobial genes........................................................................... 7 3.3 Assays for antimicrobial activity ................................................................................................ 9 3.4 Delivery of antimicrobial proteins by live bacterial vectors ..................................................... 10 3.5 Delivery of multiple antimicrobial proteins.............................................................................. 11 4. Results and Discussion .................................................................................................................... 13 4.1 Expression of antimicrobial genes ............................................................................................ 13 5. Implications...................................................................................................................................... 29 6. Recommendations............................................................................................................................ 30 References ............................................................................................................................................ 31

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TablesTable 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. Table 10. Table 11. Table 12. Table 13. Table 14. Table 15. Table 16. Table 17. Amino acid sequence of the mature form of bacteriocins used in this study ........................ 6 Amino acid sequences of defensins targeted for gene construction ...................................... 7 Reported characteristics of selected defensins ...................................................................... 7 DNA sequence of constitutive promoters used in this study................................................. 8 Secretion signal sequences used in this study ....................................................................... 8 Transcription terminator regions used in this study .............................................................. 9 Properties of suitable live E. coli vectors to use for delivery .............................................. 10 Antimicrobial activity of chemically synthesized defensins against indicator strains ........ 14 Plasmid constructs created in this study .............................................................................. 16 Protective potential of the bacteriocins when delivered by the E. coli vectors. Trial 1062-2A, Mar-Apr 2006............................................................................................. 20 Protection from NE via delivery of P126 with CCEC31rn. Trial 1137-2, Apr-May 2006 . 22 Screening of E. coli vector strains for effect on necrotic enteritis. Trial 1137-3, Sept-Oct 2006...................................................................................................................... 23 Influence of different feed sources on trial outcomes. Trial 1137-4, Oct-Nov 2006 .......... 23 Protection from NE via delivery of P126 with CCEC101rn. Trial 1137-5, Feb-Mar 200724 Performance of bacteriocins in face of improved challenge. Trial 1193-3, Aug-Sep 2007 24 Trial results from chickens dosed with cycled and uncycled E. coli vector. Trial 1062-2B, April 2007................................................................................................... 25 Trial results from chickens dosed with cycled and uncycled E. coli vector and exposed to a more severe NE challenge. Trial 1193-3, Aug-Sep 2007 ............................... 26

FiguresFigure 1. Plasmid map of construct containing multiple antimicrobial proteins, pWarhead.............. 12 Figure 2. Activity of selected bacteriocins expressed from E. coli vectors. ....................................... 13 Figure 3. Activity of antimicrobial proteins against C. perfringens. .................................................. 14 Figure 4. Plasmid organisation of the promoter probe vector, pPromProbe....................................... 17 Figure 5. Strength of the selected promoters demonstrated by GFP fluorescence intensity............... 17 Figure 6. Sensitivity of P126-resistant C. perfringens to the defensin antimicrobial proteins. .......... 18 Figure 7. Sensitivity of indicator strains justifying the multi warhead approach. .............................. 19 Figure 8. Antimicrobial activity of two bacteriocins expressed from a multi-warhead construct. ..... 20 Figure 9. C. perfringens counts isolated from the ileum .................................................................... 21 Figure 10. In vitro bacteriocin production from E. coli vectors carrying pRM1503. ........................... 22 Figure 11. C. perfringens counts isolated from the ileum of chickens ................................................. 26 Figure 12. Antimicrobial activity within the ceacal contents of bacteriocin treated birds.................... 27

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Executive SummaryWhat the report is about This report details a research project that was undertaken to provide proof-of-principle evidence that appropriately engineered benign bacteria could be used as live vectors to deliver an antimicrobial peptide to the gut of chickens. Such a product could be used to control the clinical symptoms and production implications of necrotic enteritis. Who is the report targeted at? This report outlines in some technical detail the molecular biology work that has been carried out towards the development of an alternative control strategy for necrotic enteritis. As such it is mainly targeted at the scientific community who may find the approaches we have taken interesting and useful and may be able to suggest ways of overcoming the hurdles that have been encountered. Background The intensive animal production industries are under pressure to reduce the use of in-feed antibiotics because of the perceived implications of ongoing use in the spread of antibiotic resistant bacteria in the human population. Real or perceived problems associated with in-feed antibiotic use include (i) antibiotic residues in meat, (ii) amplification in animals of drug resistant human pathogens, (iii) amplification in animals of drug resistance gene cassettes that can jump to human and animal pathogens, and (iv) antibiotic residues in the environment. Stopping the prophylactic use of in-feed antibiotics, without the use of alternative practices is likely to lead to a reduction in animal health and welfare and increased cost of production due to ill health, slower growth rates, and poorer feed conversion. Specifically, the chicken industry has identified that some pathogens (in particular Clostridium perfringens, the causative agent of necrotic enteritis) may increase in prevalence if in-feed antibiotics are withdrawn and hence are proactively looking for alternative control strategies. Necrotic enteritis is a disease of chickens that can cause high mortality rates and low productivity, leading to severe economic loss. The manifestations of disease can be very variable, from mild subclinical infections with few signs to severe outbreaks causing mortalities of greater than 3% per day. Although C. perfringens is the agent that causes the disease there are also other predisposing factors that play important roles in the disease. These include protein-rich diets, coccidial infection, housing, and management practices. Currently in Australia necrotic enteritis is largely controlled by in-feed antibiotics and good husbandry practices, but with the increasing pressure to reduce in-feed antibiotics, the industry has anticipated that it may become a major issue. One possible solution is the use of antimicrobial proteins that are able to kill the causative agent, C. perfringens, and thus reduce the disease. In a previous RIRDC project (CSA-12A) it was demonstrated that recombinant antimicrobial proteins (called bacteriocins) could kill C. perfringens. In that project we designed and cloned bacteriocin genes, showed that we could express them in lab strains of bacteria and in potential live vector strains, and developed a series of assays to measure in vitro activity. We demonstrated that the expressed bacteriocins could be used in a purified form to significantly reduce Listeria monocytogenes numbers in a mouse infection model and in a live vectored form to reduce L. monocytogenes in a chicken infection model. In a subsequent RIRDC project (CSA-29A) we have developed a reliable necrotic enteritis disease induction model that can now be used to assess and further develop the bacteriocin based products. A related RIRDC funded PhD project (CSA-20A) has been instrumental in identifying and developing new isolates of benign E. coli that have the potential to be used as the live vector strain to deliver the bacteriocins and other antimicrobial proteins. The present study draws all these threads

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together to develop bacterial strains that can be used to inoculate chickens to protect against necrotic enteritis. Bacteriocins are antimicrobial peptides that are produced by bacteria. Some bacteriocins, in particular Nisin, have been used for many years as food preservatives and hence are recognised as safe for human consumption. Because these antimicrobials are small peptides they are likely to be unstable and have very short half-lives if delivered simply as purified protein either intravenously or orally. This is a major reason why they have not previously been used to control disease in animals. The key to their successful use in the treatment of disease is likely to be in the way they are delivered. Live delivery in a bacterial vector may represent a very effective way of delivering therapeutic doses of antimicrobial proteins to invading pathogens. Aims/objectives This project was designed to provide proof-of-concept information regarding the potential to develop a cost effective, user-friendly, environmentally sustainable biological strategy to control necrotic enteritis in meat chickens. The aim was to colonise the gut location in which C. perfringens resides with benign bacteria expressing an antimicrobial protein. In this way high local concentrations of the antimicrobial should be achieved, sufficient to control C. perfringens growth, and hence abolish disease. Methods used In this report we describe work with a number of antimicrobial proteins, including several bacteriocins, to identify the most suitable antimicrobial proteins active against C. perfringens, clone these genes and insert actively expressed genes into benign bacteria that could be used to infect animals and hence deliver the expressed antimicrobial. We designed and synthesized genes for four bacteriocins and three defensins (which are intrinsically different in design and origin compared to the bacteriocins). Defensins are a class of molecule found in plants and animals with a wide spectrum of antimicrobial activity, whereas bacteriocins are produced by bacteria, and generally have a more restricted range of activity. To assess the activity of the cloned antimicrobials we employed a plate zoning assay that allowed rapid testing for activity, and liquid killing assays, which differentiated between bactericidal and bacteriostatic activity. Engineered bacteria expressing antimicrobial protein were introduced into chickens and their ability to protect against a C. perfringens challenge was measured. Results/key findings A variety of antimicrobial proteins of the bacteriocin and defensin types were targeted for investigation and a range of expression elements including various promoters, secretion signals, and transcription terminators were prepared to provide an interchangeable Toolbox of components to be used in engineered constructs for introduction into bacterial vectors. Three different bacteriocins, Piscicolin 126 (P126), Enterocin P and Pediocin PA1, were successfully produced in constitutive expression vectors suitable for use in the bacterial delivery vectors. A number of constructs expressed large amounts of the active bacteriocins in in vitro tests and all three of these bacteriocins showed excellent killing activity against C. perfringens. Four defensin-like peptides were investigated, initially by chemically synthesising small quantities for activity assays; three had activity against C. perfringens. We were unable to develop suitable expression constructs for the defensin-like peptides, probably because the E. coli vectors were susceptible to internally produced peptide. Therefore the in vivo efficacy tests were restricted to the bacteriocin constructs. The effects of the cloned antimicrobial proteins were assessed by testing in our previously established necrotic enteritis disease induction model. A series of chicken trials were conducted in which groups of chickens were dosed with an E. coli vector carrying various antimicrobial proteins, and then challenged with C. perfringens. The best results were obtained when a specific P126 expression

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plasmid, pRM1503, was used in the E. coli live delivery vectors. Other bacteriocin expression plasmids gave only intermittent or no evidence of protection in the disease challenge model. The ultimate goal of the live vectoring approach was to deliver a construct that could express several antimicrobial proteins. By delivering multiple active proteins, with different specificities, the likelihood of spontaneous resistant mutants arising would be greatly reduced because of the very low possibility of double mutants. A multi-warhead construct that could express two bacteriocin proteins with activity against C. perfringens was developed and tested. This is a prototype for the sort of construct that would ultimately be used in an optimised live vector strategy for the control of necrotic enteritis in chickens. When using the pRM1503 expression plasmid in our live delivery strategy we were successful in a number of trials in reducing or eliminating necrotic enteritis lesions in the test groups of birds However, this treatment was not always successful and we saw little sign of success with other constructs. There is some critical element of the interaction between the bird, the live bacterial treatments and the C. perfringens pathogen in our disease challenge model that we dont understand and have not controlled. There seems to be some sort of critical tipping point that determines whether the treatment works or not. Until we can understand and control the factors that are causing the variability in performance the strategy we have developed is not ready to be considered for commercial development. Implications for relevant stakeholders If this issue of reproducibility could be overcome then delivery of a successful biological control strategy would offer the Australian poultry industry several potential benefits, including both an immediate solution to the control of necrotic enteritis and a longer term solution for the predicted health and productivity issues that may arise after the reduction of in-feed antibiotic usage. The first of these is the controlled, biological, enteric delivery of natural anti-C. perfringens peptides which are able to specifically stop necrotic enteritis in chickens. With the delivery of these peptides by live bacterial vectors, this treatment would be capable of effective delivery at time of hatch and the product could be easily administered orally, allowing for a practical means of application to large numbers of chickens. The approach should also allow for both prophylactic and therapeutic treatment. Another major potential benefit is that with delivery of a commensal, live bacterial vector, a general improvement in gut health may improve productivity. This may be in the form of growth promotion or a more productive feed conversion ratio of the chickens. This is an important aspect of any potential product that would need to be carefully assessed, firstly to confirm that possible perturbations in the intestinal microflora do not adversely affect productivity, and secondly to look for evidence that a reduction in C. perfringens and other sensitive bacteria (e.g. Listeria) has a positive effect on productivity. The proposed treatments are designed to be environmentally benign, leaving no harmful residues in bird or the environment, thus alleviating concerns of the industry and the general public. The proposed treatments are based on a genetically manipulated organism and this is a serious issue that would have to be carefully addressed. Recommendations If the promise of this technology is to be realised the causes of the variability of outcomes following treatment with the prototype products needs to be understood. Further work should be directed specifically at addressing this issue and ensuring consistency in the protection from disease. If this issue can be overcome then the prototype products offer a viable method to protect birds from necrotic enteritis which is anticipated, from international experience, to be a major emerging disease threat if the use of antibiotics in feed is reduced or stopped. The ability to deliver biologically active proteins to the gut of chickens using live bacterial vectors opens up other interesting possibilities for the development of products to benefit the poultry industry. For example, direct delivery of cytokines could be used to enhance the immune status of birds and live delivery of vaccine antigens could protect against other diseases and reduce the carriage of human

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pathogens such as Campylobacter and Salmonella. These possibilities represent potentially valuable extensions of the work reported here. This report shows the potential of controlling disease by the delivery of selected antimicrobial proteins via live bacterial vectors, and with further development and recruitment of a commercial partner, this approach could provide to Australian poultry growers a viable alternative for the control of necrotic enteritis.

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1. Introduction1.1 Background to the projectThere is an international trend towards a reduction in the use of in-feed antibiotics in the intensive animal production industries. The withdrawal of these products is likely to lead to a requirement for alternative methods for the maintenance of animal health and productivity. In the poultry industry necrotic enteritis caused by Clostridium perfringens has been identified as a specific threat that could increase in importance following a reduction in the use of in-feed antibiotics. This report describes the development of one possible alternative strategy for controlling the disease.

1.2 Concept to be testedThe aim of this project was to provide a basic proof-of-principle that the live vectored delivery of an antimicrobial peptide could effectively control the clinical symptoms and production implications of necrotic enteritis. Several previous RIRDC projects have provided the underpinning lead-up research to allow this strategy to be implemented and assessed.

1.3 In-feed antibioticsThe supplementation of feed with low levels of antibiotics is widely practiced in the broiler industry. The purpose of this supplementation is to prevent and control disease but it also results in improved growth rates and feed conversion efficiencies. Because of the improvements in production efficiency the in-feed antibiotics are often called growth promoters or digestive enhancers. These products play an important role in the poultry growers ability to produce a healthy and nutritious product at an affordable price. In the last decade or so there have been increasing concerns expressed about the potential impact of infeed antibiotic use on the development and spread of antibiotic resistant bacteria of importance in human health. The increasing prevalence, in the human population, of antibiotic resistant bacterial pathogens, including multi-drug resistant strains, is a major public health issue. It is likely that the emergence of these resistant bacteria has been driven by a variety of factors including over prescription and inappropriate use in humans. It has been hypothesised that the widespread use of in-feed antibiotics in the animal production industries may have contributed to the problem by selecting for resistant bacteria that can either be directly transferred to humans or provide the genetic potential, antibiotic resistance genes, that can be transferred to human bacterial pathogens. The World Health Organisation has extensively reviewed the area and has recommended restrictions in the use of antibiotics in food production systems (World Health Organisation, 1998). Many national governments around the world have also reviewed the problem of the emergence of antibiotic resistant pathogens and have likewise recommended a reduction of their use in animals and the development of alternative methods for disease control in animals. In Australia the government commissioned the Joint Expert Technical Advisory Committee on Antibiotic Resistance (JETACAR) to investigate the matter. This Committee reported on their findings in 1999, in The use of antibiotics in food-producing animals: antibiotic-resistant bacteria in animals and humans. The Committee made a number of recommendations including minimising the use of antibiotics in humans and animals and increasing support for work looking at alternatives. Restrictions on the use of in-feed antibiotics have been legislated for in a number of jurisdictions around the world, with some European countries introducing complete bans. The European countries

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that have significantly reduced in-feed usage have generally claimed good ongoing maintenance of animal health and productivity, although anecdotal reports suggest that the prevalence of some diseases, such as necrotic enteritis, have increased substantially. There is clearly a need to develop new products and strategies to address the disease and productivity impacts associated with a reduction or withdrawal of antibiotics for in-feed use.

1.4 Necrotic enteritisNecrotic enteritis is a disease of chickens which manifests itself in a number of different ways, ranging from a mild reddening of the gut, with little clinical implication, to severe disease in which there is extensive necrosis of the gut lining and high mortality rates of greater than 1% per day. The determinants of disease are multifactorial, but C. perfringens has been shown to play a central role. Other factors such as protein-rich diet, coccidial infection, housing, and management practices are all believed to play potential roles in the development of the disease. In mild outbreaks the disease can cause some diarrhoea and the gut can be reddened, thin-walled and fragile. Although there is little mortality there can be quite significant reductions in the performance of the birds with poor food conversion rates and low weight gains. If the disease progresses the intestine can show foci of necrosis or ulceration and there can be disease involvement in the liver leading to carcass condemnation at slaughter. In the most severe disease there is very extensive necrosis throughout the gut with virtually the complete intestinal surface sloughing off. This sort of disease can result in mortality rates of greater than 3% per day and lead to severe economic loss. At present the disease is controlled by the use of in-feed antibiotics and environmental controls included in management practices. There are a number of reports in the literature of model systems used to reproduce the disease. These include direct infusion of broth cultures of C. perfringens (Al-Sheikhly and Truscott, 1977a) and crude culture supernatants (Al-Sheikhly and Truscott, 1977b) into the duodenum, oral challenge with C. perfringens (Kaldhusdal et al., 1999), and oral challenge with the addition of dietary or coccidian predisposing factors (Craven, 2000; Baba et al., 1997; Hofacre et al., 1998). A previous RIRDC project (CSA-29A) established a reproducible necrotic enteritis induction model based on dietery factors and oral challenge with C. perfringens NE18 (Sheedy et al., 2004), and is the most suitable to induce the disease in sufficient numbers of birds in small experimental trials.

1.5 Clostridium perfringensC. perfringens is a spore forming gram-positive bacterium that is ubiquitous in the environment. It is a normal constituent of the intestinal microbiota of animals. Chickens usually have a resident population by day three post-hatch. The C. perfringens that colonises is likely to come from the litter or feed. C. perfringens spores are resistant to varied environmental conditions (e.g. desiccation, chemicals, temperature) and can remain viable in the environment, feed and housing for extended periods. For the first few weeks of life the clostridial numbers in the gut tend to be quite low because the oxygen content is sufficient to restrict growth. In more mature birds, anaerobic conditions prevail in the gut and results in proliferation of anaerobic bacteria, including C. perfringens. This may in part explain why disease is mainly seen in birds between 14 and 42 days of age and most typically can become a problem in birds of about 21 to 28 days of age. C. perfringens strains are divided into five toxinotypes (A-E) according to the toxin genes which they carry. In most cases necrotic enteritis in chickens is caused by type A strains. Type A strains produce -toxin but none of the other major toxins used for classification. The -toxin is a phospholipase C enzyme and was believed to be the major virulence factor responsible for disease induction for many

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years; however, a new study has recently shown that this is not the case, and in C. perfringens NE18, the main virulence factor is a novel toxin, called NetB (Keyburn et al., 2006 and 2007).

1.6 Antimicrobial peptidesOrganisms within all kingdoms of life produce antimicrobial peptides as part of their innate defense system to help them survive against invading microorganisms. Many eukaryotic organisms of the animal and plant kingdoms have been shown to produce eukaryotic antimicrobial peptides, called defensins. A vast array of defensins have been identified which have a wide variety of known actions and activities. Antimicrobial peptides that are produced by bacteria are called bacteriocins. Bacteriocins have been widely studied and evaluated for use in the food industry but little research has addressed their potential use in the control of bacterial diseases. Bacteriocins are small peptides, with specific antimicrobial activity. Bacteria use them to compete, within an ecological niche, against sensitive bacteria. The activities range from being exquisitely specific for particular strains of one species to having quite broad activity against a wide range of bacteria. Activities are predominantly against gram-positive bacteria. The bacteriocins fall into a number of different classes (Jack et al., 1995). Some classes of antimicrobial peptides include novel amino acids that are produced post-translationally whereas others are conventional unmodified peptides. It is these unmodified peptides, the type IIa bacteriocins, that have been used in a previous RIRDC project (CSA-12A) and now developed further in this study. The nature of these peptides means that it is possible to relatively easily produce recombinant versions of the peptides in a range of different host systems. The post-translationally modified peptides are likely to require co-expression of a range of other genes in a heterologous host and so are likely to be significantly more complex to express in a functional form. A number of the type IIa bacteriocins have been reported to have activity against various clostridial species including C. perfringens. The bacteriocin, Nisin, has been used for many years as a food preservative. It is recognised as a food grade compound and it is anticipated that other bacteriocins will also be accepted for use in human foods. The bacteriocins have also been used in coating for food products, in toothpaste, and more recently have been incorporated into wipes for management and treatment of mastitis in cattle. Apart from the surface treatments for mastitis there have been no other reports of the use of bacteriocins to combat bacterial infections in animals. To treat most important infections the active agent needs to be delivered to internal organs, such as gut or lung, the sites of infection. For small peptide therapeutics, such as bacteriocins, direct delivery via an intravenous or oral route is likely to be problematic. Small peptides tend to be rapidly degraded and cleared in active biological systems such as that encountered within an animal. For such molecules to be successfully delivered it is likely that alternative approaches are required. These could be in the form of some sort of highly specialized formulation chemistry or the use of a live delivery system. Due to the success of a previous RIRDC project (CSA-20A), the potential of live bacterial vectors for the delivery of antimicrobial proteins, such as bacteriocins and eukaryotic defensins, was considered worthy of further investigation.

1.7 Live deliveryLive vectors, both viral and bacterial, have been suggested to be suitable vehicles for the delivery of vaccine antigens and other biologically active proteins. It has been recognised for some time that the use of live attenuated pathogens is often a more effective way of producing appropriate protective immune responses than vaccination with killed organisms or subunit vaccines. Attenuated strains of Salmonella have been used commercially for some time. Such attenuated pathogens can also be used to deliver heterologous recombinant antigens from other organisms. Some of these genetically modified organisms (GMOs) have been widely used (e.g. vaccinia - rabies glycoprotein (VRG)

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recombinant virus vaccine (Raboral) is a live viral vaccine presently used in France, Belgium and in the USA.). More recently, live vectors have also been developed for the delivery of DNA vaccines and proteins of therapeutic value, such as cytokines. For this later purpose live delivery has been recognised to offer a number of potential advantages over intravenous or oral delivery of active proteins. Vectored delivery has the potential to deliver more uniform doses to the desired site of action over a prolonged period. Live-vectored products are also likely to be cheaper and easier to produce than alternatives such as recombinant proteins or purified subunits. Live-vectored products are also unlikely to require the same degree of sophisticated product formulation technology as the alternatives. In the poultry production setting live vector delivery via feed, water, or aerosol is likely to offer a cost-effective and realistic means of administration. This project aimed to use a live delivery vector bacterium to harness these advantages for the delivery of therapeutic doses of an anti-clostridial antimicrobial protein, such as a bacteriocin or defensin.

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2. ObjectivesThe overall objective of the project was to develop a therapeutic treatment for necrotic enteritis based on the use of antimicrobial proteins, such as bacteriocins or defensins, delivered to chickens by live bacterial vectors. The outcome of the project would be a product to control necrotic enteritis in broiler chickens, which addresses the industrys need for alternative pathogen control strategies in the face of the anticipated reduction in the in-feed use of conventional antibiotics. Deliverables of the proposed research would include a bacterial strain expressing antimicrobial proteins that has been shown to protect chickens against experimentally induced necrotic enteritis. The strain would be patent protected and have had sufficient background work done on it to be an attractive proposition when presented to prospective commercial partners.

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3. Methodology3.1 Identification of antimicrobial genes to express and deliverIn a previous RIRDC project (CSA-12A), an extensive list of bacteriocins was compiled from the scientific literature based on the following characteristics: 1. the complete protein or gene sequence data was available for that bacteriocin and thus, could be easily manipulated and transferred to a range of expression systems and host bacteria 2. the peptide required no post-translational modifications, and so did not require additional enzymes for heterologous expression 3. these peptides were reported to have activity against Clostridia or related species of bacteria. Three bacteriocins were identified as possible candidates able to inhibit the growth of C. perfringens and thus, could potentially be delivered to the gut of chickens to help control necrotic enteritis. A fourth bacteriocin, Divercin V41, which meets the above requirements, was also included in this study. The primary amino acid sequence of the mature forms of each of the bacteriocins is shown in Table 1. This information was used to design synthetic genes for incorporation into expression constructs.Table 1. Amino acid sequence of the mature form of bacteriocins used in this study Bacteriocin Piscicolin 126 (P126) (Jack et al., 1996) Amino acid sequence KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTGGAAGWNKG

Pediocin PA-1 (PedPA1) (Nieto Lozano et al., 1992) KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC Enterocin P (EntP) (Cintas et al., 1997) Divercin V41 (Metivier et al., 1998) ATRSYGNGVYCNNSKCWVNWGEAKENIAGIVISGWASGLAGMGH TKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGAIPGKC

The bacteriocins that were studied are all small proteins with molecular weights of 5 to 6 kilodaltons. Because the genes to encode such proteins are small it was decided to chemically synthesize the expression cassettes rather than clone the genes from their original host. This approach is more efficient for a number of reasons; in particular it meant that (a) it did not require a source for the various original hosts to be found, (b) it was not necessary to obtain import permits and arrange importation and most importantly, (c) the genes could be designed to have desirable features such as convenient restriction endonuclease recognition sites and optimised codon usage. The plasmid constructs were analysed by restriction endonuclease digestion and DNA sequencing to confirm that the designed genes had been successfully constructed. In addition to the four bacteriocins, four small, eukaryotic antimicrobial peptides (known as defensins) were also of interest; Citropin 1.1, an antimicrobial protein found in the skin glands of the green tree frog Litoria citropa; Phylloseptin-1, found in the Brazilian frog Phyllomedusa hypochondrialis; Temporin-1Vb, found in the Carpenter frog Rana virgatipes; and CAMEL24, a hybrid peptide made up of seven amino acids from CecropinA of the silk moth Hyalophora cecropia, and eight amino acids of melittin from honey bee venom. The amino acid sequence of these proteins are shown in Table 2.

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Table 2. Amino acid sequences of defensins targeted for gene construction

DefensinCitropin 1.1 (Giacometti et al., 2005) Phylloseptin-1 (Leite et al., 2005) Temporin-1Vb (Conlon et al., 2005) CAMEL24 (Oh et al., 2000; Andreu et al., 1992)

Amino acid sequenceGLFDVIKKVASVIGGL FLSLIPHAINAVSAIAKHN FLSIIAKVLGSLF KWKLFKHIGAVLKVL

The approach taken was to attempt expression of these proteins because the final goal for the project was to utilise multiple antimicrobial proteins in a single product. It was therefore of interest to look at the antimicrobial activity of different classes of protein. However, due to the eukaryotic origin and the reported wide spectrum of antimicrobial activity of these proteins, the specific activity against C. perfringens was unknown. Reported characteristics of the selected defensins are shown in Table 3.Table 3. Reported characteristics of selected defensins Peptide size (aa) Citropin 1.1 Phylloseptin-1 Temporin-1Vb CAMEL24 16 19 13 15 Calculated MW 1614.0 2016.14 1405.9 1779.4 Reported antimicrobial activity against Gram positive Both gram positive and negative Gram positive Both gram positive and negative

To streamline the process of identification of good targets for gene expression, the four defensins were chemically synthesized and tested against indicator strains (including C. perfringens, E. coli and Listeria innocua) on plate zoning assays before expression constructs were made. The design of the expression constructs was based on choosing the optimal codon usage for the expression hosts to encode the required amino acid sequences.

3.2 Strategy for expression of antimicrobial genesThe goal of this research was to deliver active antimicrobial proteins using live E. coli vectors. Effective delivery is best achieved by secreting the antimicrobial proteins from the host bacteria. The first effective expression construct that was previously made (see CSA-12A) used a hybrid lambda phage promoter and a PelB secretion signal to direct expression of Piscicolin 126. It was anticipated that other constructs may give more effective results, so a series of alternative expression constructs was designed. Each expression construct was designed to carry four elements; (i) a promoter to drive expression, (ii) a secretion signal sequence to direct the expressed protein to the periplasm from where it can then escape to the external environment, (iii) the gene encoding the antimicrobial protein fused to the secretion signal, and (iv) a transcription terminator to stop uncontrolled and excessive plasmid transcription. Our aim was to synthesize DNA elements containing various alternative promoters, signal sequences, antimicrobial genes, and terminators so that they could be mixed and matched to develop plasmids with optimal performance. The key consideration in choosing promoters was that they needed to be constitutively expressed. Most promoters used in biotechnological applications to express recombinant proteins are specifically

7

chosen or designed to be regulated so that expression only occurs when an inducer is added. For this projects specialized use, induction systems are impractical for in vivo application in chickens and hence promoters that express all the time (constitutive) were selected. Our goal was to be able to fine tune expression; in some cases this might mean maximizing expression however in some instances high expression may be deleterious to the host cell and so lower expression may be desirable. Six promoters were synthesized, which the literature suggested would have a range of different strengths. Predicted active sequences of minimal length were designed and constructed from synthetic oligonucleotides (Table 4). The promoters selected, in increasing order of expected strength, were derived from the following E. coli or phage genes: gyrA, purFP1, aroF, synthetic Ptac hybrid promoter, T5 N25, T7 PA1 and the sterically repressed promoter (srp).Table 4. DNA sequence of constitutive promoters used in this study

Promoter namesrp Phage T7 PA1 Phage T5 N25 Ptac hybrid E. coli aroF E. coli purFP1 E. coli gyrA

DNA sequence (5-3)GAATTGACATTGTGAGCGGATAACAATATAATGTGTGGAATTCG CTAGTATTGACTTAAAGTCTAACCTATAGGATACTTACAGCCATCGAG CTAGTTTGCTTTCAGGAAAATTTTTCTGTATAATAGATTCATAAATTTGAG CTAGTGTTGACAATTAATCATGGGCTCGTATAATGTGTGGAATTGTG CTAGTATGGATTGAAAACTTTACTTTATGTGTTATCGTTACGTCATCG CTAGTAAATCCCTACGCAAACGTTTTCTTTTTCTGTTAGAATGCGCCCCGAACAG CTAGTATAGGTTTACCTCAAACTGCGCGGCTGTGTTATAATTTGCGACCTTG

It was anticipated that particular antimicrobial proteins may have different requirements for optimal secretion depending on the exact secondary structure of the protein and secretion signal fusion. A series of secretion signal sequences were selected and codon optimized coding sequences were synthesized (Table 5) allowing expression constructs to be tested with a number of different secretion signals.Table 5. Secretion signal sequences used in this study

Name of secretion signal/ isolation sourceEnterotoxin II Bordetella pertussis phoA SSS2B yebF OmpA

Amino acid sequenceMKKTIAFLLASMFVFSIATNAQA MVAAGIGAGLLMLSSAA MKQSTIALALLPLLFTPVTKA MGKKQTAVAFALALLALSMTPAYA MKKRGAFLGLLLVSACASVFA MKKTAIAIAVALAGFATVAQA

The final variable element within the range of constructs that was designed was the transcription terminator region, a small region of sequence that forms a secondary structure that favours stopping of the transcription process by RNA polymerase. Three different terminators (Table 6) were incorporated into different constructs.

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Table 6. Transcription terminator regions used in this study

Name of terminator regionrrnB T1 T7Te RNA I

DNA SequenceTCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAATCTG AAATGTAATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG GATCCGGCAAACAAACCACCGTTGGTAGCGGTGGTTTTTTTGTTTG

Initial antimicrobial protein genes were synthesized as a cassette structure in cloning vectors (pUC18 or pBluescriptSK+), and were manipulated to give a simple fusion of the genetic regions encoding the signal sequence and the mature sequence of the antimicrobial protein. Although some difficulties were experienced in the construction process, the strategy worked well for all three of the bacteriocin constructs. As a result, plasmids pP126, pEntP and pPedPA1 were created (expressing pelB-P126, pelB-EntP, and pelB-PedPA1 respectively). The same approach was used for cloning of the other antimicrobial genes (Divercin V41, Citropin 1.1 and CAMEL24). Antimicrobial activity of the mature antimicrobial peptide was detected using plate zoning assays against various indicator strains (C. perfringens and L. innocua).

3.3 Assays for antimicrobial activityTo determine if the expression constructs were functioning correctly and producing biologically active proteins two previously described in vitro assays were employed. The plate zoning assay was the main method used to assess the activity of the recombinant antimicrobial proteins against strains of C. perfringens.

3.3.1 Plate zoning assayThe plate zoning assay gives a direct, quantifiable measure of relevant biological activity. For the assay an agar plate with a lawn of indicator bacteria was produced and then potential antimicrobial protein containing samples were placed in wells in the agar. If an active antimicrobial protein is present it diffuses from the well into the agar and kills the overlaying bacterial lawn. The killing was visualised as an area of clearing in the lawn surrounding the well. For initial activity tests the lawn bacteria used was L. innocua, as this species, or related species, were reported to be sensitive to most of the bacteriocins that have been expressed and are easier to grow. However, to be considered a potential therapeutic for the control of necrotic enteritis, antimicrobial proteins were also tested on a bacterial lawn of C. perfringens (EHE-NE18; Sheedy et al., 2004).

3.3.2 Liquid killing assayThe liquid killing assay is a method that can supply more information about the nature of the antimicrobial activity being measured. It can differentiate between bactericidal (kills the cell) and bacteriostatic (stops cell growth) activities. As the name suggests the assay is done in liquid culture rather than the solid agar surface used for the plate zoning assay. Dilutions of potential antimicrobial protein containing samples were added to liquid cultures of indicator bacteria and the growth of bacteria monitored by changes in culture density measured at a wavelength of 600 nm. The read-out of the relative growth rates between bacteria recovered from treated and untreated samples could be used as an indicator of activity. There are a number of variables within the assay that can be manipulated; most importantly the contact time between antimicrobial and culture, and the dilutions of indicator bacteria and antimicrobial solution. All these variables can have a considerable impact on the interpretation of the results.

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3.4 Delivery of antimicrobial proteins by live bacterial vectors3.4.1 Selection of live bacterial vectors for deliveryA previous RIRDC project (CSA-20A) demonstrated the feasibility of using live bacterial vectors to deliver proteins to the gastro-intestinal tract of chickens. Unlike most previously reported work on live bacterial vectoring, the strains that were identified were able to persist in the host for long periods of time and hence could provide sustained release over the life of the chicken. Details of the strains suitable to use as live delivery vectors are shown in Table 7. These vectors were ready to be used to deliver the most appropriate antimicrobial proteins (identified above) to the gut of chickens to ameliorate the effects of C. perfringens in causing necrotic enteritis.Table 7. Properties of suitable live E. coli vectors to use for delivery CCEC31rn Serotypinga

CCEC59rn E. coli OR:H10 no Rif200, Nal35, Amp100 no no no no observed effect >42 days yes yes yes yes yes

CCEC101rn E. coli OR:H10 no Rif200, Nal35, Amp100 no no no no observed effect 28 days yes yes yes yes yes

E. coli Ont:H10b

Outbreak recorded for this serotype Antibiotic resistance Verocytotoxic a Shiga toxin produceda b

no Rif200, Nal35, Amp100 no noa

Cytotoxic necrotizing factors produced Disease status in chickens Persistence in chickensb b b b b

no no observed effect >42 days yes yes yes yes yes

Isolation from lower GIT

Easy to grow in LB media

Transformable (with plasmid)

Expression of plasmid-encoded proteins (in vitro) b Colonisation of GIT by oral dose b

a. Test performed at the National E. coli Laboratory, Department of Microbiology, University of Melbourne, Australia, and reported in Sheedy (2006) b. All other results observed and reported in Sheedy (2006)

3.4.2 Expression of antimicrobials from live E. coli vectorsExpression constructs were introduced into the selected E. coli vectors (CCEC31rn, CCEC59rn and CCEC101rn) by electro-transformation, and colonies were isolated on selective media, indicating successful introduction of the constructs. Plasmid DNA was isolated from the colonies and analysed by restriction endonuclease digestion and DNA sequencing to confirm the correct genetic arrangement of the constructs. To test expression of the antimicrobial proteins from the host cells, 2 mL cultures were grown overnight and centrifuged, and the collected supernatant was filtered and loaded into wells of a plate zoning assay as described above. After anaerobic incubation, anti-clostridial activity was visualised as a zone of clearing in the bacterial lawn.

3.4.3 Isolation of resistant vector strainsEach of the constructs expressing the selected bacteriocins were able to be expressed from the E. coli vectors described above. However, the construct containing the defensin, CAMEL24, was predicted to

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be troublesome due to the reported antimicrobial activity of this protein against gram negative microorganisms. To isolate CAMEL24-resistant variants of the E. coli vector, chemically synthesized CAMEL24 was loaded into wells on the plate zoning assay and targeted against the vector strain. The aim was to isolate E. coli colonies that were growing in the zone of inhibition, and thus, were resistant to the antimicrobial.

3.4.4 Dosing of chickens with E. coli vector strainsDuring the chicken trials, chickens to be dosed with the vector strain expressing antimicrobial proteins or relevant controls were orally inoculated with 1 mL of pure culture one day after hatch. Birds were inoculated at this early time point to maximise colonisation of the vector strain in the less developed microflora of the bird.

3.4.5 Chicken trials (NE challenge model)In the challenge trials, chickens were inoculated with C. perfringens EHE-NE18 according to the necrotic enteritis challenge model developed in a previous RIRDC project (CSA-29A). Birds were necropsied at day 25 (four days after first challenge) and the gastrointestinal tract was scored for necrotic lesions. The caeca of these birds were removed, and prepared for bacterial counts and antimicrobial activity assays.

3.4.6 Isolation of higher expressing vector strainsAlthough the selected E. coli vector strains were suitable for delivery of the selected antimicrobial proteins, the aim was to determine if vector strains could be selected with even more favourable properties by repeated cycling and recovery from the key target areas of the gut, namely the duodenum and ileum. Marked E. coli strains were isolated directly from the target area of the chicken gut and this was repeated a total of three times with the recovered cell population. Assessment of whether this selection process had changed the properties of the vector E. coli was based on quantitative real time (qRT) PCR assays to quantify E. coli numbers in the target region to determine if these strains have better ability to colonise this area of the gut than the original marked strain. Any isolated higher expressing strains were also tested for delivery of antimicrobial proteins in the NE challenge model.

3.5 Delivery of multiple antimicrobial proteins3.5.1 Resistance to antimicrobial proteinsBacterial antibiotic resistance is an ongoing concern in the intensive livestock industries and in human health. In order to reduce the chance of resistant C. perfringens strains arising, the ultimate construct to deliver to the gut of chickens would include multiple antimicrobial proteins so as to greatly reduce the chances of resistant bacteria arising. During the course of this project, several antimicrobial resistant strains of C. perfringens were recovered. To determine if these resistant strains were resistant to multiple antimicrobial proteins they were tested, using plate zoning assays, against all of the antimicrobial proteins used in this study. In an additional experiment, two indicator strains (Lactobacillus brevis #2146 and L. plantarum #2119) were tested for their sensitivity to a range of bacteriocins on plate zoning and liquid killing assays, and the differential sensitivity profile of these indicator strains were used to assess expression from the multi-warhead expression vector.

3.5.2 Construction of a multi-warhead expression plasmidFor proof-of-principle of the multi-warhead approach, the two bacteriocins that were chosen for construction in the multi-warhead vector were P126 and PedPA1. The bacteriocin genes were cloned

11

from existing plasmids, pP126 and pPedPA1srp, into the new vector, pWarhead (Figure 1). Each bacteriocin was under control of a different promoter (Lambda or srp, respectively).

Lambda prom ampR

pWarhead3355 bps

pelB P126

srp prom pelB PedPA1

Figure 1. Plasmid map of construct containing multiple antimicrobial proteins, pWarhead

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4. Results and Discussion4.1 Expression of antimicrobial genes4.1.1 Expression of selected bacteriocinsAt the start of this project only one plasmid, pRM1503, was available that constitutively expressed a bacteriocin (P126) (see report for project CSA- 12A). The first modification that was made was to add a transcription terminator sequence to the plasmid after the expressed gene. This was inserted to stop unwanted transcription from progressing through the plasmid. This plasmid was modified by replacing the P126 gene with coding sequences for the bacteriocins Enterocin P and Pediocin PA1. All three of these different PelB-bacteriocin fusion constructs, under the control of the Lambda promoter, were successfully obtained and had activity against C. perfringens (Figure 2).

P126

EntP

PedPA1

CCEC31rn

CCEC101rn

Figure 2. Activity of selected bacteriocins expressed from E. Coli vectors

The bacteriocins tested (P126, EntP and PedPA1) were all active against C. perfringens and L. innocua when expressed by five different E. coli strains (Lab strain JM109, and E. coli vector strains CCEC22rn, CCEC31rn, CCEC59rn and CCEC101rn). Based on the size of the zones of inhibition on the plate zoning assay, the bacteriocins were produced in similar amounts by each of the E. coli strains and when compared to each other. Therefore, there was no logical reason to predict one bacteriocin would be more active against C. perfringens in vivo than any other.

4.1.2 Activity of chemically synthesized antimicrobial proteinsFour antimicrobial proteins (defensins) were chemically synthesised and tested for activity against C. perfringens, L. innocua and E. coli CCEC31rn by plate zoning assays (Table 8).

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Table 8. Antimicrobial activity of chemically synthesized defensins against indicator strains Synthesized antimicrobial protein Citropin 1.1 Phylloseptin-1 Temporin-1Vb CAMEL24 C. perfringens Yes No Yes Yes L. innocua Yes No Yes Yes E. coli CCEC31rn No No Yes Yes

The chemically synthesized defensins were prepared as 1 mg/mL solutions and tested for activity using the plate zoning assay. Three of the defensins (Citropin 1.1, CAMEL24 and Temporin-1Vb) had activity against C. perfringens, while the fourth, Phylloseptin-1, did not (Figure 3).

Citropin 1.1

P126

EntP

CAMEL24

negative

PedPA1

Phylloseptin1

Temporin1Vb

Figure 3. Activity of antimicrobial proteins against C. perfringens

The four chemically synthesized defensins were also tested against each of the E. coli delivery vectors (CCEC31rn, CCEC59rn and CCEC101rn), and all three E. coli vectors were shown to be resistant to Citropin 1.1. Therefore it was predicted that these strains could be used in an unmodified form to deliver Citropin 1.1. CAMEL24 was also of interest as a potential defensin to deliver to the gut of chickens; however, in order to achieve this CAMEL 24 resistant variants of the E. coli strains would have to be isolated before this antimicrobial could be successfully delivered using these vectors. Citropin 1.1 and CAMEL24 were selected for further study, and genes were designed in expression constructs, fused to a secretion signal sequence and under the control of a constitutive promoter.

4.1.3 Expression of defensinsCloning and expression of the defensin molecules from expression plasmids proved to be more difficult than expected. Due to the antimicrobial spectrum of CAMEL24 (active against both Gram positive and negative microorganisms), recovery of plasmids containing an expressed CAMEL24 gene was unsuccessful after several attempts. Plasmid manipulation and recovery was possible during construction of the plasmid without a promoter, however, as soon as an active promoter was cloned in place the desired clones could not be recovered. It is assumed that the expressed plasmid was lethal to the host (E. coli) cell. Thus no constructs expressing CAMEL24 were derived. Constructs containing the Citropin 1.1 gene under the control of various promoters and secretion signals were isolated (see Table 9) and expression was intermittently observed by plate zoning assays, but no stably expressing colonies could be recovered. This was an unexpected result because the activity assays using synthetic Citropin 1.1 indicated that the E. coli strains in which attempts were made to engineer the expressing

14

constructs were resistant to the antimicrobial protein. It is postulated that although the strains were resistant to exogenous Citropin 1.1 they could not tolerate endogenously produced protein. To overcome these problems in recovering expression plasmids for Citropin 1.1 and CAMEL24 an attempt was made to isolate host cells (E. coli) that were resistant to the synthetic versions of the antimicrobial proteins. The limited amounts of synthetic peptide that were available restricted the ability to produce large zones on plates within which spontaneously resistant mutant colonies could be identified (as had successfully been done to obtain P126 resistant colonies). Mutant colonies resistant to Citropin 1.1 or CAMEL24 could not be found and hence it was not possible to progress with the construction of expression plasmids any further. The inability to identify resistant hosts is an interesting result and demonstrates the power of these antimicrobial proteins. It suggests that they are particularly good candidates for the therapeutic approach that this project sought to develop, if only the expression issue could be overcome.

4.2 Optimized expression of antimicrobial genesThe strategy of using the toolbox of expression elements to construct a wide variety of expression constructs was designed to give wide scope to optimize a range of expression plasmids. In particular expression cassettes were needed that utilized different expression elements so that in the final multiwarhead plasmids, expressing multiple antimicrobial proteins, unrelated sequences could be incorporated in the different gene expression cassettes to remove the possibility of recombination occurring between duplicated sequences. Although the manipulations required to produce the designed plasmids were simple in principle, it was often difficult to recover the required active expression plasmids. Intermediate constructs consisting of the secretion signals fused to the antimicrobial protein genes could be readily obtained however when the promoter element was added severe difficulties were sometimes encountered and few expressing clones were recovered. The potential expression plasmids that were successfully constructed are shown in Table 9. It can be seen that many of these clones, although having the correct sequence that was designed (and confirmed by DNA sequencing), did not express antimicrobial activity.

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Table 9. Plasmid constructs created in this study Name of plasmid pP126 pP126srp pRSPR pRSPRsrp pRM1528 pEntP pEntPsrp pTYER pTYERsrp pRM1537 pRM1540 pPedPA1 pPedPA1srp pOOPT pOOPTsrp pRM1522 pDiv pRM1531 pRM1534 pCit pRM1521 pRM1525 Promoter Lambda srp rrnB srp OmpA Lambda srp Phage T7 srp OmpA rrnB Lambda srp OmpA srp Phage T7 Lambda Phage T7 rrnB Lambda Phage T7 OmpA Secretion signal PelB PelB SSS2B SSS2B SSS2B PelB PelB yebF yebF yebF yebF PelB PelB OmpA OmpA OmpA Enterotoxin II YebF SSS2B B.pertussis YebF OmpA Antimicrobial gene P126 P126 P126 P126 P126 EntP EntP EntP EntP EntP EntP PedPA1 PedPA1 PedPA1 PedPA1 PedPA1 Divercin V41 Divercin V41 Divercin V41 Citropin 1.1 Citropin 1.1 Citropin 1.1 Antimicrobial activity confirmed yes yes unstable no no yes yes unstable no unstable no yes yes unstable no no no no unstable no unstable no

It was postulated that the problem may have been related to the strength of the promoter in the constructs, and that the promoter activity may be interfering with plasmid replication and/or cell survival. To address this issue a series of constitutive promoter elements with varied strength (Table 4) was selected. When these promoters were incorporated into expression constructs, still no measurable expression of antimicrobial protein was observed. This lead to the question as to whether the minimal promoter sequences that were used were actually capable of providing a functional promoter element. To test this, the promoters were transferred into a promoter probe vector in which putative promoter fragments could be assessed for their ability to drive expression of a Green Fluorescent Protein (GFP) gene. The seven different promoters that were identified from the literature (gyrA, purFP1, synthetic Ptac hybrid promoter, T5, N25, T7 PA1, and srp) and the Lambda promoter that we ,used in the first successful expression plasmid, were cloned into the promoter probe vector (Figure 4) and their ability/strength to drive expression of GFP was assessed.

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SphI BamHI

GFP Kan-r

pPromProbeterminator

4217 bps

Figure 4. Plasmid organisation of the promoter probe vector, pPromProbe. Promoters were cloned into the SphI and BamHI restriction sites to drive expression of the GFP gene. Successful expression of the GFP gene results in green fluorescent bacteria when viewed under UV light.

All eight of the promoters were able to actively drive expression of the control protein, which resulted in green fluorescent bacteria, and after analysis of these cultures in a fluorescence reader, three of the promoters appeared to drive higher expression of this protein (Figure 5).50000 45000 Fluorescence intensity (Exc 485nm, Em 535nm) 40000 35000 30000 25000 20000 15000 10000 5000 0 Lambda srp PA1 T7 T5 N25 ptac purFP gyrA negative

Figure 5. Strength of the selected promoters demonstrated by GFP fluorescence intensity

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It was interesting to note that although all promoters expressed the GFP, the fluorescence intensity of colonies on bacterial plates could not be distinguished by simple observation, and even under the fluorescence microscope, GFP expressed from the stronger promoters did not appear any different in fluorescence than the GFP expressed from the weaker promoters. There was, however, a difference in the percentage of cells that were fluorescent, and this supports the results obtained from the fluorescence reader. With the demonstration that all the promoter sequences were active it was necessary to reassess the possible reasons why so many of the constructs detailed in Table 9 did not express antimicrobial activity. The one element common to all the successful constructs was the use of the PelB secretion signal. It would appear that the alternative secretion signals may be interfering with the expression of the bacteriocins. At the time of project completion it had not been possible to definitively analyse this apparent problem but it is suggested that more work will need to be done if suitable alternative secretion signals are to be identified. Those constructs that have been shown to express antimicrobial proteins were possible candidates for delivery to the gut of chickens, using the selected E. coli vectors.

4.3 Construction of a multi-warhead expression plasmidDuring the course of the project several C. perfringens strains had been selected for resistance to P126 or EntP. When these strains were tested for sensitivity to a different class of antimicrobial protein, namely the defensins (Citropin 1.1, Temporin-1Vb, Phylloseptin-1 and CAMEL24), they were still killed by these antimicrobial proteins (Figure 6). This is a very important result that demonstrates that there is no cross-resistance between the bacteriocins and the defensin-like antimicrobial peptides. Therefore, a multi-warhead approach to reduce bacterial numbers in the gut of chickens is worthwhile and it is a strategy that should reduce the likelihood of antimicrobial peptide resistant strains arising in the real production environment. Citropin 1.1 P126 EntP

CAMEL24

negative

PedPA1

Phylloseptin1 Temporin1Vb

Figure 6. Sensitivity of P126-resistant C. perfringens to the defensin antimicrobial proteins

The ideal multi-warhead plasmid would be engineered to express one or multiple bacteriocins plus one or multiple defensin like peptides. Unfortunately the inability to construct a viable defensin expressing plasmid meant that this ideal construct could not be attempted. Therefore, as proof of principle, a plasmid was constructed (Figure 2) that was capable of simultaneous expression of two bacteriocins. The original plan was that all the expression elements used in each of the expression cassettes would be different however, as active constructs using a secretion signal sequence other than

18

PelB had not been successfully produced, this element of each expression cassette was common to both. A pair of Lactobacillus strains that displayed differential levels of sensitivity and resistance to the two bacteriocins, Piscicolin 126 and Pediocin PA1, had previously been identified. They were chosen for use in the dual expression vector (Figure 7). All other indicator strains tested were either sensitive to both bacteriocins or resistant to both. These differentially sensitive indicator strains provided an easy way of visualising the output from the vector and confirmed that both bacteriocins were being produced (Figure 8). Therefore, expression of P126 and PedPA1, cloned into the same plasmid and under the control of two different promoters, was successful.

0.15 0.14 0.13 0.12 0.11 0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 15 30 Time (mins) 45 60

Figure 7. Sensitivity of indicator strains justifying the multi warhead approach. Results of indicator strain 1 (Lactobacillus brevis #2146) are shown with black lines; inhibited by P126 (), resistant to PedPA1 (), and media control (-).Results of indicator strain 2 (Lactobacillus plantarum #2119) are shown with grey lines; resistant to P126 (), inhibited by PedPA1 (), and media control (-).

O D600

19

A P126 EntP

B P126 EntP

PedPA1

pWarhead

PedPA1

pWarhead

Figure 8. Antimicrobial activity of two bacteriocins expressed from a multi-warhead construct. (A) Antimicrobial activity of P126, EntP, PedPA1 and pWarhead against L. brevis #2146 (B) Antimicrobial activity of P126, EntP, PedPA1 and pWarhead against L. plantarum #2119.

This multi-warhead expression vector was only able to be constructed at the end of the project period so there has not been an opportunity to test its performance in an animal disease challenge trial.

4.4 Delivery of antimicrobial proteins by live E. coli vectorsIn an earlier RIRDC project (CSA-20A) it was found that in the final trial that was carried out one E. coli vector-bacteriocin construct, CCEC31rn(pRM1503) expressing the bacteriocin piscicolin 126 (P126), gave total protection from disease and greatly reduced the carriage of C. perfringens. This current project aimed to reproduce and extend this finding. The other bacteriocins which had been successfully expressed in vitro were first investigated to determine if they were capable of providing similar protective effects. The three bacteriocins, Piscicolin 126, Enterocin P, and Pediocin PA1, were all independently tested (Table 10). In this trial each group was dosed with two E. coli vector strains (CCEC31rn and CCEC101rn) carrying the particular plasmid for that group.Table 10. Protective potential of the bacteriocins when delivered by the E. coli vectors. Trial 1062-2A, Mar-Apr 2006 Challenge Control Average Lesion Score No. affected Av. X No. (Normalised to group size of 10) 5/11 6.6 4/10 5.2 5/10 7.0 4/10 5.2 3/10 2.4 1.45 Plasmid Control 1.3 E. coli (pP126) 1.4 E. coli (pEntP) 1.3 E. coli (pPedPA1) 0.8

20

Although the group treated with the Pediocin PA1 construct had a lower overall lesion score (Av x No) the difference was not statistically significant. Although the differences in lesion scores were not significant, other evidence that the Pediocin PA1 did have a marked effect was obtained an assessment of the C. perfringens load in the ileum showed that the group had significantly lower C. perfringens levels than the other groups (Figure 9). However, despite this positive finding there was essentially no useful protection seen in this trial, not even in the P126 treated group which had previous given a protective effect.

1000 0

100 0

10 0 Relative

bacteria l 10

count1

Challenge

Vector

P126 EntP Treatment Group

PedPA1

Figure 9. C. perfringens counts isolated from the ileum (group average with standard deviation shown)

The P126 expressing construct used in this trial was slightly different to the construct originally used to show protection. The pP126 plasmid has a transcription terminator signal added and was the same in overall structure as the Pediocin PA1 and Enterocin P expressing plasmid. It was because of this matching structure that the pP126 plasmid was used in this trial rather than the pRM1503 plasmid that was used in the trial that first showed protection. To address this issue, another trial was conducted with the originally used construct, CCEC31rn(pRM1503) (Table 11). Also included in this trial was a group treated with the E. coli strain that was a precursor to the CCEC31rn strain. The CCEC31rn strain has been cycled in vitro to obtain the strain marked by resistance to rifampicin and naladixic acid the CCEC31 strain is the original isolated parent strain.

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Table 11. Protection from NE via delivery of P126 with CCEC31rn. Trial 1137-2, Apr-May 2006 Challenge control Average Lesion Score No. affected Av. X No 8/10 9.6 2/10 1.2 3/10 1.8 1.2 CCEC31rn (pRM1503) CCEC31 (pRM1503)

Although not giving the total protection seen in the original trial both treatment groups had significantly fewer lesions than the challenge control group, thus confirming the utility of these constructs in controlling necrotic enteritis under these challenge conditions. The scope of the vectoring options available was investigated by including all four of the E. coli vector strains which had previously been identified as capable of long-term colonization of the chicken gut. Constructs expressed from the E. coli vector strains (CCEC22rn, CCEC31rn, CCEC59rn and CCEC101rn) carrying the plasmid pRM1503 were all capable of producing large amounts of bacteriocin in vitro, as demonstrated on the plate zoning assay (Figure 10).

Figure 10. In vitro bacteriocin production from E. coli vectors carrying pRM1503

When these strains carrying pRM1503 (expressing P126) were tested in the NE challenge model, the lesion scores in groups treated with the bacteriocin expressing strains were all lower than the challenge control (Table 12). The E. coli CCEC101rn(pRM1503) group had no disease at all; this indicated that it may be the best strain to use for protecting birds from experimentally induced necrotic enteritis.

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Table 12. Screening of E. coli vector strains for effect on necrotic enteritis. Trial 1137-3, SeptOct 2006 Challenge Control Average Lesion Score No. affected Av. X No. (Normalised to group size of 10)*This group had a number of deaths in the pen. These deaths were unrelated to the treatment or challenge but made the results for this group impossible to interpret.

CCEC22rn (pRM1503) 1.0 3/11 3.0

CCEC31rn (pRM1503) 0.82 3/11 2.46

CCEC59rn (pRM1503) 0.57 1/7* 0.57

CCEC101rn (pRM1503) 0 0/11 0

1.64 5/11 8.2

In this first series of trials the infection rate in the challenge control groups was lower than expected and this indicated a need for caution in interpreting the results. It is speculated that the infection level in the control groups was lower because of a change in the supplier of the specialized feeds needed to run the disease induction model. Over the course of this project the sourcing of the feeds has been an issue of concern, as the exact specification of the feed seems to play an important role in the disease induction model and it has been difficult to maintain continuity of supply. The possible role of different feed supplies in influencing the trial outcomes was investigated by directly comparing groups feed new batches of feed and the old supply of feed (Table 13).Table 13. Influence of different feed sources on trial outcomes. Trial 1137-4, Oct-Nov 2006 Old feed Challenge control (Av.) Average Lesion Score No. affected Av. X No. (Normalised to group size of 10) 0.88 10/44 2.2 CCEC101rn (pRM1503) 1.82 6/11 10.91 New feed Challenge control (Av.) 1.39 20/44 6.95 CCEC101rn (pRM1503) 0.2 1/10 0.2

The different batches of feed used in this trial were made to the same formulation but by different feed mills with different sources of raw materials. The amount of disease seen in the trial in the challenge control groups was again lower than expected and particularly low in the groups fed from the old source. One group of birds treated with the CCEC101rn(pRM1503) construct had very reduced NE lesions; however, a second group of birds, on a different feed ration, were not protected. With the indication that strain CCEC101rn may be the best delivery vector, the trials were repeated using other bacteriocin expressing constructs in this strain background. In the first of these trials (Table 14) no protection was seen and it even seemed that the CCEC101rn(pEntP) construct may have exacerbated disease.

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Table 14. Protection from NE via delivery of P126 with CCEC101rn. Trial 1137-5, Feb-Mar 2007 Challenge Control Average Lesion Score No. affected Av. X No. 6/11 8.4 4/11 6.9 5/11 6.0 7/11 19.9 1.55 Plasmid Control 1.38 CCEC101rn (pP126) 1.2 CCEC101rn (pEntP) 2.56

The lack of protection seen with CCEC101rn(pP126) mirrors the lack of protection seen in an earlier trial (Table 10). The picture emerging was that strains carrying the pRM1503 construct expressing P126 could give reasonably reliable protection whereas the similar construct, pP126, could not protect. This result is somewhat puzzling as the two strains produce very similar levels of active P126 protein in vitro. To investigate this point further a trial was conducted using a third P126 expressing plasmid, pP126srp. This expression plasmid is significantly different from the other two plasmids in that it has a different promoter sequence. The in vitro expression level of P126 from this plasmid is still high and similar to the other two constructs. The trial also included bacterial vectors carrying the equivalent expression constructs or Enterocin P and Pediocin PA1 (Table 15).Table 15. Performance of bacteriocins in face of improved challenge. Trial 1193-3, Aug-Sep 2007 Challenge Control Average Lesion Score No. affected Av. X No. (Normalised to group size of 10) 11/11 30.9 9/9 33.3 9/10 28.8 9/11 19.3 9/10 27.9 3.1 Plasmid Control 3.3 E. coli (pP126srp) 3.2 E. coli (pEntPsrp) 2.4 E. coli (pPedPA1srp) 3.1

It can be seen that in this trial the level of disease induced is significantly greater than in all the previously reported trials. This increased challenge was achieved by modifying the environmental conditions by reducing temperature and increasing the length of time the birds were illuminated, by modifying the way in which the challenge cultures of C. perfringens were prepared, and by putting the birds on the high protein feed earlier than was previous practice. There was no sign of protection in any of the groups treated with the bacteriocin expressing constructs. This further confirms the unexpected result that the newer expression constructs, although showing good in vitro activity, were not giving any protection in the animal challenge trials. The only constructs that have given protection in a number of trials are those carrying the plasmid pRM1503.

4.5 Isolation of enhanced vector strainsAlthough the current E. coli vector strains appear to be suitable for delivery of the bacteriocin P126 it was thought that it might be possible to recover a vector strain with even more favourable properties by repeated cycling and recovery from the key target area of the gut, namely the ileum. Therefore, during the course of trials carried out during the first year of the project the opportunity was taken to

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recover the strain CCEC31rn(pRM1503) from dosed birds and cycle it three times through chickens during the course of the other trial work that was performed. After cycling, the strain was compared to the normal uncycled strain in a disease challenge trial (Table 16).Table 16. Trial results from chickens dosed with cycled and uncycled E. coli vector. Trial 10622B, April 2007 Challenge Control Uncycled CCEC31rn (pRM1503) 3 cycled CCEC31rn (pRM1503) Average Lesion Score No. affected Av. X No. (Normalised to group of 10) 4/9 5.3 5/10 7.0 0/10 0 1.2 1.4 0

Surprisingly the normal uncycled strain, which had previously given protection from disease, did not show any protection in this trial; however, the cycled strain gave total protection from lesion formation and this was also correlated with a difference in the number of C. perfringens isolated from these groups (Figure 11). The 3 cycled vector gave a 2 log reduction in C. perfringens numbers compared to the uncycled and challenge controls.

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10000

number of bacteria (10^1)

1000

100

10

1

Challenge

Uncycled

3 cycled

Figure 11. C. perfringens counts isolated from the ileum of chickens (group average with standard deviation shown)

The second test of the cycled strain was included in the last trial, in which the challenge was much heavier than in all the previous trials. Even in the face of the severe C. perfringens challenge the group treated with the cycled vector strain had a reduction in lesion score compared to the uncycled and challenge controls and a fewer number of birds were affected (Table 17). However, the total protection seen in the previous trial was not observed.Table 17. Trial results from chickens dosed with cycled and uncycled E. coli vector and exposed to a more severe NE challenge. Trial 1193-3, Aug-Sep 2007 Challenge Control Uncycled CCEC31rn (pRM1503) 3 cycled CCEC31rn (pRM1503) Average Lesion Score No. affected Av. X No. (Normalised to group of 10) 11/11 30.9 10/11 33 7/11 13.9 3.1 3.3 2.2

The recovery of the vector strains from the cycled and uncycled groups were semi-quantitatively measured and no clear difference could be seen in the colonisation of the two strains in the ileum. This suggests that the reason for the differential results is more likely to be in the ability of the strains to secrete the active P126 in the in vivo situation. No difference in in vitro production of P126 could be detected. These results show that the principle of cycling strains through the location in which action is

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desired does have a beneficial effect on the characteristics of the strain and this strategy may be worth pursuing further.

4.6 Investigation of factors that may affect reproducibility of in vitro effects of bacteriocinsAlthough the pRM1503 constructs have given protection in a number of trials the results have been somewhat variable. The lack of complete reproducibility between trials is difficult to understand but it is likely that there is a fine balance between antimicrobial activity and conditions in the intestinal environment. Conditions may need to be just right for the generation of sufficient bacteriocin activity to reduce C. perfringens numbers and consequently reduce disease. A number of parameters were studied in an attempt to gain some insight into what influences the critical tipping point that pushes the birds to either being protected or not. Firstly, P126 activity over the pH range expected in the chicken gut was investigated. It was found that P126 was stable within this range, in agreement with the scientific literature (Jack et al., 1995). Secondly, the bacteriocidal activity of P126 against C. perfringens was studied and it was confirmed that P126 killed C. perfringens within 5 minutes incubation at 37C. If the bacteriocin had proven to be bacteriostatic rather then bacteriocidal then it may have accounted for some of the trial variability. Thirdly, the release of P126 from bacterial cells was measured via an osmotic shock assay and no additional release of P126 from the cells was found. This suggested that P126 was actively released from the bacterial cell under in vitro conditions. Unfortunately, the same test could not be conducted in vivo, as it is impossible to reproduce the exact conditions of the gastrointestinal environment of the chicken outside the bird. However, to test for antimicrobial activity within the chicken, a fourth approach was taken to look at the bacteriocin activity recoverable from caecal contents of treated birds (Figure 12). In the first trial that was previously reported (CSA-20A), it was clearly shown that birds that were treated with the P126 had antimicrobial activity in the caecal contents of these birds, whereas caecal contents of untreated birds did not.

A

B

Figure 12. Antimicrobial activity within the ceacal contents of bacteriocin treated birds. (A) control birds dosed with vector control (no P126) (B) treated birds dosed with bacteriocin (P126).

The fact that this detectable antimicrobial activity coincided with protection from disease suggested that the antimicrobial was successfully delivered to the gut of chickens by the E. coli vector, and

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moreover, was active against C. perfringens in these conditions. Possible antimicrobial activity in the caeca