Chicken Meat Program

2005-2006 Research Report

January 2007

ISBN 1 74151 332 4 ISSN 1440-6845

2005-2006 Research Report RIRDC Chicken Meat Program Publication No 06/069

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 industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Communications Manager on phone 02 6272 3186.

RIRDC Chicken Meat Program Research Manager Dr Vivien Kite RIRDC PO Box 579 NORTH SYDNEY NSW 2059

Phone: 02 9929 4077 Fax: 02 9925 0627 Email: [email protected]

RIRDC Publications Manager Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 3186 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au

Published in January 2007 Printed on environmentally friendly paper by Union Offset, Canberra

ii Foreword

This year RIRDC has produced Research in Progress, June 2006, which contains short summaries of continuing projects as well as those that were completed during 2005-2006 for all of the Corporation’s 22 program areas.

The complete report on all the programs is only available in electronic format on our website at www.rirdc.gov.au.

The following report is a hardcopy extract covering the Chicken Meat Sub-program. It contains all entries from continuing and completed Chicken Meat research projects funded by RIRDC in 2005-2006. Additional information on other activities funded by the program in 2005-2006 and projects and activities to be funded in 2006-2007 have also been included in this publication.

The objective of the Chicken Meat Program is to support increased sustainability and profitability in the chicken meat industry by focussing on research and development on those areas which will enable the industry to become more efficient and globally competitive and which will assist in the development of good industry and product images.

Research reported upon herein was funded from industry revenue which is matched by funds provided by the Federal Government.

This report is an addition to our extensive catalogue of more than 1500 research reports, videos and CD-roms of projects supported by RIRDC. Most of our publications are available for viewing, downloading or purchasing online through our website:

• downloads at www.rirdc.gov.au/fullreports/Index.htm • purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

iii

CHICKEN MEAT PROGRAM ADVISORY COMMITTEE

Chairperson: Research Manager: Mr Barry Shay Dr Vivien Kite Food Safety Consultant RIRDC Chicken Meat Program 27 Uther Street PO Box 579 CARINDALE QLD 4152 NORTH SYDNEY NSW 2059 Ph: (07) 3398 1766 Ph: (02) 9929 4077 Mobile: 0402 245 744 Fax: (02) 9929 0627 Fax: (07) 3398 6631 Email: [email protected] Email: [email protected] Committee Members:

Dr Pat Blackall Mr Rod Jenner Animal Research Institute Golden Cockerel Pty Ltd Department of Primary Industries PO Box 142 Locked Mail Bag No 4 CLEVELAND QLD 4163 MOOROOKA QLD 4015 Ph: (07) 3206 6444 Ph: (07) 3362 9498 Fax: (07) 3206 6999 Fax: (07) 3362 9429 E-mail: [email protected] E-mail: [email protected] Dr Tom Grimes Dr Ron MacAlpine Grimes Consultancy Pty Ltd Inghams Enterprises Pty Ltd 29 Tradewinds Avenue Locked Bag 4000 PARADISE POINT QLD 4216 LIVERPOOL BC NSW 1871 Ph/fax: (07) 5529 6146 Ph: (02) 9606 5666 Mob: 0416 257 524 Fax: (02) 9606 6640 Email: [email protected] Email: [email protected] Dr Margaret MacKenzie Mr Gary Sansom Inghams Enterprises Pty Ltd 82 Hawkins Road PO Box 1100 JIMBOOMBA QLD 4280 BROWNS PLAINS QLD 4118 Ph: (07) 5546 9235 Ph: (07) 3297 4644 Fax: (07) 5546 0070 Fax: (07) 3290 4471 E-mail: [email protected] Email: [email protected] work: (07) 3837 4747 Mob: 0428 155 795 Ms Margie Thomson Mr Liam Morrisroe RIRDC Bartter Enterprises Pty Ltd PO Box 4776 PO Box 616 KINGSTON ACT 2604 NORTH RYDE NSW 1670home address - use for Ph: (02) 6272 4152 docs posted from RIRDC: 90 Taylors Rd, SKYE VIC Fax: (02) 6272 5877 3977 Email: [email protected] Ph: (02) 8870 0000 Fax: (02) 8870 0093 E-mail: [email protected] Program Coordinator:

Ms Vicki Byrne RIRDC, PO Box 4776, KINGSTON ACT 2604 Ph: (02) 6271 6398, Fax: (02) 6272 5877, Email: [email protected]

iv INDEX TO PROJECT SUMMARIES

COMPLETED PROJECTS

Flock Health ...... 1 Use of cytokines to enhance vaccine efficacy in poultry ...... 1 Postgraduate scholarship – Kristie Jenkins: Improved therapeutics for Marek’s disease virus infection3 Postgraduate scholarship – Manija Asif: Cytokines and innate molecules for enhanced mucosal immunity in the chicken ...... 5 Biological control of necrotic enteritis in meat chickens ...... 7 The development of vaccination strategies to control necrotic enteritis in poultry ...... 8 Preliminary investigations into the efficacy of novel treatments and application methods for darkling beetle (Alphitobius diaperinus [Panzer]) control in Australian broiler houses ...10 Molecular evaluation of responses to vaccination and challenge by Marek’s disease viruses ...... 11 Avian leukosis subgroup-J (ALV-J): epidemiological studies for control of ALV-J in Australian broiler breeder flocks...... 13 Systematic pathotyping of Australian Marek’s disease (MDV) isolates...... 15 Typing of Pasteurella multocida...... 17 Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis...... 19 Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the apicomplexan parasite, Eimeria maxima ...... 21 Effects of organic acids, prebiotics, and enzymes on control of necrotic enteritis and performance of broiler chickens...... 22 Bird Nutrition and Feed Supply...... 23 Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets (AECL- managed project WAU-1A) ...... 23 Digestible amino acids and improved broiler performance...... 25 Efficiency of Production ...... 27 Investigation of the cause of miliary hepatitis in laying chickens...... 27 Product Quality and Safety...... 28 Baseline survey of Campylobacter and Salmonella during chicken processing ...... 28 RESEARCH IN PROGRESS...... 30 Flock Health ...... 30 Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics ...... 30 New diagnostic assays to improve control of coccidiosis in poultry ...... 31 Bird Nutrition and Feed Supply...... 32 Nutritional characterisation of sorghums from Queensland and NSW for chicken meat...... 32 Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal integrity, and performance of broiler chickens ...... 34 Early feeding of prebiotics on development of the digestive system and gut microflora of broilers ....35 Establishment of a Chair in Poultry Sciences at The University of Sydney...... 36 Assessment of the anti-nutritive effects of phytate by dephytinisation ...... 37 Early dietary and management intervention on broiler breast meat yield ...... 38 Variability in performance and physiology of broilers fed wheat- and sorghum-based diets ...... 39 Food Safety...... 40 Development of a sequence-based bacteriophage typing system for Salmonella...... 40 Campylobacter bio-replacement program to control food poisoning organisms in poultry ...... 41 Environmental Management...... 43 Evaluating risks posed by pathogen emissions from meat chicken sheds...... 43 Efficacy of windbreak walls for odour reduction...... 44 Managing litter re-use for minimal nutrient runoff to surface water ...... 45 Trials of odour control technologies for broiler farms ...... 46

v Efficiency of Production ...... 47 Postgraduate scholarship - David Stephenson: Delivery of therapeutic proteins using lactobacillus .47 Using antimicrobial proteins to control necrotic enteritis in meat chickens ...... 48 Developing novel biological management strategies for darkling beetle (Alphitobius diaperinus [Panzer]) in Australian broiler houses ...... 49 Broiler performance on pearl millet based diets ...... 50 Potassium diformate evaluation in necrotic enteritis challenge model ...... 51 Product Quality and Safety...... 52 Differential typing of Campylobacter...... 52 Molecular epidemiology of antibiotic resistance in Salmonella from chickens ...... 53 The Workboot Series - The story of chicken in Australia...... 54 Sustainable Production Environment...... 55 Pilot avian influenza surveillance program ...... 55 OTHER SUPPORTED ACTIVITIES ...... 56 SCHOLARSHIPS ...... 56 PROGRAM REVIEW AND DEVELOPMENT...... 56 COLLABORATIVE PROGRAMS...... 56 TRAVEL/CONFERENCE/WORKSHOPS ...... 56 RESEARCH TO BE SUPPORTED IN 2006/2007...... 57

COMPLETED PROJECTS

Flock Health

Project Title: Use of cytokines to enhance vaccine efficacy in poultry

RIRDC Project No.: CSA-26J Researcher: Dr. Andrew Bean Organisation: CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220 Phone: (03) 5227 5792 Fax: (03) 5227 5531 Email: [email protected]

Objectives • To develop novel vaccine formulations and therapeutics that will enhance the efficacy of vaccines currently used to control Marek's disease in poultry. This will involve the evaluation of various cytokines and an assessment of their ability to enhance vaccine efficacy and immune competence. • To enhance disease resistance and vaccine efficacy in poultry by administering therapeutic doses of chicken interferon-gamma to commercial broilers and layers

Background Marek’s disease is caused by a highly contagious oncogenic herpes virus and has significant economic impacts for the world poultry industry. Even in the face of the current successful vaccine approaches, Marek’s disease (MD) remains a concern, particularly with the development of more virulent strains of Marek’s disease virus (MDV). If this situation continues it may lead to the recurrence of MDV as a serious economic threat. Therefore, in order to maintain the highest possible levels of animal health, welfare and productivity it is vital to be proactive in the development of safe alternative vaccination strategies and therapeutics.

Research Previous studies have identified the potential for cytokines to act as therapeutics and vaccine adjuvants. In this project, chicken cytokines were evaluated for their immunoenhancing capacity during MDV infection.

Outcomes The final report on this project outlines the analysis of the immune response, with particular emphasis on the cytokine response, during MDV infection. Further to this, biologically active recombinant chicken cytokines were produced from an E. coli expression system and used to assess their potential as therapeutics and vaccine adjuvants during MDV infection. It was found that the cytokines IL-2 and IL-6 show immunostimulatory activity and with this in mind this evaluation of cytokines activity provides evidence for the rational use of chicken cytokines as therapeutics for disease.

Implications The further development of this technology will allow the Australian poultry industry to alleviate some of the concerns associated with vaccination against MDV infection. The results from this work test the feasibility of using cytokines as natural therapeutics and adjuvants and these cytokines are now available for a similar type of assessment as treatments for other immunosuppressive viral infections such as infectious bursal disease virus, chicken infectious anaemia virus and infectious bronchitis.

Publications Asif, M., Jenkins, K.A., Hilton, L.S., Kimpton, W.G., Bean, A.G.D. and Lowenthal, J.W. (2004). Cytokines as adjuvants for avian vaccines. Immunol. Cell. Biol, 82: 638- 643. Moore, R.J., Doran, T.J., Wise, T.G., Riddell, S., Granger, K., Crowley, T.M., Jenkins, K.A., Karpala, A.J., Bean, A.G.D. and Lowenthal, J.W. (2005). Chicken functional genomics. Australian Journal of Experimental Agriculture, 2005. Invited presentations (at meetings): Lowenthal, J.W., Bean, A.G.D., Tyack, S.G., Hilton, L.S., Asif, M., Jenkins,KA and Johnson, M.A.(2004). Oral delivery of novel therapeutics. World’s Poultry Congress, Istanbul, Turkey. 2004 Schat, K.A., Bean, A.G.D., Broadway, M., Asif, M., Thomas, J. and Lowenthal, J.W. (2004). In vitro propagation of dendritic-like cells from chicken spleens. Avian Immunology Research Meeting, Munich, 2004 Bean, A.G.D., Thomas, J.D., Bruce, M.P., Asif, M., Broadway, M.M., O’Neil, T.E., Jenkins, K.A., and Lowenthal, J.W. (2004). Immune system enhancement following in ovo adminstration of chicken cytokines, AIRG, Munich, 2004 Bean, A.G.D., Jenkins, K.A., Asif, M., Thomas, J., Hilton, L.S., O’Neil, T.E., Lowenthal, J.W. (2003). Innate Immunity: recognition and response, APSS, Sydney, Australia, 2003. Bean, A. G. D., Asif, M., Jenkins, K. A., Hilton, L. S., O’Neil, T. E., Lowenthal, J. W. (2003). Evaluation of the immunoenhancing capability of innate cell associated cytokines, MVADS, Dublin, Ireland, 2003. Asif, M., Thomas, J.D., Jenkins, K.A., Kimpton, W.G., Bean, A.G.D., Lowenthal, J.W. (2003) Enhancement of Mucosal Immunity by Cytokines, ASI, Perth, Australia, 2003. Jenkins, K.A., Bruce, M.P., Thomas, J.D., Kimpton, W.G., Schat, K.A., Lowenthal, J.W. and Bean, A.G.D. (2003). The Innate Immune Response to an Alpha Herpes Virus, ASI, Perth, Australia, 2003. Thomas, J.D., Bruce, M.P., Asif, M., Broadway, M., O’Neil, T.E., Jenkins, K.A., Muralitharan, M., Lowenthal, J.W. and Bean, A.G.D. (2003). In ovo Administration of Cytokines to Chickens, ASI, Perth, Australia, 2003.

Project Title: Postgraduate scholarship – Kristie Jenkins: Improved therapeutics for Marek’s disease virus infection

RIRDC Project No CSA-25J Researcher: Dr. Andrew Bean Ms. Kristie Jenkins Organisation: CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220 Phone: (03) 5227 5792 Fax: (03) 5227 5531 Email: [email protected] [email protected]

Objectives • To examine the early immune response to Marek's disease virus infection and apply this information to enhance the development of therapeutic strategies.

Background Prevention of viral infections is an important issue for the poultry industry. Understanding the innate immune system in chickens may help identify novel and effective ways to control viral infections in chickens such as Marek’s Disease Virus (MDV). An important group of receptors involved in the innate immune response are Toll-like receptors (TLRs). When this project began, chicken TLRs were relatively uncharacterised. With this is mind, the aim of this study was to identify and characterise TLRs that are involved in viral recognition in chickens. Examination of anti-viral TLRs in chickens revealed that chickens appear to contain only one functional receptor to represent the mammalian TLR9 subfamily. This receptor was subsequently cloned, characterised and shown to have the highest similarity to mammalian TLR7. Although the TLR9 subfamily gene was found to have the highest similarity to TLR7 it did display characteristics of TLR9, leading to the speculation that this receptor also recognises mammalian TLR9 ligands in chickens.

Research In vitro characterisation of the response of chicken splenocytes to TLR9 subfamily ligands found TLR7 and TLR9 ligands stimulate a response similar to that elicited by the homologous mammalian receptors. In contrast, a lack of response was induced by a huTLR8 ligand. In mammals, TLR7 and TLR9 ligands have been shown to have adjuvant potential. The analogous in vitro responses observed may also suggest TLR7 and TLR9 ligands may display adjuvant activity in chickens. To assess this, the in vivo response to these ligands had to first be determined. The avian system possesses the unique opportunity to administer substances in ovo and currently a number of poultry vaccines such as those for MDV are administered in this manner. Thus the ability of the TLR ligands to be administered in ovo was characterised to determine their potential use as adjuvants to these vaccines.

Outcomes The in ovo administration of the ligands was shown to be successful with no detrimental effects on hatchability. Administration of TLR9 ligands resulted in increased levels of IL6 mRNA detectable at 7 days post hatch. Subsequent examination of the prophylactic and adjuvant potential of TLR7 and TLR9 ligands in the face of Marek’s disease virus (MDV) revealed their ability to reduce MDV induced impact on CD4+ cells.

Implications The experiments performed in the current study revealed that chickens contain a smaller repertoire of viral recognising TLRs than mammals, with only one functional receptor identified to represent the mammalian TLR9 subfamily. Although an orthologue for TLR9 was not identified, chicken cells were found to respond to both TLR7 and TLR9 ligands in a manner analogous to mammalian cells. The current knowledge of the action of TLR ligands in mammals, in conjunction with their

3 immunomodulating ability shown in this study, draws attention to their potential use as therapeutic agents for the poultry industry.

Publications To date no publications have been produced from this work.

Project Title: Postgraduate scholarship – Manija Asif: Cytokines and innate molecules for enhanced mucosal immunity in the chicken

RIRDC Project No CSA-25J Researcher: Dr. Andrew Bean Ms. Manija Asif Organisation: CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220 Phone: (03) 5227 5792 Fax: (03) 5227 5531 Email: [email protected]

Objectives • To reduce disease by enhancing mucosal immunity in chickens with the aid of cytokine therapy.

Background To maintain high standards of poultry health and production there is a need for the continuing development of novel strategies directed towards improving health and welfare. With this in mind, there is a greater emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently, existing vaccine adjuvants may have deleterious side effects, such as inflammation, which can lead to down grading of meat quality and in turn to lower profits. Therefore, to enhance vaccination strategies, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants has attracted considerable attention in this respect. Moreover, their potential role as adjuvants has been addressed by several recent studies. As cytokines are regulatory proteins secreted by a variety of cell types that play a crucial role in controlling the immune response, they provide a useful candidate for adjuvant studies. Furthermore, the recent identification of a number of chicken cytokine genes has provided the opportunity to study their effectiveness in enhancing the immune response during infection or vaccination. One such cytokine, interleukin-6 (IL-6) has been shown in mammals to play a major role in controlling the immune response. The aim of this project was to characterise the biological functions of chicken IL-6 (ChIL-6) as a precursor to an assessment of its potential as a therapeutic or vaccine adjuvant.

Research To examine the immunoenhancing properties of ChIL-6, recombinant ChIL-6 (rChIL-6) protein was expressed and its biological activity was determined. Antibodies and enzyme linked immunoassays specific for ChIL-6 were developed to characterise the in vivo function of this cytokine. In vivo and in ovo administration of rChIL-6 significantly enhanced the in vitro proliferation of lymphocytes in response to ConA. Furthermore, adjuvant potential assessment studies revealed that rChIL-6 administration with an antigen increases secondary IgY responses. Since IL-6 is a pro-inflammatory cytokine in mammals, the inflammatory role of ChIL-6 in a disease model, infectious bronchitis virus, was assessed using a susceptible (S-line) and resistant (HWL) line of SPF birds. Significant over expression of IL-6 was observed in S-line birds suggesting a possible exacerbation of the disease associated with IL-6 in this susceptible line.

Outcomes The experiments performed in these studies show that ChIL-6 predisposes lymphocytes to enhanced proliferative potential when administered to the embryo or adult birds. Furthermore, ChIL-6 was found to act as an adjuvant by increasing antibody responses to an antigen.

Implications These functions of ChIL-6 suggest that it may have the potential to be used as an

5 adjuvant or therapeutic agent for the poultry industry. This potential needs to be explored in further investigations.

Project Title: Biological control of necrotic enteritis in meat chickens

RIRDC Project No.: CSA-29A Researcher: Dr. Robert Moore Organisation: CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220 Phone: (03) 5227 5760 Fax: (03) 5227 5555 Email: [email protected]

Objectives • To complete the development of a reproducible necrotic enteritis (NE) disease model that can be implemented in PC2 containment conditions, thus allowing the testing of disease treatments, such as bacteriocin products and recombinant vaccines developed in previous projects.

Background An earlier RIRDC project, CSA-12A, developed potential treatments for NE based on antimicrobial proteins called bacteriocins. To test the efficacy of these treatments an NE disease model was required but could not be completed in the time available. This project was aimed at completing the development of a suitable disease induction model.

Research A series of chicken infection trials were undertaken in order to optimise the challenge procedure. A collection of recent field isolates of Clostridium perfringens, the bacterium responsible for the disease, was tested in chickens and a number were found to be capable of eliciting necrotic lesions in the gut. A range of factors, including room temperature, coccidia treatment, bacterial growth conditions, gut motility and feed changes, were assessed for their impact on the development of NE disease under experimental conditions. Final trials briefly looked at the impact of one bacteriocin based treatment.

Outcomes Optimised procedures and conditions to induce reliable levels of mild to moderate disease were defined. The most important factors appear to be the growth conditions of the challenge bacteria, feed withdrawal over the challenge period, a reduction in housing temperature just prior to challenge, and the particular isolates of C. perfringens used. Methods for assessing and recording disease outcomes were also refined.

A quantitative polymerase chain reaction (qPCR) method was established which is capable of high throughput analysis of bacterial numbers in gut samples. This provides another level of analysis beyond the simple scoring of gut lesions. In one trial, one of the groups treated with the bacteriocin was fully protected, showing no signs of disease. In a subsequent trial this finding was not repeated; despite this the initial observation makes us optimistic that the bacteriocin treatments can be successfully developed.

Implications This project has developed the disease induction process to the point were it is now suitable to be used to assess the performance of intervention strategies being developed to combat NE. It can be used to test bacteriocin treatments, vaccines, and other possible therapeutics such as cytokines.

Publications To date no publications have been produced from this work.

Project Title: The development of vaccination strategies to control necrotic enteritis in poultry

RIRDC Project No.: RMI-11A Researcher: Prof. Peter Coloe Organisation: RMIT University Plenty Road BUNDOORA VIC 3083 Phone: (03) 9925 7104 Fax: (03) 9925 7110 Email: [email protected]

Objectives • To lay a foundation for understanding and controlling necrotic enteritis (NE) by developing a model for the reproduction of necrotic enteritis in chickens. • To develop and evaluate a vaccine, based on toxoid, for the control of necrotic enteritis that can be delivered orally, that is cost effective to manufacture and that can be delivered within established farming practices.

Background A major aim of the poultry industry is to produce high quality product without the need for supplementation with antimicrobial agents. To achieve this objective, over recent years there has been a strong move towards the use of vaccination strategies to replace long term chemical use for the control of disease. Effective vaccination of poultry against necrotic enteritis (NE) would not only illustrate the industry’s proactive stance with respect to replacing chemical control measures for disease, but should enhance the productivity of flocks. Since an outbreak of NE (which is caused by the pathogen, Clostridium perfringens) can result in between 2% to 10% mortality, as well as causing significant losses in flock productivity, effective vaccination will have a major cost benefit outcome for the industry. Effective vaccination to control C. perfringens alphatoxin will also ensure the development of a healthy, effective gut microflora. This microflora will limit the possibility of other undesirable organisms colonising the chicken and posing a threat to human health after processing.

Research The approach taken in this project was to establish a reproducible model for NE, to produce and test a killed vaccine based on toxoid and, if this proved successful, to clone and deliver a live modified vaccine.

A variety of attempts were undertaken to reproduce NE in poultry. These ranged from varying the challenge strain, varying the diet of the birds and varying the dates of challenge as well as co-infecting birds with Eimeria spp and C. perfringens. In addition, birds were also pre-treated with antibiotics to reduce the microbial flora prior to challenge.

The C. perfringens toxin was cloned in E. coli and purified toxin was used in mouse proof of concept studies to show the toxin was antigenic. A range of truncates of the toxin were also prepared and tested in mice.

Outcomes Unfortunately, a reproducible model for the reproduction of NE was not successfully established. Necrotic enteritis was achieved at a low level using a variety of approaches, but the incidence of disease was inconsistent and not reproducible enough to be used as a model. Nevertheless, the C. perfringens alphatoxin was cloned into E. coli and a variety of truncates prepared. The purified toxin was shown to protect against an intra-muscular challenge in mice. Truncates of the C. perfringens toxin were less effective than the whole toxin.

8 However, without an effective chicken model to test the purified toxin in this work did not proceed further. The availability of cloned alphatoxin will enable evaluation of this as a protective antigen when a model of NE in chickens becomes available.

Implications The development of an efficacious vaccine for necrotic enteritis would be a significant step forward for the chicken industry. A vaccine that is cost effective and can be delivered orally would enhance the productivity of the industry and also enhance disease control and flock welfare.

However, it is necessary for an effective disease model to be developed in order that the efficacy of any vaccine or new treatment method can be properly evaluated. Such a model will require a much higher incidence of disease and reproducibility than was achieved in this project, before it can be used to evaluate efficacy against necrotic enteritis.

Know-how on production and purification of toxin gained in the course of this project will facilitate future progress towards a toxoid vaccine approach and provide material for alternative vaccine approaches. However, additional work is required on a disease model so that vaccine approaches can be effectively evaluated.

Project Title: Preliminary investigations into the efficacy of novel treatments and application methods for darkling beetle (Alphitobius diaperinus [Panzer]) control in Australian broiler houses

RIRDC Project No.: DAQ-319A Researcher: Mr. Trevor Lambkin Organisation: Department of Primary Industries & Fisheries (Qld) Entomology Building, Indooroopilly Research Centre 80 Meiers Road INDOOROOPILLY QLD 4068 Phone: (07) 3896 9434 Fax: (07) 3896 9446 Email: [email protected]

Objectives • To undertake preliminary, mainly laboratory based investigations into the efficacy of a number of novel litter treatments in controlling darkling beetle (Alphitobius diaperinus).

Background A long history of applications of contact insecticides to the floors of broiler houses in Australia for the control of darkling beetle may have been largely ineffective and has resulted in the development of strong insecticide resistance in beetle populations, particularly in eastern Australia. Recent research undertaken in Australia has identified that control strategies should mostly be targeted to the treatment of litter for management of this pest. Therefore, in 2003/04, preliminary investigations commenced to laboratory trial a number of novel litter treatments and to field trial a new broiler house floor application, to determine their efficacy in controlling all stages of the beetle.

Research Imidacloprid 200SC and granules, beta-cyfluthrin 25 g/L, spinosad and diatomaceous earth were laboratory tested as litter treatments, with spinosad also field trialled as a broiler house floor treatment. Efficacy in suppressing development of progeny (larvae) was measured for each treatment.

Outcomes The most effective litter treatment overall was the imidacloprid bait granule, but beta- cyfluthrin was also very effective in suppressing progeny. Diatomaceous earth reduced progeny significantly but at doses much higher than the other two compounds. Spinosad was found to be ineffective as a litter treatment but gave significant suppression of populations when applied to the floors of broiler houses.

Implications The four formulations trialled for broiler house control of A. diaperinus offer the chicken meat industry an alternative for beetle control, but only if certain issues are addressed. These are the need for OH&S and bird safety implications to be resolved for diatomaceous earth and imidacloprid respectively, for all formulations be integrated into an integrated system of rotation (or use of two products concurrently), and for a cost effective method is found to apply treatments to litter. Each formulation, if used appropriately, is a realistic option for beetle control. If used together in the form of an integrated system, it is possible that a lower dose of each compound might be used.

Publications To date no publications have been produced from this work.

Project Title: Molecular evaluation of responses to vaccination and challenge by Marek’s disease viruses

RIRDC Project No.: RMI-12J Researcher: Prof. Greg Tannock Organisation: Royal Melbourne Institute of Technology Department of Biotechnology and Environmental Biology PO Box 71 BUNDOORA VIC 3083 Phone: (03) 9925 7142 Fax: (03) 9925 7110 Email: [email protected]

Objectives • To develop and refine a QPCR test that will be suitable for estimating Marek's disease virus DNA copy number in cell cultures infected with virulent and vaccine viruses. • To extend the use of QPCR to measure viral loads in various organs of infected chickens after infection with virulent and vaccine viruses. • To determine similar loads in organs of the developing chick embryo in an attempt to determine alternative markers for virulence that may not involve inoculation of chickens. • To measure the distribution of serotype 1 and 3 vaccine viruses after inoculation to chick embryos under conditions used for in ovo vaccination. • To carry out protection experiments after in ovo and day 1 vaccination in which the endpoints will be measured from the challenge virus load in critical target organs.

Background Despite the significance of Marek’s disease (MD) as a major cause of economic loss to the chicken industry, comparatively little is known about the mode of action of vaccines that are widely used. Most data have been obtained using cell culture to measure the presence of Marek’s disease virus (MDV) together with other traditional methods based on the capacity of the virus to cause death, morbidity and disease- specific lesions. All of these have limitations and the project attempted to overcome them by the application of newer molecular techniques for the enumeration of the MDV genome in infected tissues. The animal model so developed was then applied as a means of studying the disease and for an evaluation of optimal methods of vaccination.

Research Early stages of the project concerned the optimisation of methods for estimating MDV DNA in cultures and chicken tissues by QPCR.

The technique was initially used to optimise methods for the isolation of field isolates of MDV in cell culture. In later studies, QPCR was used to estimate viral loads in various tissues of infected chickens and chicken embryos after inoculation with virulent strains of MDV and commonly used vaccine strains. The resulting animal challenge model was used to estimate the efficacy of two commonly used Australian vaccines; the Rispens strain and HVT.

Outcomes Optimum methods for the isolation of field viruses, as defined by QPCR, include the use of chicken kidney (CK) culture and inoculation of freshly prepared CK cells in suspension and culture for six days. Highest viral DNA levels were observed in the spleen and thymus of birds inoculated with the virulent type 1 strain MPF57. Levels in the same organs of chickens inoculated with the Rispens and HVT vaccines were 1:10,000-1:100,000 lower. When day-old chickens were vaccinated with the Rispens vaccine and challenged with MPF57, reductions in MPF57 in the spleen occurred

11 over nine weeks that were consistent with clinical measures of protection that were observed in parallel. A similar challenge experiment was conducted in hatched chickens that had been inoculated with HVT in ovo at days 11 and 17 of embryonic development. Overall responses to challenge were comparable. In all challenge experiments, vaccination was shown to reduce levels of challenge virus in the spleen but did not lead to the induction of sterilising immunity

Implications The challenge model developed in this project provides a useful objective measure of immunity to MDV, which could be very useful in the evaluation of new vaccines. The model has shown quite unequivocally that vaccine evaluation based on clinical endpoints alone provides no indication of their incapacity to eliminate MDV from a flock. Accordingly, effective decontamination of litter, in conjunction with vaccination, will remain an essential strategy to reduce the exposure of young immunologically immature birds to challenge virus.

Publications Tan, J., Cooke, J., Clarke, N., Crisp, F.and Tannock, G.A. (2004). Proceedings of the 7th International Symposium on Marek's Disease, Oxford, UK, July 2004.

Project Title: Avian leukosis subgroup-J (ALV-J): epidemiological studies for control of ALV-J in Australian broiler breeder flocks

RIRDC Project No.: UM-68A Researcher: Dr. Trevor Bagust Organisation: The University of Melbourne Faculty of Veterinary Science, Building 404 Cnr Park Drive & Flemington Road PARKVILLE VIC 3010 Phone: (03) 8344 9676 Fax: (03) 8344 9675 Email: [email protected]

Objectives • To undertake a structured epidemiological survey to determine the current prevalence and distribution of ALV-J in Australian broiler breeder stocks at Great Grandparent (GGP) and GP levels. • To undertake molecular characterisation and comparative pathotyping of recent Australian strains of ALV-J isolated from large commercial poultry breeding organisations. • To provide the tools that would allow industry to control ALV-J in Australian broiler breeder flocks.

Background Avian leukosis virus subgroup J (ALV-J) causes tumours, mortalities and sub- optimal production in the broiler industry. The presence of this virus has previously been confirmed in Australia. In an earlier RIRDC project (UM-49A Avian leukosis virus subgroup J (ALV-J): Developing laboratory technologies for diagnosis in Australia), the molecular technology was developed to culture and detect ALV-J. This technology is now applied in this project to help industry control ALV-J.

Research A detailed structural epidemiological survey was carried out to determine the prevalence of ALV-J in the Australian industry. Further, Australian isolates of ALV-J were characterised antigenically and by sequence analysis to determine their likely origin and the extent of antigenic drift. Investigation of isolation procedures for ALV-J from field and experimentally infected birds was undertaken to establish the optimal samples for detection of ALV-J. Selected isolates were also pathotyped in both major strains of birds used in the Australian industry.

Outcomes The structured epidemiological survey of Australian flocks identified flocks positive for ALV-J and these were removed by industry. Over the course of this project a decline in the prevalence of ALV-J is reported with the likely eradication of ALV-J from the Australian industry. The presence of ALV-A was reported with instances of co-infection occurring with both ALVJ and ALV-A. Australian isolates of ALV-J showed significant antigenic drift from UK and US prototypes and sequence analysis indicated that Australian isolates of ALV-J were imported from both European and US sources. Pathotyping of Australian isolates of ALV-J demonstrated these isolates induced myeloid leukosis and caused significant decreases in body weight.

Implications This project has demonstrated the control and likely eradication of ALV-J from the Australian poultry industry. Furthermore, it also revealed the presence of ALV-A. Iif steps are not taken to remove ALV-A from Australian stocks the risk of further ALV recombinations increases. Investigation of sampling procedures indicated the dangers in relying solely on the one sample type when screening for ALV-J. Australian isolates of ALV-J have been introduced to this country from both European and US sources, thus highlighting the need to screen imported stocks to ensure their freedom from ALV-J.

13 Publications Bagust, T., Fenton, S. and Reddy, M. (2004). Australian Veterinary Journal, 82: 701- 706. Fenton, S., Reddy, M. and Bagust, T. (2005). Avian Pathology, 34: 48-54. Bagust, T.J., Fenton, S.P. and Reddy, M.R. (2003). Proceedings of WVPA congress Denver USA 2003. Fenton, S.P., Bagust, T.J. and Reddy, M.R. (2003). Proceedings of The Avian Veterinarian Poultry Association conference Melbourne, November 2003. Fenton, S., Bagust, T. and Reddy, M. (2004). Proceedings of the Avian Poultry Assoc. Asia Pacific Conference #5. Gold Coast Qld 20-23 April 2004.

Project Title: Systematic pathotyping of Australian Marek’s disease (MDV) isolates

RIRDC Project No.: UNE-83J Researcher: A/Prof. Stephen Walkden-Brown Organisation: University of New England Animal Science School of Rural Science and Natural Resources ARMIDALE NSW 2351 Phone: (02) 6773 5152 Fax: (02) 6773 3922 Email: [email protected]

Objectives • To evaluate current Australian strains of MDV for their ability to induce disease and overcome the effects of vaccination. • To do this in a way that facilitates comparisons with similar studies overseas.

Background In the USA there is clear evidence of evolution of Marek’s disease virus (MDV) towards greater virulence (ability to induce disease) over time, possibly as a response to vaccination. A feature of this increased virulence is the successive failure of different vaccines against MDV to provide effective protection against the disease. Australia suffered a serious outbreak of MDV from 1991-1996 and part of the response to this has been to introduce routine in ovo vaccination of broiler chickens with potential to facilitate such an evolution in virulence. Previous Australian studies had shown that there were highly virulent strains of MDV in the country.

Research Between 2002 and 2005, 533 field samples were submitted for isolation of MDV, with 655 isolations on cell culture attempted. Unfortunately isolation rates were low and only six isolates (four new and two old) grew sufficiently for inclusion in formal pathotyping experiments while a further 11 grew sufficiently to infect chickens in screening experiments. These isolates showed a wide range of virulence in SPF (specific pathogen free) chickens with the more virulent strains inducing pathology typical of very virulent isolates overseas. A new early mortality syndrome induced by MDV was observed and detailed for the first time in Australia. There was no clear evidence of a trend towards increased virulence over the last decade although the number of isolates tested was relatively small. In unvaccinated SPF chickens, virulence was well predicted by a range of measurements on chickens as soon as two weeks after challenge.

Outcomes The research undertaken confirmed the presence of very virulent MDV in Australia. It did not confirm evolution in virulence of Australian MDV over time. Isolation of MDV in cell culture proved to be impractical, and isolation directly in chickens should be pursued instead. New methods for screening for virulence in unvaccinated SPF chickens were developed. The work also demonstrated that virulence in unvaccinated chickens is not necessarily related to the ability of MDV to overcome the effects of vaccination.

Implications The work has allowed for an improved understanding of current status of Australian MDV. HVT, the main vaccine used in the broiler industry, induces low levels of protection against several isolates of MDV. This is suggestive of some evolution in virulence occurring in Australian MDVs, although this was not able to be confirmed in the current project. Ongoing monitoring for MDV virulence and vaccine efficacy is worthwhile. The project has provided good data on how such ongoing monitoring might be conducted.

Publications Hussain, Z., Islam, A.M.F.M., Burgess, S.K., Reynolds, P.S. and Walkden-Brown, S.W. (2005). Isolation of Marek's disease virus from dust samples from commercial chicken farms. Proc. Aust. Poult. Sci. Symp., 17: 100-104.

Project Title: Typing of Pasteurella multocida

RIRDC Project No.: UQ-100J Researcher: Prof. Linda Blackall Organisation: The University of Queensland Department of Microbiology & Parasitology ST LUCIA QLD 4072 Phone: (07) 3365 4645 Fax: (07) 3365 4620 Email: [email protected]

Objectives • To establish a Multi-locus Sequence Typing (MLST) system for Pasteurella multocida. • To facilitate the rapid, accurate typing of P. multocida isolates that will allow any isolate to be directly compared with any previous isolate already typed and allow an understanding of the epidemiology of fowl cholera outbreaks. • To provide typing tools which will support improved prevention and control programs for fowl cholera.

Background Fowl cholera remains one of the major bacterial diseases of poultry. The importance of P. multocida as a pathogen of birds and many other species is such that the complete chromosome of this pathogen was recently sequenced, allowing the development of typing systems based on this sequence knowledge. MLST is a new generation typing method that has considerable advantages over all other typing methods. The technique is highly reproducible, highly discriminatory and is totally portable. This last feature means that once an MLST typing scheme has been established any laboratory can compare their isolate with any previously typed isolate by simply comparing the DNA sequences. All typed isolates stored in the central Web data-bank are accessible to anyone with Web access. The rapid progress in DNA sequencing technology means that this form of DNA-based typing is now feasible (and indeed already functioning for major human pathogens such as Neisseria meningitidis and Campylobacter jejuni.)

Research A total of 63 isolates of P. multocida from Australian poultry – all associated with fowl cholera outbreaks - as well as three international reference strains, representing the three subspecies within P. multocida, were used. The complete sequence of seven different house-keeping genes was determined. The sequences were then analysed using a specialised suite of software. This analysis showed that 39 different MLST Sequence Types (STs), representing 39 distinct allelic profiles, were present in the study. One particular ST (ST-2) was found to be the predominant ST (18.18%) in these isolates.

A dendogram showing the genetic relatedness of the isolates was produced. This dendogram showed a strong correlation with the dendogram created using the earlier phenotypic-based technology of multi-locus enzyme electrophoresis (MLEE). In particular, both MLST and MLEE have shown that there are two major sub-divisions within the species P. multocida. In accordance with the prior MLEE and ribotyping data, MLST has confirmed that a diverse range of P. multocida is associated with fowl cholera in Australia. A retrospective analysis confirmed that MLST has the power to be a useful typing tool , allowing outbreaks of fowl cholera to be followed and understood.

Outcomes The major outcome of this project was the first web based MLST scheme for P. multocida, with this scheme being constructed on Australian poultry isolates. This is a significant advance, both in the Australian and international context. The scheme will enhance the management and control of fowl cholera outbreaks, both in

17 Australia and around the world. The data-base generated in this study has been provided to the major MLST Web site http://www.mlst.net/.

Implications The development of the MLST database and its placement on the central website serviced by the Oxford University means that laboratories around Australia and around the world can now directly compare isolates and build a truly international picture of the diversity within this important pathogen of poultry and other commercial livestock.

Publications To date no publications have been produced from this work.

Project Title Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis

RIRDC Project No.: UTS-4J Researcher: A/Prof. Nicholas Smith Organisation: University of Technology, Sydney Institute for the Biotechnology of Infectious Diseases Westbourne Street GORE HILL NSW 2065 Phone: (02) 9514 4013 Fax: (02) 9514 4201 Email: [email protected]

Objectives • To test the immunogenicity of recombinant versions of gametocyte antigens of Eimeria maxima. • To test the efficacy of recombinant versions of Eimeria maxima gametocyte antigens as a subunit, maternally-delivered vaccine against coccidiosis.

Background Coccidiosis caused by species of Eimeria costs the poultry industry an estimated $1 billion per year worldwide. The resistance of the parasite to currently used drugs, the high cost of research and development of new anticoccidial agents ($100 million for each new drug) and the demands by the public for chemical-free agricultural practices drive the quest for a vaccine to control this disease. A vaccine using native proteins from the gametocytes of Eimeria maxima is currently marketed in several countries as CoxAbic®. We have cloned and expressed recombinant versions of the gametocyte antigens to succeed the native antigens as a second generation anticoccidial vaccine. The immunogenicity and efficacy of the recombinant proteins, however, needs to be tested. A recombinant version of the vaccine may allow simplification of production and quality control because it will avoid cycling of parasites through chickens. The recombinant vaccine may potentially also be cheaper since the high costs of maintaining chickens pathogen free to ensure the safety and quality of the native vaccine can be avoided.

Research The genes encoding two immunodominant antigens of Eimeria maxima, gam56 and gam82, have been cloned into a bacterial expression vector and the proteins expressed and purified. The testing of the recombinant gametocyte antigens involved a systematic sequence of evaluations. Different recombinant proteins were compared in a variety of different doses and combinations. Immunogenicity and efficacy were determined relative to negative controls and positive controls (birds vaccinated with the proven native gametocyte vaccine formulation).

In the first stage of evaluation, immunogenicity of the new products was tested. This involved initial injection of the proteins, booster injections, and collection of sera (at various times after immunisation) to test for antibody reactivity evaluated by a previously described enzyme linked immunosorbent assay (ELISA).

The second stage of the evaluation involved immunisation of broiler breeder hens housed on floor litter, with evaluation of transfer of antibodies from hens to yolks to hatchlings and efficacy evaluated in hatchlings by counting oocysts excreted by individual birds.

Outcomes Both recombinant proteins were recognised by protective chicken antibodies that were raised to the native 56 and 82 kDa gametocyte proteins, by

19 immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of protective antibodies to the native proteins by 76%, which was comparable to the inhibition of 98% observed when the native antigens themselves were used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised the native proteins by ELISA, and gametocytes by immunoblotting. The antibodies induced by immunisation with the recombinant 56 kDa protein were efficiently transferred by Cobb500 hens to their egg yolks and absorbed by the hatchlings, resulting in reduced oocyst excretion.

Implications A recombinant version of the 56 kDa gametocyte protein of E. maxima appears to be a very promising potential substitute for the native protein components of CoxAbic®. It induces a strong, and competitive, anti-native protein antibody response in hens that is efficiently transferred into egg yolks and, thence, to hatchlings, affording them a level of protection against challenge infection with Eimeria. This result can, however, only be regarded as preliminary and it is recommended that repeated trials via maternal immunisation be carried out to confirm the encouraging results reported here.

Publications Belli, S.I., Mai, K., Skene, C., Gleeson, M.G., Witcombe, D.M., Katrib, M., Finger, A., Wallach, M.G. and Smith, N.C. (2004). Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima. Vaccine, 22: 4316-4325.

Project Title: Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the apicomplexan parasite, Eimeria maxima

RIRDC Project No.: UTS-6A Researcher: A/Prof. Nicholas Smith Ms. Kelly Mai Organisation: University of Technology, Sydney Institute for the Biotechnology of Infectious Diseases Westbourne Street GORE HILL NSW 2065 Phone: (02) 9514 4013 Fax: (02) 9514 4026 Email: [email protected] [email protected]

Objectives • To identify and characterise structural proteins and enzymes involved in oocyst wall formation in Eimeria maxima.

Research and Compositional analysis of oocyst walls (isolated from bleached unsporulated oocysts Outcomes of E. maxima) using gas chromatography and mass spectrometry indicated that the oocyst wall is composed of peptides, carbohydrates (mainly glucose and ~ 1% novel carbohydrates) and, surprisingly, no lipid. These results are currently being confirmed.

Bioinformatic analysis of the protein sequence of the E. maxima antigen, gam56, showed that the protein contains disorder regions characteristic of ‘unstructured’ proteins. Unstructured proteins are proteins that lack a fixed tertiary structure, essentially being partially or fully unfolded; other such proteins have a variety of important and unexpected biological functions. Structural analysis of gam56 by NMR (nuclear magnetic resonance) will be performed to confirm the results of the bioinformatic analyses.

A recombinant version of gam56 - designated r56 – was constructed and produced. It was subsequently demonstrated that this recombinant protein can be crosslinked in vitro using exogenous peroxidase in the presence of peroxides. R56 proteins were crosslinked into high molecular weight oligomers including dimers, tetramers and polymers as detected by SDS-PAGE gel electrophoreses and Western Blotting. In addition, biochemical data (HPLC analysis) showed that high concentrations of dityrosines were detected in crosslinked protein samples. These results support the hypothesis that crosslinking of proteins originating in the wall forming bodies of the gametocytes of E. maxima is mediated by the formation of dityrosine bridges through oxidation of tyrosine residues in proteins.

Implications These data establish a molecular model to explain a fundamental developmental phase in the Eimeria lifecycle, namely the formation of the oocyst and, most particularly, its distinctive protective wall, which is essential for transmission of this genus of parasites.

Publications To date no publications have been produced from this work.

Project Title: Effects of organic acids, prebiotics, and enzymes on control of necrotic enteritis and performance of broiler chickens

RIRDC Project No.: UNE-75A Researcher: Prof. Mingan Choct and Dr Andreas Kocher Organisation: School of Rural Science and Agriculture The University of New England ARMIDALE NSW 2351 Phone: (02) 6773 5121 Fax: (02) 6773 3050 Email: [email protected]

Objectives • To produce data and demonstrate the effect of using organic acids, prebiotics and enzymes in broilers as alternatives to antibiotic growth promotants to maintain feed efficiency and general bird health. • To evaluate the effect of these alternative products on the prevention of necrotic enteritis.

Background Most chicken meat producers in Australia use antibiotics, such as zinc bacitracin, to control outbreaks of necrotic enteritis. However, continued use of antibiotics in feed may encourage the development of resistance to antibiotics by some organisms. In order to examine the effectiveness of alternative products to antibiotics in controlling necrotic enteritis (NE) and other diseases, a reliable experimental model must first be established.

Research Thirteen experiments were conducted to establish a reliable necrotic enteritis model and to test organic acids, prebiotics and enzymes as alternatives to antibiotics in broilers. Some examples or probiotics were also tested.

Outcomes The first 24 months of the project focused on the establishment for a reliable necrotic enteritis model, whereas during the last 18 months’ experiments were conducted to test the efficacy of a number of organic acid products, xylanases, a probiotic (B. subtilus) and oligosaccharides using the previously established NE model. The NE model was reproduced consistently with 1-3% NE-related mortalities (lesion scores 1-4), and clear reductions in growth and feed conversion efficiencies of broilers. Enzymes worked most effectively in maintaining bird performance and reducing the number of Clostridium perfringens in the small intestine. Organic acids were intermediate in their effects on these parameters, whereas the effect of oligosaccharides was not consistent.

Implications Under a subclinical infection, the effect of NE on bird performance can be alleviated by the use of appropriate enzymes or organic acids, but under the circumstances of a clinical outbreak of NE, it is doubtful that any of these alternatives can control or prevent the damage caused by it.

Publications Kocher, A., Anderson, G., Choct, M. and Carter, R.R. (2002). An Australian model for experimental reproduction of clostridial enteritis in broilers. Proceedings of the Australian Poultry Science Symposium, 14: 193. Kocher, A., Choct, M., Teo, A., Tan, H.M. and Carter, R.R. (2004). Effectiveness of alternative feed supplements to broiler diets using a necrotic enteritis challenge model. Proceedings of the Australian Poultry Science Symposium, 16: 84. Kocher, A., Rodgers, N.J. and Choct, M. (2004). Efficacy of alternatives to AGP's in broilers challenged with C. perfringens. Proceedings of the Australian Poultry Science Symposium, 16: 130-133.

Bird Nutrition and Feed Supply

Project Title: Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets (AECL-managed project WAU-1A)

RIRDC Project No.: UWA-76J Researcher: Dr. Ian Williams Organisation: The University of Western Australia Faculty of Natural and Agricultural Sciences CRAWLEY WA 6009 Phone: (08) 6488 3780 Fax: (08) 6488 1040 Email: [email protected]

Objectives • To improve the nutritional value of whole and dehulled lupins so that they can replace soybean meal in diets for broilers and layers, with major savings in feed costs. • Breakdown of thick cell walls of whole and dehulled lupins by expansion and enzymatic treatments. • To increase the inclusion rates of whole and dehulled lupins up to 20% in broiler and layer diets by the above treatments without significant losses in productivity.

Background Australia produces 87% of the world’s lupins which are an excellent source of protein and energy. While the world faces a shortage in plant protein meals, feed manufacturers and poultry producers cannot use more than about 5% lupins in broiler and 7% in layer diets. The main reason is because lupins contain complex cell-wall polysaccharides (33%) that are indigestible. The main component of cell walls is pectin which varies from 33 to 71%. Pectin increases the viscosity of digesta in the bird’s digestive tract, increases water intake and wet droppings and, consequently, reduces food intake and efficiency of feed utilisation. Poultry cannot digest pectin because they don’t secrete the appropriate enzymes so their use of lupins is limited. However, by treatment of lupins with exogenous enzymes and mechanical heat treatment (expansion), it might be possible to increase the nutritive value of lupins for poultry.

Research Three experiments were carried out to investigate whether exogenous pectinases and expansion might increase the nutritive value of lupins for broilers and layers. The first involved the inclusion of 10 and 20% whole and dehulled lupins using pectinase (endo-polygalacturonase) for layers. The second experiment evaluated enhancement of pectinase activity through expansion of lupins before including them at 10 and 20% in the diets for broilers. The third experiment investigated the in vitro breakdown of pectin in dehulled lupins by two pectinases, endo-polygalacturonase (PG) and pectin methyl esterase (PME).

Outcomes Low levels of PG (0.6 g/kg diet) improved the nutritive value of both whole and dehulled lupins for laying hens while higher doses appeared detrimental to performance. Higher levels (0.8 g/kg diet) of PG improved the nutritive value of whole and dehulled lupins for broilers. Expansion was effective in breaking down cell walls of lupins but this was not reflected in improved performance of birds. PG on its own broke down about 20% of the pectin in cell walls but a combination of pectinases broke down 50% of the pectin. Initially, PME removed the methyl ester radicals to allow PG to break down more of the glycosidic bonds of pectin.

23 Implications Treatment of lupin-based diets with PG should allow feed manufacturers to incorporate at least 10% lupins in both broiler and layer diets and, depending on the level of wet droppings that can be tolerated, may allow up to 20% inclusion. Layers can tolerate higher levels of lupins than broilers. Although expansion breaks down the cell walls of lupins it cannot be recommended as a process for improving nutritional value because of negative effects. On the in vitro evidence, a combination of two pectinases, PG and PME, offers much greater potential to improve the nutritive value of lupins and, at the same time, control the problem of wet droppings than PG on its own.

Publications Ali, A., Williams, I.H., Martin, G.B. and Sipsas, S. (2005). Hydrolysis of lupin pectin by pectinases for broilers. Proceedings of the Australian Poultry Science Symposium, 17: 219 – 222. Ali, A., Williams, I.H., Martin, G.B. and Sipsas, S. (2006). The optimal dose of pectinase in lupin-based diets for laying hens. Proceedings of the Australian Poultry Science Symposium. 18: 218. Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. (2006). Expansion and pectinase treatment of lupins for broilers. Proceedings of the Australian Poultry Science Symposium. 18: 62.

Project Title: Digestible amino acids and improved broiler performance

RIRDC Project No.: UQ-107A Researcher: Prof. Wayne Bryden Organisation: The University of Queensland School of Animal Studies GATTON QLD 4343 Phone: (07) 5460 1257 Fax: (07) 5460 1236 Email: [email protected]

Objectives • To delineate the digestible amino acid supply from the grain portion of the diet. • To estimate the ileal digestible amino acid requirements of broilers fed wheat/sorghum based diets. • To estimate the availability and requirements of digestible amino acids such as lysine and methionine for lean tissue deposition. • To improve broiler performance by integration of the above information into feed formulation practices.

Background The supply of protein and amino acids in poultry diets is a significant cost of production. Digestible amino acid values are becoming important as the basis of poultry feed formulations. However, for industry to optimise the digestible amino acid approach to feed formulation, it must be confident in estimates of broiler requirements for digestible amino acids.

Research Studies were conducted to determine ileal digestible lysine, methionine and threonine requirements in broiler chickens from 1 to 21 and 22 to 42 days of age. Responses of sex and genotype of broilers to diets formulated on total and digestible amino acid values were investigated along with the dietary supply of ileal digestible amino acids from Australian wheat and sorghum.

Outcomes A series of studies that examined the lysine, methionine and threonine requirements of broiler chickens in both the starter and grower periods were conducted. The influence of the sex and strain of the broiler chickens on digestible amino acid requirements was also evaluated. It was also demonstrated that the supply of digestible amino acids could be estimated from the protein content of wheat and sorghum.

Implications The data derived in this project for amino requirements matches nicely with previous data generated by the authors on ileal digestible amino acid supply from feed ingredients. Moreover, further refinement of the digestible amino acid system for both supply and requirements will be required if the industry is to minimise protein and amino costs in the future. To achieve this goal, ongoing research is required on amino acid digestion and utilisation.

Publications Bryden, W.L. and Li., X. (2003). Prediction of amino acid digestibility of complete broiler diets. Proc. Aust. Poult. Sci. Sym., 15: 67. Li., X., Sun, Y., Huang, K., Hyde, M.L., Muir, W.I., Gill, R.J. and Bryden, W.L. (2003). Broiler performance and methionine and lysine concentrations in diets formulated using digestible amino acid values. Proc. Aust. Poult. Sci. Sym., 15: 71. Bryden, W.L. and Li., X. (2003). Estimating amino acid availability from digestibility coefficients: application to poultry diets. Proc. Nutr. Soc. Aust., 27: 22. Bryden, W.L., Li., X., Nigusti Ayu, M., Huang, K.H. and Pym, R.A.E. (2004). Growth performance of broilers and fed diets formulated using digestible amino acid values. WPC 2004 XXII World’s Poultry Congress. Turkey.

25 Lemme, A., Ravindran, V. and Bryden, W.L. (2004). Ideal digestibility of amino acids in feed ingredients for broilers. Wld’s Poul. Sci. J. 60: 421-435. Shini, S., Li., X., and Bryden, W.L. (2005). Methionine requirement and cell- mediated immunity in chicks. Asia Pacific Journal of Clinical Nutrition (2005) 14 (suppl). s123. Shini, S., Li., X., Mulyantini, N.G.A., Zhang, D., Shini, A., Kumar, A., Hosking, B.J. and Bryden, W.L. (2005). Methionine requirements and immune function in broiler chicks. Recent Advances in Animal Nutrition in Australia 15: 13a. Li., X., Mulyantini, N.G.A., Zhang, D., Gurney, K. and Bryden, W.L. (2006). Apparent ileal amino acid digestibility of Australian sorghum for broiler chickens. Aust. Poult. Sci. Symp. 18: 48. Zhang, D., Li., X., Huang, K., Huynh, H.T, Mulyantini, N.G.A., Kumar, A. and Bryden, W.L. (2006). The response of broiler to dietary digestible lysine levels in the starter phase. Aust. Poult. Sci. Symp. 18: 66. Bryden, W.L., Zhang, D. and Li., X. (2006). Relationship between total crude protein content and apparent ileal amino acid digestibility of wheat for broilers. XII European Poultry Conf. (in press). Zhang, D., Li., X. and Bryden, W.L. (2006). Dietary digestible lysine contents and broilers performance during the starter period. XII European Poultry Conf. (in press).

Efficiency of Production

Project Title: Investigation of the cause of miliary hepatitis in laying chickens

RIRDC Project No.: DAV-226J Researcher: Dr. W. Mike Forsyth Organisation: Department of Primary Industries (Vic) Primary Industries Research Victoria, Attwood Site 475 Mickleham Road ATTWOOD VIC 3049 Phone: (03) 9217 4200 Fax: (03) 9217 4399 Email: [email protected]

Objectives • To determine whether the origin of the microorganism causing miliary hepatitis is the liver or the intestine. This would be a starting point for further work to use more advanced cultural techniques such as strict anaerobiasis, egg inoculation and tissue culture. • To find a model of miliary hepatitis in laying chickens and to test two bacterial isolates in this model to see if they are a cause of miliary hepatitis.

Background This was a pilot trial to establish a model so that laboratory manipulation can establish the cause of the disease.

Research Laying hens at their peak of production were inoculated by mouth with extracts of intestines and liver, and by two bacteria previously collected from affected birds. They were then observed for ten days to establish whether the disease, miliary hepatitis, could be reproduced.

Outcomes The disease was not reproduced in this trial by the tissues or by the two bacterial inoculums given by mouth.

Implications Understanding of the cause of miliary hepatitis has been advanced but the disease remains an enigma to the industry. The evidence from these studies indicates that two bacteria isolated in the course of investigation of natural disease appear not to be primary pathogens.

Publications To date no publications have been produced from this work.

Product Quality and Safety

Project Title: Baseline survey of Campylobacter and Salmonella during chicken processing

RIRDC Project No.: FSA-3A Researcher: Gary Dykes Organisation: Food Science Australia P O Box 3312 TINGALPA DC QLD 4173 Phone: (07) 3214 2037 Fax: (07) 3214 2050 Email: [email protected]

Objectives • To establish levels of Campylobacter and Salmonella on chicken carcasses at ten points during processing in five Australian facilities and use these data to determine the impact and relative importance of the different processing points.

Background There are currently a wide range of practices and levels of process control across the Australian industry with respect to the various steps in chicken processing. Information on the effectiveness of these steps in reducing pathogens during processing is available within individual companies. No independent survey of both prevalence and levels of Campylobacter and Salmonella across the range of industry practices has been previously undertaken in Australia.

Research Facilities across three states, representing both medium and large processors (based on throughput) were selected for sampling. At each of ten processing points in each facility ten chicken carcasses from a single flock were taken. Whole bird rinses were prepared and the presence and levels of both Salmonella and Campylobacter on carcasses were determined by established methods. Environmental and processing parameters associated with each of the facilities, including water temperatures, overflow rate and chlorine levels, were recorded. Data were analysed and results between plants with respect to processing parameters were compared.

Outcomes Despite variability in processing parameters, numbers of both Salmonella and Campylobacter on positive carcasses at the initial (~3 and ~7.5 log cfu carcass-1, respectively) and final (~1 and ~3 log cfu carcass-1, respectively) sampling points did not differ significantly between plants. Prevalence of both Salmonella and Campylobacter at the final sampling point did, however, vary substantially between plants. In most cases the reason for the effect on prevalence was not easily identified or consistent but some evidence for a role of temperature and chlorine levels in the washing/spin chilling step was apparent. The scalding and spin chilling steps were the only two points that clearly reduced prevalence and levels of Salmonella and Campylobacter. Higher temperatures in the scalder, in particular, appeared to result in a greater reduction in numbers of both pathogens. Subsequent processing neutralised the effect of scalding in most but not all cases, indicating that scalding may be a useful control point if reduced levels of pathogens can be subsequently maintained.

Implications This study demonstrated that most of the individual processing points, with two

28 exceptions, appeared to have no influence on levels and prevalence of pathogens on carcasses. Control of parameters associated with scalding and washing/spin chilling are critical to achieving reduction in prevalence and levels of both Salmonella and Campylobacter on chicken carcasses. The results obtained represent variations on industry practice but it cannot be assumed that this is the same across the entire industry and results cannot be extrapolated to all plants in Australia.

Publications To date no publications have been produced from this work.

RESEARCH IN PROGRESS

Flock Health

Project Title Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics

RIRDC Project No.: CSA-30J Start Date: 01/07/03 Finish Date: 31/07/06 Researcher: Dr. Sandra Sapats Organisation: CSIRO Livestock Industries Private Bag No 24 GEELONG VIC 3220 Phone: (03) 5227 5769 Fax: (03) 5227 5555 Email: [email protected]

Objectives • To monitor genetic changes in circulating IBDV strains in order to determine if field strains are changing significantly. • To optimise the already developed Reverse Genetic system for IBDV for the genetic modification and the molecular characterisation of biological properties of IBDV. • To apply the Reverse Genetics system for the construction of recombinant IBDV to ascertain the importance of specific mutations in VP2 gene related to virulence and antigenicity and in particular how these effect virulence of endemic strains.

Current Progress A total of 124 samples from 21 farms located in Queensland, NSW, WA, SA and Victoria were tested for the presence of infectious bursal disease virus (IBDV) antigen. Fifty five samples tested positive for IBDV covering 13 farms located in WA (1/2), NSW (5/7), Queensland (2/2) and Victoria (5/5), with birds ranging in age from 26-35 days of age. In contrast, all five farms from SA tested negative for IBDV.

Sequence analysis indicated that most field isolates were similar to strains isolated previously in each of the states including ‘classical’ like strains in WA, NSW and Queensland, and variants in Victoria. As seen in the previous two years, isolates from NSW appear to be showing the highest number of amino acid substitutions. One Queensland isolate was shown to possess an amino acid substitution which has been associated with vvIBDV (isoleucine 256) but showed no increase in pathogenicity.

Using the reverse genetics system developed for IBDV, a number of amino acid changes were introduced into the genome of an Australian IBDV, focusing on residues potentially involved in the virulence of IBDV. These recombinants are currently being passaged through specific pathogen free (SPF) chickens to generate virus stocks to enable pathogenicity testing in SPF birds.

Project Title New diagnostic assays to improve control of coccidiosis in poultry

RIRDC Project No.: DAQ-316A Start Date: 01/01/2004 Finish Date: 31/12/2006 Researcher: Dr. John Molloy Organisation: Department of Primary Industries & Fisheries (Qld) Animal Research Institute Locked Mail Bag No. 4 MOOROOKA QLD 4105 Phone: (07) 3362 9413 Fax: (07) 3362 9429 Email: [email protected]

Objectives • To develop and validate new DNA-based quantitative assays for the diagnosis of poultry coccidiosis. • To develop and validate new serological assays for the diagnosis of poultry coccidiosis.

Current Progress Real time PCR probes specific for each of the seven Eimeria species have been designed using sequence data generated in the first year of the project and these probes are now undergoing evaluation.

Considerable progress has been made towards understanding the antibody response to infection in chickens and developing specific serological tests. Immunoblot tests specific for E. tenella and E. necatrix have been developed and have undergone initial evaluation. Immunoblots with parasite antigens prepared from the gut of infected birds at varying times after infection suggested that the antibody response recognised exclusively gametocyte antigens. This was confirmed using immunoperoxidase and fluorescent antibody tests on sections of gut that demonstrated strong antibody binding to gametocytes but not to merozoites or oocysts. Gametocytes of E. tenella vaccine strain have now been purified from the gut of infected birds in useful quantities. The gametocyte antigen has been used in immunoperoxidase and fluorescent antibody tests to demonstrate the specificity of monoclonal antibodies developed previously. At least three monoclonal antibodies have been shown to bind to gametocytes. The purified gametocyte antigen is also being evaluated for development of a generic ELISA and species-specific ELISAs. Initial results suggest that the generic ELISA using gametocyte antigen could be much more sensitive and specific than an ELISA developed previously using oocyst antigen.

Bird Nutrition and Feed Supply

Project Title Nutritional characterisation of sorghums from Queensland and NSW for chicken meat

RIRDC Project No.: DAQ-326A Start Date: 01/10/04 Finish Date: 30/02/07 Researcher: Dr Rider A. Perez-Maldonado Organisation: Department of Primary Industries and Fisheries Poultry Research and Development Centre (PRDC) PO Box 327 CLEVELAND Qld 4163 Phone: (07) 3824 3081 Fax: (07) 3824 4316 Email: [email protected]

Objectives • To characterise and evaluate the variability of sorghums sourced from various regions of Queensland and NSW in term of their (a) nutritional value for chicken meat production; (b) chemical; (c) apparent metabolisable energy (AME) on a dry matter basis and corrected for N; (d) digestible AA values; (e) starch digestibility; (f) condensed tannin (CT) content and digestibility; (g) sorghum ergot, alkaloid and aflotoxin content. • Determine the cause of reduced chicken meat performance, including feed conversion efficiency (FCR), and carcass variability (breast yield and fat pad) in broilers fed sorghum-based diets. • Ensure this project provides a link to evaluations undertaken on sorghum as part of the Premium Grain for Livestock Program (PGLP).

Current Progress Apparent metabolisable energy (AME) evaluations undertaken in broilers compared Poultry Research and Development Centre (PRDC) and Premium Grains for Livestock Program (PGLP) methodologies, and results indicated that (a) the AME of sorghum was about 0.8 MJ/kg lower when using the PRDC method (which is based on studies in younger birds, between 15-21 days of age), and (b) the AME and bird feed intake (BFI) values between sorghums were similar for four diet preparations (mash, cracked grain hot pellet, cracked grain cold pellet and whole grain cold pellet).

During October-November 2005, a semi-commercial floor experiment evaluated five sorghums against a control wheat diet using six dietary treatments with six replicates each and 30 birds (15 males and 15 females)/pen. Results at 21 days of age indicated that BFI on sorghum 11 (Pacific Buster, Gunnedah) was significantly depressed (1049 g) compared to that of birds on sorghum 7 (1160 g, Pioneer bonus, Dalby), sorghum 8 (1125 g, Pioneer 85G, Dalby), sorghum 4 (1117 g, Pacific buster Yelarbon), or a commercial sorghum (1123 g) which had similar (P>0.05) BFI as the control wheat diet (1163 g). Live weight gain (LWG) at 21 d was also lowest in sorghum 11 (710 g) with wheat (832 g) and sorghum 7 (808 g) diets being superior followed by sorghum commercial (801 g), sorghum 8 (791 g) and sorghum 4 (781 g). Feed conversion ratio (FCR) was numerically better on the wheat diet (1.397).

However, during the 21-42 days of age period, BFI in all sorghums (including sorghum 11) were similar to the control wheat. During the 21-42 days of age

32 period, live weight gain (LWG) was also similar among sorghums, but sorghum 8 (1829 g) and the commercial sorghum (1843 g) performed significantly (P<0.05) better than the control wheat (1768 g). FCR tended to be better for all sorghums compared with wheat. It was concluded that performance of broilers consuming sorghum diets is affected during the starter phase but this effect disappeared during the grower/finisher period with sorghums performing similarly or better than birds consuming the wheat diet. Further experiments are being undertaken to gain a better understanding of the negative effects of sorghums observed during the starter phase.

Project Title: Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal integrity, and performance of broiler chickens

RIRDC Project No UNE-86A Researcher: Prof. Mingan Choct Mr. Nicholas Rodgers Organisation: University of New England School of Rural Science and Agriculture ARMIDALE NSW 2351 Phone: (02) 6773 5121 Fax: (02) 6773 3050 Email: [email protected]

Objectives • To elucidate the mechanisms by which grain processing and feed constituents affect gut physiology of birds from early life, which in turn affects life-long productivity.

Current Progress Remaining results on digesta transit time have been obtained and analysed. Broilers fed a diet based on whole vs fine or commercially ground hammer-milled sorghum had a slower liquid phase passage. There were no significant transit time differences for the solid phase of digesta.

A semi-commercial trial was conducted in March/April at Bartter Enterprises’ Hanwood trial facility to determine the effects of wheat particle size and processing type (roller-milling vs hammer-milling) on broiler males and females under commercial conditions on deep litter.

No significant performance differences were seen, but numerically, birds fed the whole or intermediate roller-milled wheat diets were more efficient over the six week period.

Feeding roller-milled wheat to broiler on deep litter reduces total ileal anaerobes and increases gizzard lactobacilli. Few differences were seen in gut development at three and six weeks of age, however feeding roller-milled wheat over all hammer-milled and whole wheat diets tended to increase villi height to crypt depth ratio in the jejunum at six weeks of age. It was postulated that consumption of litter may have reduced differences in gut development between birds on different dietary texture treatments. Unfortunately, an infectious bursal disease outbreak occurred in the last week of the experiment which would have altered the performance.

Project Title: Early feeding of prebiotics on development of the digestive system and gut microflora of broilers

RIRDC Project No.: UNE-93A Researcher: Dr. Paul Iji Start Date: 01/05/05 Finish Date: 31/05/08 Organisation: The University of New England Animal Science School of Rural Science & Agriculture ARMIDALE NSW 2351 Phone: (02) 6773 2347 Fax: (02) 6773 3922 Email: [email protected]

Objectives • To evaluate whether provision of low-molecular weight carbohydrates (oligomers of mannose, galactose, xylose or glucose) from hatch up to seven days of age will affect development of the digestive system, gut microflora and the immune function of broilers.

Current Progress Day-old chicks at about 24 hours post-hatch were raised on a commercial diet and provided with water with or without four sugar supplements, each at two levels (5 or 10 g/kg body weight). The supplements did not significantly affect feed or water intake over 7 or 14 days of test. Over the first 7 days of test, low levels of maltose and trehalose and both levels of palatinose marginally improved body weight gain. Over two weeks, the same supplements improved body weight gain by about 5 %. Feed conversion efficiency was increased by 1-3 % in the palatinose-supplemented group and the lower level of trehalose. The supplements had no significant effects on the activities of digestive enzymes, but any gross changes in the intestinal structure are still being analysed. Further studies will be conducted on chicks within less than 24 hours post-hatch, to be able to draw conclusions as to the effectiveness of the supplements. Later in the project, the route of administration (water or feed) of the best supplement(s) will be evaluated.

Project Title Establishment of a Chair in Poultry Sciences at The University of Sydney

RIRDC Project No.: US-127A Start Date: 01/01/2004 Finish Date: 30/12/2007 Researcher: Prof. Tom Scott Organisation: The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570 Phone: (02) 4655 0612 Fax: (02) 4655 0693 Email: [email protected]

Objectives • To establish a Chair in Poultry Science at the University of Sydney in order to maintain a high level research capability for the industry at that facility. • To assist in the development of a centre of excellence in poultry science at the university of Sydney. • To develop a targeted research program in poultry nutrition, health and safety focused on the needs of the Australian poultry industries.

Current Progress The Poultry Science Chair has progressed two RIRDC supported research projects and one Australian Poultry CRC research supported project. Full applications have been submitted for support from RIRDC (Chicken Meat and New Animal Industries Programs), ARC (Australian Research Council), and the Australian Egg Corporation Ltd (AECL). The Australian Poultry Science Symposium for 2006 was hosted February 20-22, 2006 and highlighted issues pertaining to (a) physical effects of feed processing on digestion and performance; and (b) optimising egg and chick quality. The APSS organising committee is currently finalising plans for the 2007 meeting and will focus on factors pertaining to regulation of body temperature and osmoregulation, and losses in broilers due to metabolic disease. Three fourth year honours thesis projects have been developed and are currently underway; these include: alternative protein sources and sustainable chicken production, growth and feeding of ducks, and dietary intervention for early hatched broilers. Two PhD students have initiated studies; one will work in the area of pre-conditioning broilers to best withstand high temperatures, the other will determine if peptides from meat/bone by-product meal have potential as nutraceuticals. A MSc student will complete a program in course work. The Chair in Poultry Science will attend the Poultry Science Association meetings in Canada in July and has been asked to address the World’s Poultry Science Association – Canada Branch during the meeting. The Chair has also accepted faculty duties as (a) postgraduate coordinator (Camden); and (b) sub-dean international (representing the faculty on The University of Sydney’s international recognition initiatives).

Project Title Assessment of the anti-nutritive effects of phytate by dephytinisation

RIRDC Project No.: US-133A Start Date: 01/01/2005 Finish Date: 31/01/2008 Researcher: Dr Peter Selle Organisation: The University of Sydney Faculty of Veterinary Science 425 Werombi Road CAMDEN NSW 2570 Phone: (02) 9351 1697 Fax: (02) 9351 1693 Email: [email protected]

Objectives • To define the negative ecological and nutritive effects of phytate on chicken meat production via the experimental use of dephytinised feed ingredients.

Current Progress Initially, a series of investigations was completed to determine the extent to which small samples (1 kg) of sorghum and soyabean meal (SBM) could be dephytinised by hydro-thermal pre-treatment with exogenous phytase. It was found that with the addition of citric acid to a 1:1 slurry of feed ingredient and water plus liquid phytase substantially enhanced dephytinisation of both feed ingredients (94.6% sorghum; 94.1% SBM). It is likely that citric acid, a chelating agent, strips cations (particularly Mg2+) from mineral phytate complexes and renders phytate more susceptible to hydrolysis by phytase. This phase of the project has been reported1. Subsequently, larger batches of 20 kg of sorghum and SBM have been treated and the effects of dephytinised, sham- treated and standard feedstuffs on AME and nitrogen (N) retention have been compared in broilers. In the most recent study, both a dephytinised and sham- treated blend of SBM and sorghum significantly depressed N retention. It would appear that the dephytinisation procedure is adversely affecting SBM protein quality. The mixing and drying methods of 20 kg batches are now being reassessed in terms of dephytinisation, residual phytase activity and protein dispersibility indices (SBM) with the intention of developing a more innocuous dephytinisation procedure. In association with Dr Rob Caldwell a HPLC method of phytate analysis (IP6) has been developed.

1Selle, P.H. (2006). Citric acid enhances enzymatic hydrolysis of phytate. Proceedings, Australian Poultry Science Symposium 18: 91.

Project Title Early dietary and management intervention on broiler breast meat yield

RIRDC Project No.: US-134A Start Date: 01/07/2004 Finish Date: 30/12/2007 Researcher: Prof. Tom Scott Organisation: The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570 Phone: (02) 4655 0612 Fax: (02) 4655 0693 Email: [email protected]

Objectives • To increase the yield of high value breast meat from broiler chicks by developing early dietary and/or managemnet intervention programs to stimulate initial muscle cell fibre numbers and subsequent breast muscle yield at an optimum market weight. • To maximise the capacity of ‘supply’ organs (gut, respiratory, curculatory systems) to supply the nutrients and metabolic homeostasis of the ‘demand’ tissues (meat, feathers and maintenance). • To monitor (and expand methods to monitor) the effect of early dietary and/or management programs on the development of growth related metabolic disease, and on broiler flock uniformity and market efficiency. • To train a graduate with a focus on muscle development and meat quality of broiler chickens.

Current Progress Protocols for tissue sampling (gut and breast), incubation and bioassay trials have all been established. Protocols for histology and the digitisation of the mounted sections have also been performed utilising a digital image analysis program. Refinements to this protocol have included the acquisition of a wide- objective microscope lens to facilitate less handling of slides during the digitisation process. A thorough analysis of gut tissue has been conducted on samples acquired to date, yielding approximately 4,000 measurements. Analysis of this data is continuing.

To date, seven incubation trials incorporating four floor pen, one bioassay and two incubation only studies have been conducted. Approximately 30 treatments have been analysed for their performance and potential as in ovo stimulants to broiler meat and/or gut development.

An in ovo study is currently being performed with an early nutrition study planned to follow, commencing in June 2006. The in ovo study, for which 1,000 eggs have been set, will include individual measurements for egg weight, egg length, candle percentage (fertility/early embryonic deaths), hatch weight, chick length and numerous day seven performance indices, to provide a holistic data set (from egg weight at setting through to seven days post hatch chick performance). The early nutrition study will focus on combinations of ingredients already identified for their potential to increase muscle development and gut maturation post hatch. Information from the studies to date has been incorporated in a presentation to industry and academics (Australian Poultry Science Symposium, February, 2006).

Project Title Variability in performance and physiology of broilers fed wheat- and sorghum-based diets

RIRDC Project No.: US-137A Start Date: 01/07/2005 Finish Date: 30/12/2006 Researcher: Prof. Tom Scott Organisation: The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570 Phone: (02) 4655 0612 Fax: (02) 4655 0693 Email: [email protected]

Objectives • To define what characteristics are critical to determining/predicting the feeding value of wheat and sorghum grains, with an emphasis on wheat. • To provide an approach the will contribute to a more complete understanding of feeding value for these cereal grains, including the impact on gut physiology/microbiology.

Current Progress Our definition of feed value now includes measurements of variation in nutrient intake that have been directly correlated with growth. Within cereal grains, variation in intake can be in excess of 20% and is not related to energy or protein levels of the diets. In the present study, 45 grains (34 wheats, 8 triticales and 3 sorghums) were ground and included (750 g/kg) in mash diets (wheat and triticale diets had a different basal diet from sorghum-based diets); all diets contained 1% acid insoluble ash marker. The diets were then split and one portion was supplemented with 0.5 g/kg xylanase and phytase (Danisco Animal Nutrition, UK). The 90 diets (45 cereal-based diets with or without enzyme) were each full fed to one cage of six male broilers (Cobb 500) in four consecutive bioassays from 4 to 17 days of age. At 16 days of age a 6 hour excreta collection was frozen, freeze-dried, ground and analysed to determine the apparent metabolisable energy (AME; kcal/kg diet). Feed intake, growth and mortality were recorded from 4 to 17 days of age and used to calculate feed conversion ratios. Fifteen of the 34 wheat samples had been previously tested in another bioassay; diets contained no enzyme(s) and birds were five weeks of age; AME was determined by total collection.

The results of the present study supported earlier work that demonstrated measurements of feed value must not ignore the variation in intake, growth and efficiency. Variation in feed intake of wheat-based diets (~20%) was positively correlated (r>0.70) with body weight, while correlations between feed intake and AME were positive (r=0.30; P<0.01). Variation in performance of triticale samples was similar to wheat, while no enzyme response was observed for sorghum-based diets due to low levels of soluble non starch polysaccharides. The 15 wheat samples measured in two different bioassays had significant positive correlations for measures of performance, but no relationship for measures of AME. This indicates that factors within these common grains consistently influenced performance in a similar manner, but not AME. Further work is required to understand the cause of variation or limitation in intake and how this can be minimised.

Food Safety

Project Title Development of a sequence-based bacteriophage typing system for Salmonella

RIRDC Project No.: IMV-5A Start Date: 01/07/2003 Finish Date: 01/07/2006 Researcher: Dr. Michael Heuzenroeder Organisation: Institute of Medical & Veterinary Science Infectious Diseases Laboratories PO Box 14 Rundle Mall PO ADELAIDE SA 5000 Phone: (08) 8222 3275 Fax: (08) 8222 3543 Email: [email protected]

Objectives • To continue to provide conventional typing (serotyping and phage typing) to the industry on an ongoing basis for the purposes of organism tracing and the monitoring of Salmonella serovars for public health and research objectives. • To use AFLP and PFGE typing (where appropriate) for typing industry Salmonella strains when conventional typing methods fail to offer sufficient discriminatory power. • To establish an objective sequence-based molecular typing system that would enhance and extend the current classical phage typing system. • To characterise genes in endogenous phages in Salmonella to determine the predicted impact of these genes on current typing methods.

Current Progress An average of 6,183 samples per annum was received from industry during the project. Salmonella enterica II serovar Sofia remains the most common serovar isolated from Australian chickens. There were no significant changes in Salmonella serovar distribution from humans or chickens. Phage type distribution within S. Typhimurium has revealed that PT135 and PT135a are isolated from both sources in significant numbers. Notably, PT108 (designated PT170 by some laboratories) has emerged as a type of significance in both humans and chickens.

A novel PCR-based typing method for the discrimination of Salmonella, called multiple amplification of phage loci typing (MAPLT), was developed. This method has shown to be superior to PFGE and MLST techniques. A publication and a provisional patent have been major outcomes from this work.

Comparisons of MAPLT to multilocus VNTR analysis (MLVA) were also undertaken for S. Typhimurium and S. Enteriditis. Both methods showed similar discrimination for serovar Typhimurium but MAPLT showed superior discrimination for serovar Enteritidis. It was concluded that a combination of approximately seven MAPLT and MLVA primer sets could provide sufficient discrimination of Salmonella. Both methods have been used to compare Salmonella strains from a number of outbreaks. Further development and evaluation of both systems is continuing.

Project Title Campylobacter bio-replacement program to control food poisoning organisms in poultry

RIRDC Project No.: UG-7A Start Date: 01/07/2003 Finish Date: 31/07/2007 Researcher: Dr. Victoria Korolik Organisation: Griffith University School of Health Sciences, Gold Coast Campus PMB 50 GOLD COAST MAIL CENTRE QLD 9726 Phone: (07) 5552 8321 Fax: (07) 5552 8908 Email: [email protected]

Objectives • To develop methods for controlling Campylobacter spp in poultry by utilising a collection of non pathogenic Campylobacter strains as bio- replacement organisms to exclude pathogenic Campylobacter spp from commercial chicken flocks. • To determine the overall effect of colonisation by super-colonising Campylobacter strains on the general health of chickens.

Current Progress This project aims to minimise the risk of human exposure to pathogenic campylobacters and involves the ‘bio-replacement’ of potentially virulent Campylobacter strains in the chicken gut by a non-virulent C. jejuni strain.

Available data relating to the most likely time for colonisation of commercial chicken flocks with campylobacters suggests that the greatest risk of introduction of campylobacter strains into chicken flocks occurs around 28-35 days of age (around the time of the first pick-up) and also that most flocks become positive by the time of final collection at 56 days of age. The ability of a potential ‘bio-replacement’ strain to out-compete other highly colonising strains was therefore tested by superinfecting ten individual birds (marked) in each experimental flock with these strains, which were colonised by the potential ‘bio-replacement’ strain 331 at either day 35 or day 42. This was followed by a second introduction of 331 one week later. Secretion of campylobacteria was monitored from cloacal swabs from ten marked and ten randomly chosen in contact birds on days 28, 35, 42, 49 and 55 of the experiment. The identity of resident C. jejuni strains was established by PCR/FlaA typing.

The results of this trial showed that, in all cases, ‘bio-replacement’ strain 331 out-competed other, highly colonising challenge strains, with the exception of birds challenged on day 45 where small numbers of one of the challenge strains were recovered from marked birds.

In addition, ten birds were randomly chosen from the control group challenged only with ‘bio-replacement’ strain 331 on days 28, 35 and 42 and sacrificed. The birds were processed using standard processing plant procedures. Five birds from each group were immediately examined for carcass contamination by campylobacteria and the other five birds were frozen, kept frozen for one week, thawed out and the carcass examined for contamination by campylobacteria. This experiment showed that all birds taken on day 28 had C. jejuni strain 331 on their carcases. The birds examined immediately following processing carried on average 700 viable cells per gram of meat and frozen birds carried 100 cells per gram on average. All five freshly processed birds sacrificed on day 35

41 carried on average 80 viable cells per gram of meat; however, of the five frozen birds only two were found to carry C. jejuni with 5 viable cells on average. All five freshly processed birds sacrificed on day 42 carried on average 83 viable cells per gram of meat; however, no viable C. jejuni cells could be detected on any of the five frozen birds. The results of this experiment indicate that C. jejuni 331 does no survive well during standard carcass processing.

Environmental Management

Project Title Evaluating risks posed by pathogen emissions from meat chicken sheds

RIRDC Project No.: DAQ-318A Start Date: 01/01/2004 Finish Date: 31/12/2006 Researcher: Dr. Pat Blackall Organisation: Department of Primary Industries & Fisheries (Qld) Animal Research Institute Locked Mail Bag No 4 MOOROOKA QLD 4105 Phone: (07) 3362 9498 Fax: (07) 3362 9429 Email: [email protected]

Objectives • To quantify emission rates for pathogenic bacteria (Salmonella spp, Escherichia coli, Campylobacter jejuni/coli, Staphylococcus) from typical tunnel ventilated meat chicken sheds. • To evaluate the potential for human health effects caused by these emissions, evaluated by combining measured emissions, gaussian air dispersion models, bacterial survival models and applying quantitative risk assessment methodology.

Current Progress During the period covered by this report, two approaches were taken to this project. In the first approach, the focus was on the presence of E. coli and non- pathogenic bacteria (fungi, total bacteria, staphylococci). This work involved sampling weekly from day 0 to day 55 of the batch. Samples were collected at 2, 10, 30, 50, 75 and 100 metres in front of the fans, 2 and 20 metres from the back of the shed and at 2 and 20 metres on both sides of the shed. Two full cycles (batches) have been studied.

In the second approach, there is a focus on enumerating the levels (if present) of Salmonella and Campylobacter, as well as E. coli, in air samples inside the broiler shed as well as external to the shed (10 metres in front of the fans). Large volumes of air (4,000 litres) were collected. Litter, a major source of bacteria in aerosols, was sampled and tested for Salmonella, Campylobacter and E. coli levels. One complete growth cycle has been studied to date with a further growth cycle now being studied.

These studies are providing the first knowledge of the levels of bacteria (pathogenic and non-pathogenic) in the emissions from Australian meat chicken sheds.

Project Title Efficacy of windbreak walls for odour reduction

RIRDC Project No.: DAQ-321A Start Date: 01/05/04 Finish Date: 31/10/06 Researcher: Mr Mark Dunlop Organisation: Department of Primary Industries & Fisheries (Qld) PO Box 102 TOOWOOMBA QLD 4350 Phone: (07) 4688 1280 Fax: (07) 4688 1192 Email: [email protected]

Objectives • To identify the value of windbreak walls for improving dispersion of exhaust air from tunnel ventilated chicken sheds. • To evaluate the use of windbreak walls as an odour reduction strategy for meat chicken sheds.

Current Progress This study aimed to evaluate the efficacy and value of windbreak walls for improving the dispersion of odours from tunnel ventilated chicken sheds. Tracer gas methods were initially identified as the most suitable method for this evaluation. Following several trials using a tracer gas, it became apparent that these methods were not suited for use in this project.

Visualisation of exhaust air movement over the windbreak wall using smoke was then trialled. A windbreak wall was constructed and fitted to a meat chicken shed. Smoke was released from this shed as well as a neighbouring chicken shed where no windbreak wall was present. Smoke was generated using coloured smoke flares. Smoke was released under a range of atmospheric and ventilation conditions. Photographs were taken during the smoke releases for accurate comparison of plume movements and dispersion. Approximately 40 comparative smoke releases were undertaken producing approximately 1,000 photographs. Visualisation of plume movement using smoke proved to be very successful.

In addition to the smoke observations, computational fluid dynamics (CFD) modeling is being undertaken to confirm the observations made during the smoke releases. It is hoped that CFD modeling will also assist in quantifying the influence of a windbreak wall for improving the dispersion of odorous air emitted from tunnel ventilated meat chicken sheds.

Project Title Managing litter re-use for minimal nutrient runoff to surface water

RIRDC Project No.: DAQ-323A Start Date: 01/09/04 Finish Date: 30/09/07 Researcher: Mr. Jarl Devereux Organisation: Department of Primary Industries and Fisheries (Qld) PO Box 102 TOOWOOMBA QLD 4350 Phone: (07) 4688 1096 Fax: (07) 4688 1192 Email: [email protected]

Objectives • To establish accurate estimates of meat chicken re-use contributions to catchment nutrient loads at case study farms under conditions of appropriate management, helping to ensure that a realistic representation of this form of nutrient re-cycling is available for local government planners and regulators. • To establish effective management techniques, likely nutrient load outcomes, and costs through application of existing state-of-the-art modelling techniques to case study litter re-use enterprises in sensitive locations. • To extend effective management practices to litter end-users and industry, and provide updates to the National Chicken Industry Environmental Management System.

Current Progress Research is currently being directed towards collecting information on environmental indicators in order to validate initial model estimates of nutrient export. Initial mapping and soil surveys at the case study sites have been completed, along with hydraulic assessment of the sites to allow for initialising the chosen model.

The initial rainfall simulation used to investigate the hydraulic parameters at the study sites indicated that dissolved reactive phosphorus (DRP = P <0.45 μm) and sediment transport were likely to be the major sources of nutrients reaching surface waters. While sediment transport from the study sites was low, the sediment contained high concentrations of phosphorus, and therefore led to significant nutrient loss. High concentrations of DRP were found in runoff, especially immediately following litter application.

As our research indicates that DRP and P contained in sediment are the major sources of contamination to surface water, the following management practices are being investigated with a view to minimising this problem: • Matching the litter application rate to the crop phosphorus requirement, thereby reducing the amount of surplus P in the agronomic system. • Liming the litter applied. A number of studies have indicated that liming of litter reduces the solubility of phosphorus and consequently the concentration of DRP in runoff. • Filter strips and sediment traps may prove useful where sediment is the major source of phosphorus to surface water. • Application of polyacrylamide to cultivated soils to reduce sediment transport. These management techniques will be assessed on the basis of environmental and economic costs and benefits to identify effective management practices for the industry.

Project Title Trials of odour control technologies for broiler farms

RIRDC Project No.: DAV-213A Start Date: 01/07/2003 Finish Date: 31/11/2006 Researcher: Dr. Julie Simons Organisation: Department of Primary Industries (Vic) Primary Industries Research Victoria, Attwood Site 475 Mickleham Road ATTWOOD VIC 3049 Phone: (03) 9217 4358 Fax: (03) 9217 4111 Email: [email protected]

Objectives • To identify, assess and quantify the performance of technology options to control odour from broiler sheds. • To define the conditions and application methods in which they work. • To monitor the extent of odour emissions as they occur on a number of farms. • To develop test protocols for the testing the efficacy of future odour control technologies as they become available.

Current Progress Field trials commenced in 2005 and are ongoing to examine the effectiveness of four odour control technologies. One litter additive, one feed additive, and two air-additives are being assessed for their effectiveness in reducing odour emitted from commercial broiler sheds. Each technology is being tested on four farms spread across Victoria and over a number of different batches. The result will be that each technology will have been tested in different seasonal conditions, farm and shed conditions, and topography. The trials aim to define the conditions for when the technologies may be most effective, and methods for ongoing testing of other technologies into the future.

The primary indicator that is being used to measure a reduction in odour is olfactometry. This analytical technique is based on taking air samples at the trial site and assessing the odour concentration using a panel of trained assessors. The panel members are presented with increasing concentrations of sample. The concentration at which the sample can be accurately detected by the panel is the odour concentration. The olfactometry is conducted in accordance with Australian Standard for odour assessment (AS4323.3, 2001).

The trials will also examine the relationship of litter moisture and litter pH to odour concentration and will consider other potential impacts such as stocking density and mortality. The trials are due to be completed in August 2006.

Efficiency of Production

Project Title Postgraduate scholarship - David Stephenson: Delivery of therapeutic proteins using lactobacillus

RIRDC Project No.: ANU-72A Start Date: 01/04/2006 Finish Date: 31/05/2009 Researcher: Mr. David Stephenson Organisation: Australian National University School of Biochemistry and Molecular Biology CANBERRA ACT 0200 Phone: (02) 6258 9843 Fax: (02) 6125 0313 Email: [email protected]

Objectives • To deliver therapeutic proteins to the gastrointestinal tract of chickens targeting Clostridium perfringens using strains of Lactobacillus, by (a) isolating and characterising a large number of Lactobacillus strains from the chicken gastrointestinal tract; (b) identifying persistent strains by tracking marked strains in chickens; (c) genetically engineering persistent strains to produce antimicrobial peptides and other potentially therapeutic proteins; and (d) testing the performance of these engineered strains in animal disease models.

Current Progress A pilot study has been conducted to compare different sampling techniques for the isolation of lactobacilli from the tissue and contents of the duodenum, jejunum and ileum of chickens. Preliminary genetic analysis suggests that the same strains are isolated from both tissue and contents, within and among these sections of the small intestine.

A high-throughput method is required for genetic identification and characterisation due to the large number of isolates to be collected and screened. The ability of three repetitive element PCR (Rep-PCR) protocols to distinguish different species and strains of Lactobacillus were compared, with the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) producing profiles with species- and apparent strain-specific patterns. Currently the ERIC- PCR profiles for different strains are being confirmed using pulse field gel electrophoresis (PFGE), which is the gold-standard for Lactobacillus strain identification.

Antibiotic resistant derivatives have been produced through direct plating and successive culturing in increasing concentrations of antibiotic in broth. On average, a mutation rate of 1 in 108-9 has been obtained for the strains examined. Optimisation for high-throughput processing is in progress.

Several transformation protocols are currently being compared, from which an optimised protocol for medium-throughput transformation will be developed.

Project Title Using antimicrobial proteins to control necrotic enteritis in meat chickens

RIRDC Project No.: CSA-32A Start Date: 02/10/2005 Finish Date: 01/10/2007 Researcher: Dr. Robert Moore Organisation: CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220 Phone: (03) 5227 5760 Fax: (03) 5227 5555 Email: [email protected]

Objectives • To develop therapeutic treatments for necrotic enteritis based on the use of antimicrobial proteins such as bacteriocins, delivered to birds by live bacterial vectors.

Current Progress New antimicrobial protein (AMP) expression vectors have been designed for use in the group’s bacterial vector strains. These vectors are designed to individually express three different bacteriocins.

A range of different promoters are being tested to determine the optimal ones for constitutive expression of AMPs. These promoter sequences are predicted to cover a range of different strengths and should allow fine tuning of the performance of the expression vectors. There are some indications to date that very strong promoters may be detrimental to the group’s existing constructs.

Currently, the leading bacterial vector strain is being cycled through chickens and the best performing derivatives are being selected. By doing this, it is anticipated that it will be possible to select for improved colonisation in the target areas of the gut which are most often affected by necrotic enteritis.

The potential to use a different class of AMP to broaden the killing potential of engineered strains is being examined. To date it has been shown that a number of defensin-like peptides have activity against Clostridium perfringens. The work has also shown that C. perfringens isolates that have been selected for resistance to the lead bacteriocin are still sensitive to several of the defensin-like AMPs. This demonstrates the usefulness and desirability of including multiple AMPs in the vector strains.

Project Title Developing novel biological management strategies for darkling beetle (Alphitobius diaperinus [Panzer]) in Australian broiler houses

RIRDC Project No.: DAQ-330A Start Date: 01/09/2005 Finish Date: 31/07/2006 Researcher: Mr Trevor Lambkin Organisation: Department of Primary Industries and Fisheries (Qld) Entomology Building, Indooroopilly Research Centre Locked Mail Bag No 4 MOOROOKA QLD 4105 Phone: (07) 3362 9606 Fax: (07) 3362 9429 Email: [email protected]

Objectives • To develop protocols for laboratory inoculation and assessment of virulence of entomopathogenic fungi (bio-pesticide) for darkling beetle control, including assessment of field isolates. • To undertake preliminary investigations of entomopathogenic nematodes for beetle control. • To determine, in the laboratory, if diatomaceous earths synergise the activity of entomopathogenic fungi.

Progress Now that the pest’s insecticide resistance status, ecology, and population dynamics in Australian broiler houses is better understood, more effective and strategically targeted control practices can be trialed and implemented. This project is investigating safer and more environmentally friendly control strategies for darkling beetles in Australian broiler houses using entomopathogenic fungi and nematodes.

Preliminary laboratory investigations were undertaken to assess the susceptibility and efficacy of entomopathogenic fungi and nematodes to a range of life stages of darkling beetle. In addition, screening of a number of isolates of each biological agent was undertaken to determine the most effective isolates. Some litter applications of fungi with and without diatomaceous earth were undertaken in the laboratory to determine their suitability as litter treatments.

In laboratory litter trials, larval stages of darkling beetle were found to be highly susceptible to two species of fungi, with Beauvearia bassiana having the highest efficacy at very low rates (91% progeny reduction at a litter surface dose of 0.55g/m2). Some increase in efficacy was recorded with the addition of diatomaceous earth. Three species of nematodes have been investigated with Steinernema carpocapsae the most effective in vitro against all stages of darkling beetle. A laboratory method to assess the efficacy of this nematode as a litter or earth floor treatment is now being formulated.

Preliminary results therefore indicate that that enteropathomorphic fungi and nematodes show efficacy in the laboratory in suppressing survival, and the development of progeny, in darkling beetles. Prior to considering the use of these types of biological control agents for control of darkling beetles in the field, a suitable method for applying the agents to litter or earth floors must be identified. This research will also help to determine if the use of these biological agents for suppression of darkling beetles in the field is feasible.

Project Title Broiler performance on pearl millet based diets

RIRDC Project No.: DAQ-337A Start Date: 01/04/2006 Finish Date: 30/05/2007 Researcher: Mr. Danny Singh Organisation: Department of Primary Industries and Fisheries (Qld) Poultry Research and Development Centre PO Box 327 CLEVELAND QLD 4163 Phone: (07) 3820 0515 Fax: (07) 3824 4316 Email: [email protected]

Objectives • To ascertain the effect on broiler performance of a peal millet based diet. • To ascertain the effect on chicken meat quality when pearl millet based diets are fed to birds. • To promote the benefits and advantages of pearl millet as a feed grain to both grain growers and meat chicken industry.

Current Progress Two open pollinated varieties of pearl millet (NPM3 and NPM4) were grown at Biloela Research Station. A second batch of NPM3 was collected from a grain grower in Central Queensland. NPM3 had a higher protein content and lower starch content than NPM4. The fat content of the pearl millet varieties was similar (at 6%) which was twice that of sorghum (3%). The AME content of pearl millet was 1.4MJ/kg higher than sorghum (16.1 compared to 14.6 MJ/kg on an as is basis). The amino acid profile of NPM3 pearl millet was superior to that of NPM4 and sorghum.

A feeding experiment was conducted to compare pearl millet and sorghum based diets. Replacing sorghum with pearl millet had no significant effect on the bodyweight of the broilers compared to the sorghum control when measured at either 21 or 42 days of age, although birds on the sorghum diet had numerically lower bodyweights (P>0.05). Feed intake of birds during the starter and grower periods was not significantly different. However feed intake of birds on sorghum diets over the whole period (0-42 days) were numerically higher than those on pearl millet based diets. Birds on the sorghum diet had the poorest feed conversion efficiencies compared to birds on pearl millet diets in both growth periods.

Project Title Potassium diformate evaluation in necrotic enteritis challenge model

RIRDC Project No.: US-142A Start Date: 01/09/2005 Finish Date: 30/08/2006 Researcher: Dr. Peter Selle Organisation: The University of Sydney Faculty of Veterinary Science 425 Werombi Road CAMDEN NSW 2570 Phone: (02) 9351 1697 Fax: (02) 9351 1693 Email: [email protected]

Objectives • To evaluate the potential of the feed additive, potassium diformate, to control losses due to necrotic enteritis (NE) in broiler chickens caused by Clostridium perfringens.

Current Progress This project was conducted in association with Dr Lene Mikkelsen at the University of New England (UNE).

Potassium diformate (KDF) is a chemical complex, which dissociates into formic acid and potassium formate in the gut. At an inclusion rate of 2.25 g/kg, KDF numerically reduced necrotic enteritis (NE) mortality rates in challenged birds by 31% (20.7 versus 30.0%) and, at 4.5 g/kg KDF, the 58% reduction in NE mortalities (12.7 versus 30.0%) was statistically significant. Thus it would appear that KDF has real potential to control NE losses in broiler chickens.

Product Quality and Safety

Project Title Differential typing of Campylobacter

RIRDC Project No.: DAQ-334A Start Date: 01/09/2005 Finish Date: 31/08/2008 Researcher: Ms. Jillian Templeton Organisation: Department of Primary Industries and Fisheries (Qld) Animal Research Institute Locked Mail Bag No 4 MOOROOKA QLD 4105 Phone: (07) 3362 9520 Fax: (07) 3362 9479 Email: [email protected]

Objectives • To establish the technique of single nucleotide polymorphism (SNP) analysis for typing of C. jejuin/coli. • To extend the current Campylobacter collection to include poultry isolates from other research groups and industry and to obtain non-poultry isolates from sources such as diary cattle, pets and humans. • To establish the SNP types of the extended Campylobacter collection. • To compare and contrast the SNP types of the poultry C. jejuni with the non- poultry types (including human isolates from OzFood that have already been examined by SNP typing by the Queensland node of the CRC for Diagnostics). • To compare and contrast the capacity of SNP typing with pulsed field gel electrophoresis (PFGE) and flaA typing. • To establish an on-going typing technique that will function as a national reference service on a user pays basis.

Current Progress Extension of the current DPI&F Campylobacter culture collection has commenced to obtain poultry isolates from other research groups and to obtain non-poultry isolates from dogs, cats and feedlot cattle.

Isolates have been obtained from a line survey representing several poultry processing plants. Of the 84 isolates obtained, 45 were C. jejuni and 24 were C. coli.

A survey has commenced on healthy dogs and cats where pet owners have voluntarily submitted samples. Of the 59 dogs samples collected 29 were Campylobacter-positive. The majority of samples were from adult dogs. Of the 29 isolates, one C. jejuni isolate from a puppy and 16 C. upsaliensis isolates were obtained. Of the 19 adult cat samples collected, three were Campylobacter-positive. Of the three isolates obtained two were C. upsaliensis. A detailed survey was completed for each animal tested. Information including the diet of the pet was recorded, in particular if the animal had been fed raw chicken in the past week before the sample was collected. A survey of feedlot cattle has commenced. Samples have been collected from three commercial feedlots in south east Queensland on two sampling occasions. 137/223 samples were Campylobacter-positive. Of the 137 isolates PCR identification showed there were 52 C. jejuni, 1 C. coli, 1 C. fetus and 83 C. hyointestinalis.

Project Title Molecular epidemiology of antibiotic resistance in Salmonella from chickens

RIRDC Project No.: IMV-6A Start Date: 01/07/2005 Finish Date: 31/07/2007 Researcher: Dr. Michael Heuzenroeder Organisation: Institute of Medical & Veterinary Science Infectious Diseases Laboratories PO Box 14 Rundle Mall PO ADELAIDE SA 5000 Phone: (08) 8222 3275 Fax: (08) 8222 3543 Email: [email protected]

Objectives • To develop an improved understanding of the epidemiology of antibiotic resistance of Salmonella involved in poultry production by (a) determining the molecular epidemiology of resistance genes in Salmonella in humans and chickens; (b) clarifying the role of bacteriophages in the transfer of antibiotic resistance in Salmonella and other enteric organisms such as Escherichia coli, and (c) determining if antibiotic resistance can be transferred in the chicken gastrointestinal tract by bacteriophages.

Current Progress PCR primers have been designed to detect streptomycin, ampicillin, tetracycline and chloramphenicol resistance genes commonly found in Salmonella isolates. Reproducible PCR and DNA sequencing reactions have been achieved with bla- tem1 gene (ampicillin resistance).

It has been shown that phage lysates from antibiotic resistant Salmonella isolated from chickens and pigs will plaque (multiply) on S. Typhimurium and S. Enteritidis strains that are sensitive to antibiotics from human sources. In addition these phage lysates are capable of plaquing on Shigella flexneri and Shigella sonnei. Furthermore, transduction of ampicillin and streptomycin resistance via bacteriophage lysates to other Salmonella serovars and Shigella species was demonstrated. Attempts to transduce tetracycline resistance with a number of phage lysates was successful at low frequency. It appears that tetracycline resistance may be more readily transferred by conjugation.

Characterisation of induced bacteriophages has begun, with the production of high titre lysates. It is proposed that these will be examined by electron microscopy and eventually genomic DNA will be extracted for simple characterisation by restriction analysis.

In vivo studies have commenced, and although the strains administered to the chickens could be readily recovered during the course of the experiment, transfer of antibiotic resistance could not be demonstrated. As this was a single experiment, it is envisaged that more strains will be tested in the near future.

Project Title The Workboot Series - The story of chicken in Australia

RIRDC Project No.: KDI-30J Start Date: 01/07/2003 Finish Date: 31/11/2006 Researcher: Ms. Kim Field Organisation: Kondinin Group PO Box 913 CLOVERDALE WA 6105 Phone: (08) 9478 3343 Fax: (08) 9478 3353 Email: [email protected]

Objectives • To raise awareness of the nutritional value of chicken meat and the importance of the Australian poultry industry, particularly among children, by gathering accurate and up-to-date information about the Australian chicken meat industry, from producer to consumer, and presenting the information as a high-quality educational book which will be marketed nationally.

Current Progress The Story of Chicken in Australia is currently in the final design phase. Illustrations, diagrams and images are being developed and placed in the document. Some feedback from industry has yet to be forthcoming and feedback from educational specialists is to be incorporated.

Sustainable Production Environment

Project Title Pilot avian influenza surveillance program

RIRDC Project No.: MS056-45 Start Date: 01/03/2006 Finish Date: 30/06/2007 Researcher: Dr. Vivien Kite Organisation: Rural Industries Research and Development Corporation Chicken Meat Program PO Box 579 NORTH SYDNEY NSW 2059 Phone: (02) 9929 4077 Fax: (02) 9925 0627 Email: [email protected]

Objectives • To develop and refine strategies and protocols for a possible future ongoing active surveillance program for AI in the Australian chicken meat industry which would be compliant with OIE guidelines. • To generate data to confirm the chicken meat industry’s negative status with respect to notifiable AI. • To test and confirm the adequacy of and allow confidence in the industry’s biosecurity programs and existing AI surveillance activities.

Current Progress Blood samples have been collected from over 60 meat chicken farms, representative of all regions of Australia and all significant companies operating in each region, for testing for antibodies to H5 and H7 avian influenza viruses. Meat chicken flocks were sampled at last pick-up and samples were collected at the processing plant. Flocks selected for inclusion in the survey were those considered to be at a higher risk of exposure to avian influenza virus

Blood samples have also been collected from in excess of 50 meat breeder flocks from across Australia. Flocks sampled were older flocks (generally 40 weeks and over) to maximize the opportunity that they have had to have been previously exposed to an avian influenza virus.

Serological testing of samples collected as part of this survey will be performed by the Australian Animal Health Laboratory. Serological testing will be by haemagglutination inhibition (HI) test for antibodies to H5 and H7 avian influenza viruses.

OTHER SUPPORTED ACTIVITIES

SCHOLARSHIPS

CSA-25J Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus infection UTS-6A Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the apicomplexan parasite, Eimeria maxima UNE-86A Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal integrity, and performance of broiler chickens ANU- 72A Postgraduate scholarship - David Stephenson: Delivery of therapeutic proteins using Lactobacillus

PROGRAM REVIEW AND DEVELOPMENT

MS023-28 Steering committee meetings with Marek's disease researchers MS045-01 Environmental management steering committee activities and workshops MS034-02 Campylobacter research groups co-ordination MS056-53 RIRDC/Poultry CRC necrotic enteritis workshop

COLLABORATIVE PROGRAMS

CPO-1A CRC for the Australian Poultry Industries funding GRD-3A Premium grains for livestock program (stage 2) CAB-1J Implementation and validation of an avian influenza virus TaqMan assays on various instrument platforms in different state veterinary diagnostic laboratories

TRAVEL/CONFERENCE/WORKSHOPS

TA045-17 British WPSA 28th Poultry Science Symposium, Bristol, September 2005 - Mr Scott Sheedy TA056-01 13th International Workshop on Campylobacter, Helicobacter and Related Organisms 2005 (CHRO 2005), Gold Coast, September 2005 - support for 4 international speakers TA056-10 Support for 13th Australian Poultry Convention, Gold Coast, October 2005 TA056-11 2006 Australian Poultry Science Symposium, Sydney, February 2006 - Dr Robert Renema TA056-25 Poultry Information Exchange (PIX) 2006, Gold Coast, April 2006 - various speakers TA056-31 XII European Poultry Conference, Italy, 7-17 September 2006 - Dr Robert Pym TA056-32 Poultry Science Association 2006 Annual Meeting, Edmonton, Canada, 16-26 July 2006 – Professor Tom Scott MS056-33 2005 Poultry CRC Postgraduate Workshop

RESEARCH TO BE SUPPORTED IN 2006/2007

Flock Health Project Title Project No Principal Investigator Organisation Phone No Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates CSA-24J Dr Hans Heine CSIRO Livestock Industries (03) 5227 5278 Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics CSA-30J Dr Sandra Sapats CSIRO Livestock Industries (03) 5227 5769 New diagnostic assays to improve control of coccidiosis in poultry DAQ-316A Dr John Molloy Department of Primary Industries and (07) 3362 9494 Fisheries (Qld) Molecular evaluation of responses to vaccination and challenge by Marek's disease virus RMI-12J Prof Greg Tannock Royal Melbourne Institute of (03) 9925 3088 Technology Molecular techniques for monitoring Marek's viraemias in broilers and layers UJC-10J Dr Graham Burgess James Cook University (07) 4781 5472

Bird Nutrition and Feed Supply Project Title Project No Principal Investigator Organisation Phone No Nutritional characterisation of sorghums from QLD and NSW for chicken meat DAQ-326A Dr Rider Perez-Maldonado Department of Primary Industries and (07) 3824 3081 57 Fisheries (Qld) Early feeding of prebiotics on development of the digestive system and gut microflora of UNE-93A Dr Paul Iji University of New England (02) 6773 2347 broilers Digestible amino acids and improved broiler performance UQ-107A Prof Wayne Bryden The University of Queensland (07) 5460 1253 Establishment of a Chair in Poultry Sciences at The University of Sydney US-127A Dr Tom Scott The University of Sydney (02) 4655 0612 Assessment of the anti-nutritive effects of phytate by dephytinisation US-133A Dr Peter Selle The University of Sydney (02) 9351 1697 Early dietary and management intervention on broiler breast meat yield US-134A Prof Tom Scott The University of Sydney (02) 4655 0612 Variability in performance and physiology of broilers fed wheat- and sorghum-based diets US-137A Prof Tom Scott The University of Sydney (02) 4655 0612

Food Safety Project Title Project No Principal Investigator Organisation Phone No Development of a sequence-based bacteriophage typing system for Salmonella IMV-5A Dr Michael Heuzenroeder Institute of Medical & Veterinary (08) 8222 3275 Science Development and validation of Campylobacter microarrays for virulence detection and strain RMI-14A Prof Peter J Coloe Royal Melbourne Institute of (03) 9925 7104 differentiation in poultry products Technology Campylobacter bio-replacement program to control food poisoning organisms in poultry UG-7A Dr Victoria Korolik Griffith University (07) 5552 8321

RESEARCH TO BE SUPPORTED IN 2006/2007

Environmental Management Project Title Project No Principal Investigator Organisation Phone No Evaluating risks posed by pathogen emissions from meat chicken sheds DAQ-318A Dr Pat Blackall Department of Primary Industries and (07) 3362 9498 Fisheries (Qld) Efficacy of windbreak walls for odour reduction DAQ-321A Mr Mark Dunlop Department of Primary Industries and (07) 4688 1280 Fisheries (Qld) Managing litter re-use for minimal nutrient runoff to surface water DAQ-323A Mr Jarl Devereux Department of Primary Industries and (07) 4688 1096 Fisheries (Qld) Trials of odour control technologies for broiler farms DAV-213A Dr Julie Simons Dept of Primary Industries (Vic) (03) 9217 4358

Efficiency of Production Project Title Project No Principal Investigator Organisation Phone No Postgraduate scholarship - David Stephenson: Delivery of therapeutic proteins using ANU-72A Mr David Stephenson Australian National University (02) 6258 9843 lactobacillus Using antimicrobial proteins to control necrotic enteritis in meat chickens CSA-32A Dr Robert Moore CSIRO Livestock Industries (03) 5227 5760 Improved control measures for infectious bursal disease virus (IBDV) CSA-33J Dr Sandra Sapats CSIRO Livestock Industries (03) 5227 5770 Developing novel biological management strategies for darkling beetle (Alphitobius diaperinus DAQ-330A Mr Trevor Lambkin Department of Primary Industries and (07) 3362 9606 [Panzer]) in Australian broiler houses Fisheries (Qld)

58 Broiler performance on pearl millet based diets DAQ-337A Mr Danny Singh Department of Primary Industries and (07) 3820 0515 Fisheries (Qld) Further investigations into miliary hepatitis of laying hens DAV-232A Dr W Mike Forsyth Department of Primary Industries (03) 9217 4200 (Vic) Investigation of IBH outbreaks in Australian meat breeder/broiler flocks and establishment of UM-73A Dr Amir H Noormohammadi The University of Melbourne ((03) 9731 2229 an avian adenovirus typing facility and service Potassium diformate evaluation in necrotic enteritis challenge model US-142A Dr Peter Selle The University of Sydney (02) 9351 1697 Improvement of lupins and lathyrus for broilers and egg layers by enzyme treatment Dr Ian Williams The University of Western Australia (08) 6488 3780

Product Quality and Safety Project Title Project No Principal Investigator Organisation Phone No Differential typing of Campylobacter DAQ-334A Ms Jillian Templeton Department of Primary Industries and (07) 3362 9520 Fisheries (Qld) Molecular epidemiology of antibiotic resistance in Salmonella from chickens IMV-6A Dr Michael Heuzenroeder Institute of Medical & Veterinary (08) 8222 3275 Science Quality aspects of antimicrobial interventions used in chicken processing DAV-234A Ms Heather Haines Department of Primary Industries (03) 9217 4332 (Vic) Expansion and refinement of a molecular typing system for Salmonella IMV-7A Dr Michael Heuzenroeder Institute of Medical & Veterinary (08) 8222 3275 Science The Workboot Series - The story of chicken in Australia KDI-30J Ms Kim Field Kondinin Group (08) 9478 3343 Investigation of the prevalence of chlamydiosis in the Australian chicken meat industry UM-74A Dr Amir H Noormohammadi The University of Melbourne (03) 9731 2229 Use of bacteriophage and phage products to control campylobacter in chickens USA-16A Prof Mary Barton The University of South Australia (08) 8302 2933

RESEARCH TO BE SUPPORTED IN 2006/2007

Sustainable Production Environment Project Title Project No Principal Investigator Organisation Phone No Monitoring mechanical ventilation rates in poultry buildings for application of odour/dust control DAQ-340A Mr Mark Dunlop Department of Primary Industries and (07) 4688 1280 technologies Fisheries (Qld) Evaluation of "add-on" technologies for control of odour and dust from chicken sheds DAQ-341A Mr Mark Dunlop Department of Primary Industries and (07) 4688 1280 Fisheries (Qld) Pilot avian influenza surveillance program MS056-45 Dr Vivien Kite Rural Industries Research and (02) 9929 4077 Development Corporation Field exercise – use of carbon dioxide for euthanasia of poultry AHA- 2A Dr Karin Ahrling Animal Health Australia (02) 6203 3912 Other Project Title Project No Principal Investigator Organisation Phone No CRC for the Australian Poultry Industries funding CPO-1A Prof Mingan Choct CRC for the Australian Poultry (02) 6773 5121 Industries

RESEARCH TO BE SUPPORTED IN 2006/2007