Chicken Meat Program - Agrifutures Australia · UM-45J Determination of the genomic sequence of...

2002-2003 Research Report

Chicken Meat Program

September 2003 RIRDC Publication No 03/068

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© 2003 Rural Industries Research and Development Corporation All rights reserved.

ISBN 0 642 58469 9 ISSN 1440-6845

“2002-2003 Research Report RIRDC Chicken Meat Program” Publication No 03/068 The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. 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 1, AMA House 42 Macquarie Street 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 September 2003 Printed on environmentally friendly paper by Union Offset, Canberra

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Foreword This year RIRDC has produced Research in Progress, June 2003, which contains short summaries of continuing projects as well as those that were completed during 2002-2003 for all of the Corporation’s 20 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 Sub-Program 3.1. It contains all entries from continuing and completed Chicken Meat research projects funded by RIRDC in 2002-2003. Additional information on other activities funded by the program in 2002-2003 and projects and activities to be funded in 2003-2004 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 900 research report, 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/reports/Index.htm • purchases at www.rirdc.gov.au/eshop Simon Hearn Managing Director Rural Industries Research and Development Corporation

http://www.rirdc.gov.au/

http://www.rirdc.gov.au/reports/Index.htm

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CHICKEN MEAT PROGRAM ADVISORY COMMITTEE

Chairperson: Mr Barry Shay Food Safety Consultant 27 Uther Street CARINDALE QLD 4152 Ph: (07) 3398 1766 Fax: (07) 3398 6631 Email: [email protected]

Research Manager: Dr Vivien Kite RIRDC Chicken Meat Program PO Box 579 NORTH SYDNEY NSW 2059 Ph: (02) 9929 4077 Fax: (02) 9929 0627 Email: [email protected]

Committee Members: Mr Ian Farran Agribiz Engineering PO Box 279 GEELONG VIC 3220 Ph: (03) 5229 7300 Fax: (03) 5229 7566 Email: [email protected]

Dr Tom Grimes Grimes Consultancy Pty Ltd 4 Henry Street LEWISHAM NSW 2049 Ph: (02) 9569 7436 Fax: (02) 9569 4183 Email: [email protected]

Dr Ron MacAlpine Inghams Enterprises Pty Ltd PO Box 4 LIVERPOOL NSW 2170 Ph: (02) 9606 5666 Fax: (02) 9606 6640 Email: [email protected]

Dr Margaret MacKenzie Inghams Enterprises Pty Ltd PO Box 1100 BROWNS PLAINS QLD 4118 Ph: (07) 3297 0222 Fax: (07) 3297 0578 Email: [email protected]

Dr Pat Blackall Animal Research Institute Department of Primary Industries Locked Mail Bag No 4 MOOROOKA QLD 4015 Ph: (07) 3362 9498 Fax: (07) 3362 9429 E-mail: [email protected]

Mr Gary Sansom 82 Hawkins Road JIMBOOMBA QLD 4280 Ph: (07) 5546 9235 Fax: (07) 5546 0070 E-mail: [email protected]

Dr Jeff Davis RIRDC PO Box 4776 KINGSTON ACT 2604 Ph: (02) 6272 4152 Fax: (02) 6272 5877 Email: [email protected]

mailto:[email protected]





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INDEX TO PROJECT SUMMARIES

COMPLETED PROJECTS

Flock Health CSA-16A Evaluation of fowl pox strains free of reticuloendotheliosis virus as vaccines for use

in Australian poultry flocks.................................................................................................1 CSA-27A Characterisation of IBV from broilers ................................................................................3 UM-45J Determination of the genomic sequence of Mycoplasma gallisepticum ...........................4 DAQ-259J Attenuation and characterisation of chicken Eimeria for live vaccines .............................5 DAQ-273A Investigations into the development of a sustainable management strategy for the

darkling beetle, Alphitobius diaperinus (Panzer) in Australian broiler houses ..................7 CSA-10J Postgraduate scholarship – Ms Louise Hilton: Therapeutic applications of cytokines

in poultry ............................................................................................................................9 CSA-11J Molecular epidemiology of Newcastle disease in Australia ............................................11 CSA-15J Diagnostic tools for differentiation of vvIBDV and characterisation of local IBDV

strains ..............................................................................................................................13 CSA-12A Biological control of necrotic enteritis in meat chickens..................................................15

Bird Nutrition and Feed Supply DAQ-264J Canola meal and cottonseed meal in broiler and layer diets ..........................................16

Food Safety IMV-3A Salmonella typing and colonisation of chickens by characterised Salmonella Sofia ......19 UG-3A Development of campylobacter bio-replacement program and establishment of

campylobacter reference laboratory................................................................................21 UG-4A Isolation of genes responsible for Campylobacter jejuni colonisation of the chicken

intestinal tract ..................................................................................................................23 Environmental Management

SAR-33J Reduction of dust emissions from broiler and caged layer sheds...................................25

RESEARCH IN PROGRESS

Flock Health CSA-24J Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates...................27 CSA-18J The effect of Newcastle disease vaccination with strain V4 on the course of

infections with the Peats Ridge strain of Newcastle disease virus .................................28 CSA-13J Postgraduate scholarship - Jacqueline Kattenbelt: Analysis of virulence determinants

of Newcastle disease virus..............................................................................................29 UM-49A Avian leukosis-J (ALV-J) in Australia: laboratory technologies and research needs......30 UQ-100J Typing of Pasteurella multocida ......................................................................................31 UMU-29J Control of intestinal spirochaete infections in chickens...................................................32 UTS-4J Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis .........33 UTS-6A Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall

formation in the apicomplexan parasite, Eimeria maxima ..............................................34 UNE-83J Systematic pathotyping of Australian Marek's disease (MDV) isolates ..........................35 UJC-10J Molecular techniques for monitoring Marek's viraemias in broilers and layers...............36 RMI-12J Molecular evaluation of responses to vaccination and challenge by Marek's disease

viruses .............................................................................................................................37 CSA-26J Use of cytokines to enhance vaccine efficacy in poultry .................................................38 CSA-21J Postgraduate scholarship - Manija Asif: Cytokines and innate molecules for

enhanced mucosal immunity in the chicken....................................................................39 CSA-25J Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's

disease virus infection .....................................................................................................40 CSA-20A Postgraduate scholarship - Scott Sheedy: Live vectoring of therapeutic and

prophylactic proteins and pathogenesis in necrotic enteritis...........................................41

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RMI-11A The development of vaccination strategies to control necrotic enteritis in poultry..........42 UNE-75A Effects of organic acids, prebiotics, and enzymes on control of necrotic enteritis and

performance of broiler chickens ......................................................................................43 Bird Nutrition and Feed Supply

DAQ-302A Evaluation of new millet varieties as a poultry feed ingredient .......................................44 UWA-76J Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets 45 UNE-82J The net energy values of the Australian feed ingredients for poultry..............................46 DAQ-277A Estimating lysine availability by slope-ratio chick assay .................................................47 UNE-86A Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain

quality, intestinal integrity and performance of broiler chickens......................................48 US-104A Use of dietary fatty acids to increase protein accretion in broilers..................................49 UQ-107A Digestible amino acids and improved broiler performance .............................................50 GRD-3J Premium grains for livestock program (stage 2) .............................................................51

Food Safety RMI-14A Development and validation of Campylobacter microarrays for virulence detection

and strain differentiation in poultry products ...................................................................52 DAQ-282A On-farm reduction strategies for Campylobacter spp. ....................................................53 UA-61A Tannins to control microbial pathogen colonisation of broiler chickens..........................54

Environmental Management FSE-2A Meat chicken EMS: transfer to industry ..........................................................................55 JSC-2A Sustainability improvements in the Victorian chicken meat industry (Phase 2)..............56

OTHER SUPPORTED ACTIVITIES

SCHOLARSHIPS ......................................................................................................58

PROGRAM REVIEW AND DEVELOPMENT............................................................58

TRAVEL/CONFERENCE/WORKSHOPS .................................................................58

CHICKEN MEAT PROGRAM – COMPLETED PROJECTS

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COMPLETED PROJECTS

Flock Health Project Title:

Evaluation of fowl pox strains free of reticuloendotheliosis virus as vaccines for use in Australian poultry flocks

RIRDC Project No.:

CSA-16A

Researcher: Dr. David Boyle Organisation: CSIRO Livestock Industries

Private Bag 24 GEELONG VIC 3220

Phone: (03) 5227 5018 Fax: (03) 5227 5555 Email: [email protected] Objectives

• To undertake vaccine efficacy, safety and adventitious agent testing on

reticuloendotheliosis free fowl pox virus strains derived from Australian fowl pox isolates.

Background The only fowl pox vaccine stain currently available for use in Australia has proven

ineffective in controlling fowl pox disease in some circumstances. Although suitable for day old vaccination, this strain has not proven effective for revaccination of broiler breeder flocks coming into production. Earlier studies undertaken by this research group have shown that the previously available FPV S vaccine is contaminated with REV, with the REV provirus being integrated into the FPV genome. RIRDC previously funded a small project to remove the REV provirus from the FPV S vaccine and two field strains. In that project, this CSIRO research group successfully removed the integrated REV genome from FPV S vaccine and from two field strains, FPV 59vac and 62vac. Preliminary in vivo testing of these strains has shown them to be free of REV contamination. The current project was aimed at undertaking vaccine efficacy, safety and adventitious agent testing prior to the conduct of a field trial for the registration of one of these strains for use in Australian poultry flocks.

Research A vaccination and challenge experiment was conducted with the FPV strains in chickens to select a strain for further development. Intervet Pty. Ltd., the commercial partner in this project, prepared master and working seeds and pilot vaccine lots of this strain. Safety and potency tests and CIT (adventitious agent tests) as per “European Pharmacopoeia, Fourth Edition September 2001” were conducted on master and working seeds and pilot vaccine lots of the candidate fowl pox vaccine.

Outcomes The FPV vaccine based on FPV 85a (a REV free derivative of field strain FPV 62vac) was shown to meet the “European Pharmacopoeia, Fourth Edition, September 2001” requirements for safety, potency and freedom from adventitious poultry pathogens (CIT test).

Implications The FPV strain that has been developed will need to be evaluated in field trials to be conducted by Intervet Pty. Ltd. to ascertain if it is suitable for use by the Australian poultry industry for the control of fowl pox disease. Subject to the suitability of the candidate vaccine for use in control of fowl pox in Australia being demonstrated in


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these field trials and the vaccine subsequently being registered for use for this purpose, then the Australian poultry industries will have available an alternative, more immunogenic vaccine for the control of fowl pox in the future.

Publications Hertig, C.H., Coupar, B.E.H., Gould, A.R. and Boyle, D.B. (1997) Field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus. Virology 235: 367-376.


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Project Title:

Characterisation of IBV from broilers

RIRDC Project No.:

CSA-27A

Researcher: Dr. Jagoda Ignjatovic, Dr.Sandra Sapats and Ms. Gaylene Gould Organisation: CSIRO Livestock Industries



• To attempt the isolation of infectious bronchitis virus (IBV) from samples

collected from broilers with suspected IBV involvement. • To sequence the S1 glycoprotein of any isolated IBVs.

Background Respiratory disease with elevated mortality was observed on a number of broiler farms during a three month period from December 2002 to February 2003 in the state of New South Wales. Infectious bronchitis virus was suspected to be the cause.

Research Samples were obtained from 23 broiler farms in three states. Virus isolation was attempted from all farms by passaging field samples in embryonated specific pathogen free chicken eggs. Virus isolation was confirmed using monoclonal antibodies in an antigen ELISA. Sequencing of the S1 gene of isolated IBVs was then undertaken.

Outcomes IBV was isolated from 12/23 broiler farms in NSW that were suspected of having respiratory problems due to IBV. In the majority of cases, IBV was the only agent present in the sample, although Newcastle disease virus was also detected in 2/11 IBV-positive samples. In all the NSW cases the same genetic type of IBV virus was involved. Sequencing of the protective S1 antigen of this virus and of four IBV vaccines used on these farms indicated that the NSW isolate is a new type of IBV, not seen previously. Additionally, this virus differs significantly at a genetic level from all vaccine viruses available. This suggests that the currently available IBV vaccines would not control infections caused by this virus.

Implications Although the sequencing data strongly suggest that a new vaccine would be needed for control of this virus, only cross-protection studies would establish this for certain.


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Project Title:

Determination of the genomic sequence of Mycoplasma gallisepticum

RIRDC Project No.:

UM-45J

Researcher: A/Prof. Glenn Browning and Dr. Philip Markham Organisation: The University of Melbourne

School of Veterinary Science Cnr Park Drive and Flemington Road PARKVILLE VIC 3052


• To lay a foundation for subsequent studies to improve the performance of

Mycoplasma vaccines and to improve diagnosis of mycoplasmosis by determining the complete genomic sequence of Mycoplasma gallisepticum.

Background Although mycoplasmosis has been controlled with the current generation of vaccines,

future control programs may need to focus on improvements in these vaccines so that they are compatible with eradication programs or so that they are effective in different strains of bird. In the last few years a number of human pathogens have been fully sequenced, including four human mycoplasmas, and parallel information for avian mycoplasmas would enable rapid application of findings on important human mycoplasma genes to improving avian mycoplasma vaccines.

Research The complete sequence of the genome of the virulent R strain of M. gallisepticum was determined. The genome was found to be 996,422 base pairs and was predicted to contain 742 genes encoding proteins. Some function could be assigned to 469 of these. A further 150 predicted genes are similar to genes in other bacterial species and 123 are thus far unique to Mycoplasma gallisepticum. A total of 80 genes are predicted to encode lipoproteins which are likely to be involved intimately in interactions with the host and a number of other putative virulence factors have been identified.

Outcomes These data lay a strong basis for ongoing studies on the basis of virulence in M. gallispticum. Once the work has been published the data will be publicly available at http://cevr.uconn.edu/.

Implications The capacity to enhance protection against mycoplasmosis in poultry will be enhanced by the use of these data to identify genes involved in virulence and thus which may be suitable as targets for developing novel attenuated vaccines.

Publications Papazisi, L., Gorton, T.S., Kutish, G., Markham, P.F., Browning, G.F., Nguyen, K.D., Swartzell, S., Madan, A., Mahairas, G. and Geary, S.J. (2003) The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow. Microbiology (in press).


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Project Title:

Attenuation and characterisation of chicken Eimeria for live vaccines

Project No:

DAQ-259J

Researcher: Dr. Wayne Jorgensen and Dr. Glenn Anderson Organisation: Department of Primary Industries (Qld)

Animal Research Institute Locked Mail Bag No 4 MOOROOKA QLD 4105


• To develop attenuated lines of E. mitis, E. brunetti and E. praecox for

incorporation in an efficacious, live vaccine protective against all seven species of Eimeria in Australian chickens.

• To develop a trial technique to evaluate coccidiostat resistance.

Background Coccidiosis is one of the more economically important disease problems in Australia’s intensive poultry industries. Control of the disease is based on the use of chemicals and drugs that cost the poultry industries more than $10 million pa. Vaccination, used alone or in combination with coccidiostats, is currently the most sustainable avenue for control of coccidiosis. Live vaccines have been available for a number of years in some countries, but until this vaccine development work was initiated, similar vaccines had not been available to the Australian industry. The benefits of using live coccidiosis vaccines include long term, economical protection against disease, ability to manage existing and developing chemical resistance, and provision of an alternative to chemical control to minimise residue and withholding period problems. Seven species of Eimeria can cause coccidiosis in poultry. The previous two stages of this series of projects have been successfully completed with the development and evaluation of vaccine strains of E. maxima, E. acervulina, E. tenella and E. necatrix suitable for use in a live poultry coccidiosis vaccine. Vaccine strains of the remaining three species of Eimeria that can cause coccidiosis in poultry (E. mitis, E. brunetti and E. praecox) therefore remained to be developed to provide comprehensive cover against coccidiosis.

Research Vaccine candidate strains of E. mitis, E. brunetti and E. praecox were isolated from relatively small, non-commercial flocks that were not regularly exposed to coccidiostats. The strains were purified by single oocyst passage and demonstrated, in trials, to be susceptible to at least two of the following coccidiostats at doses recommended by the manufacturer - Sulphaquinoxaline, Toltrazuril or Amprolium. The Jorgensen strain of E. mitis was passaged for precocious development. The Bowden strain of E. brunetti and Jorgensen strain of E. praecox were not modified for precocious development because low pathogenicity was demonstrated in trials with the parent strains. Each of the three vaccine strains was demonstrated to provide protection against challenge with two virulent field isolates. A trial format for evaluating resistance to the in-feed coccidiostats narasin and monensin was developed and evaluated using Eimeriavax 4M (vaccine) and three field isolates. The vaccine is susceptible to both coccidiostats based on decreased oocyst output in treated groups. One field isolate, the Kelly strain of E. praecox, was not susceptible to either coccidiostat. The trial design appears suitable for detecting resistance to prophylactic coccidiostats.

Outcomes This project has developed vaccine strains of E. mitis, E. praecox and E. brunetti and evaluated them for coccidiostat susceptibility, pathogenicity, and protection against virulent field isolates. In all cases the strains were demonstrated to be suitable for use


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as vaccine strains and have been transferred to commercial partners, Eimeria Pty Ltd, for incorporation into new custom vaccines. A trial design to test resistance to in-feed (prophylactic) coccidiostats has been evaluated and demonstrated resistance in field isolates. The trial format can be used by industry to monitor developing coccidiostat resistance.

Implications The final outcome of the three stages of the project series is the availability, to the Australian poultry industry, of live precocious vaccines against the seven species of Eimeria that cause poultry coccidiosis. Vaccination is now being used routinely to protect flocks in the USA and some European countries including Britain, and the availability of this new vaccine product here in Australia will afford the Australian industry similar alternative control possibilities.

Publications Anderson G.R. and Jorgensen W.K. (2001) Development of Australian live vaccines against coccidiosis: characterisation of attenuated lines using standardised trials. Proceedings of the VIIIth International Coccidiosis Conference pp. 70.

Anderson, G.R. and Jorgensen, W.K. (2002) A comparison of virulent and precocious coccidiosis vaccines. Proceedingsof the 7th WPSA Asia Pacific Federation Conference and 12th Australian Poultry Convention pp. 103-105.

Anderson, G.R., Jeston, P.J., Blight G.W. and Jorgensen, W.K. (2003) Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia. Australian Veterinary Journal (submitted).

Jeston, P.J, Blight, G.W., Anderson, G.R., Molloy, G.B. and Jorgensen, W.K. (2001) Comparison of infectivity of Eimeria tenella oocysts maintained at 4, 12 or 28°C for up to 10 months. Australian Veterinary Journal 80: 74-75.

Jorgensen, W.K. and Anderson, G.R. (2001) Development of Australian live vaccines against coccidiosis: selection, isolation and attenuation. Proceedings of the VIIIth International Coccidiosis Conference pp. 101-102.

Lew, A.E, Anderson, G.R, Minchin, C.M, Jeston, P.J. and Jorgensen, W.K. (2003) Inter- and intra-strain variation in the internal transcribed spacer 1 (ITS-1) sequences of Australian species and isolates of Eimeria from chickens. Veterinary Parasitology 112: 33-50.


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Project Title:

Investigations into the development of a sustainable management strategy for the darkling beetle, Alphitobius diaperinus (Panzer) in Australian broiler houses

RIRDC Project No.:

DAQ-273A

Researcher: Mr. Trevor Lambkin Organisation: Department of Primary Industries (Qld)

Entomology Building Indooroopilly Research Centre 80 Meiers Road INDOOROOPILLY QLD 4068


• To investigate sustainable management practices for the darkling beetle,

Alphitobius diaperinus (Panzer), in broiler houses; these practices to be aimed at reducing pest numbers, identifying current inefficiencies in insecticide applications, developing novel control strategies, and providing sustainable management of current registered insecticides and a better understanding of pest population dynamics.

• To deliver an overall reduction in associated poultry disease and insecticide use within the broiler industry.

Background A long history of consistent applications of insecticides has proved inadequate for the

long-term control of A. diaperinus in Australian broiler sheds. Earlier research has shown that resistance to the insecticide fenitrothion is widespread in eastern Australian beetle populations and, moreover, this research has indicated that the size of pest populations in broiler houses often has no positive correlation to insecticide resistance. Therefore, in 2000 this three year research project was initiated to determine and quantify insecticide resistance in farm populations of A. diaperinus, to study the bionomics of the pest, to determine the field efficacy of another insecticide, cyfluthrin, and to investigate potential alternative control strategies.

Research Insecticide resistance to cyfluthrin in farm populations of A. diaperinus was quantified and its efficacy in controlling beetle populations investigated. The bionomics of the pest, including its spatial and temporal distributions in a range of broiler house types, was studied. Diatomaceous earth was investigated in the laboratory as a potential non-chemical litter treatment. A field trial was completed to determine and compare the efficacy of floor applications of cyfluthrin and Spinosad® on beetle numbers in the litter and floor. A study tour to the USA was undertaken to hold discussions with international scientists and observe A. diaperinus management strategies in broiler houses.

Outcomes Insecticide resistance to cyfluthrin in farm populations of A. diaperinus was found to have developed over the last three years. Results showed that the majority of beetles in broiler houses occur under feed pans in litter and that conventional earth floor houses have the highest numbers of beetles and concrete floor houses the least. Cyfluthrin was found to have little efficacy in controlling A. diaperinus in the litter and in earth floors of broiler houses. Spinosad® and diatomaceous earth appeared to have some potential for beetle control. Management strategies used for control of beetles in the USA were documented and anecdotal evidence suggested that they are also inadequate. The results from all investigations undertaken corroborate well, and have identified that floor applications of insecticides in broiler houses do little to control A. diaperinus in the earth floors of the houses at the beginning of each batch, and


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A diaperinus in the litter during the subsequent period of the batch. This lack of control has been further exacerbated by the marked increase in resistance to cyfluthrin (the industry standard insecticide) that has been demonstrated to have occurred over the past three years in some populations of A. diaperinus from southeast Queensland.

Implications The results of this project indicate that a managed strategy for control of this pest is needed. A suitable strategy for control of A. diaperinus in earth floor broiler houses could be based on disinfestation of the earth floor prior to placement of litter and the subsequent treatment of poultry litter with a range of effective insecticide and non-insecticide litter treatments. This strategy could possibly also require the application of these treatments only to litter under feed pans.

Publications Lambkin, T.A. and Cameron, M.C. (2000) Darkling beetle control in Australian broilers-A new direction. Proceedings of the 2000 Poultry Industry Exchange pp. 97-102.

Lambkin, T.A. and Cupitt, D.A. (2002) Darkling beetle in Australian meat chickens-a better understanding. Proceedings of the 2002 Poultry Industry Exchange pp. 93-99.

Lambkin, T.A. and Cupitt, D.A. (2002) Darkling beetle [Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae)] control in Australian broilers-enhancing food safety. Proceedings of the Fifty-First Western Poultry Disease Conference pp. 32-36.


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Project Title:

Postgraduate scholarship – Ms Louise Hilton: Therapeutic applications of cytokines in poultry

RIRDC Project No.:

CSA-10J

Researcher: Dr. John Lowenthal and Ms. Louise Hilton Organisation: CSIRO Livestock Industries



• To enhance disease resistance and vaccine efficacy in poultry by administration

of cytokine therapy.

Background The Australian poultry industries are reducing their usage of antibiotics and other chemicals to control disease. To facilitate this process, safe natural alternatives are being developed. The search for alternative strategies has focused on the potential use of natural therapeutics. Cytokines are proteins that are normally produced by the body’s immune system following infection. Cytokines protect against disease by controlling the immune response to infection or vaccination and therefore represent excellent, naturally occurring therapeutics. One such cytokine, interleukin-2 (IL-2), has been shown in mammals to play a major role in the induction of the immune response and IL-2 has been studied widely as an immuno-therapeutic due to its pleiotropic properties and pivotal role in initiating T cell proliferation. The aim of this study was to characterise the biological functions of chicken IL-2 (ChIL-2) as a prelude to its assessment as a vaccine adjuvant or therapeutic agent.

Research The focus of this project was to assess the potential of ChIL-2 as a therapeutic. To examine the immunoenhancing capability of ChIL-2, recombinant protein was expressed in several prokaryotic and eukaryotic expression systems and its biological activity confirmed. Antibodies and enzyme-linked immunoassays specific for ChIL-2 were developed to characterise its in vivo function, in particular its effects on modulating T cell populations and enhancing T cell activation following its administration to chickens. The effect of ChIL-2 on growth promotion was also measured.

Outcomes Treatment of birds with ChIL-2 protein resulted in both the activation (measured by expression of cell surface IL-2 receptors) and proliferation (as measured by DNA synthesis) of T cells. Injection of recombinant ChIL-2 protein increased the proportion of CD4+ cells, CD8+ T cells and the proportion of T cells expressing the ChIL-2 receptor in peripheral blood within 48 h. The increase in T cell subsets was shown to be a result of cell proliferation. These results suggest that treatment with ChIL-2 is effective in modulating the immune response and may thereby enhance immunity and protection against a variety of viral and parasitic diseases. An initial trial showed that treatment of broilers with ChIL-2 during the first week post-hatch resulted in enhanced weight gain over a six week period of growth under commercial conditions. ChIL-2 treatment increased the body weight of chickens (~4%) and also increased the efficiency of feed utilization.

Implications The implications of this work for the Australian poultry industry are significant. The introduction of cytokines as therapeutics may in part compensate for performance losses associated with the reduction in the use of antimicrobial growth promotants and antibiotics for disease control. Cytokines offer a two-pronged attack. They can strengthen and control immune responses resulting in an increase in the general health of animals. Secondly, they can improve the efficacy of vaccines, resulting in


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effective long term protection against specific pathogens. Having established this technology and models, there is now an opportunity to assess other types of cytokines that have different types of activities, for their therapeutic and immuno-enhancing capability in poultry.

Publications Hilton, L.S., Bean, A.G.D., Kimpton, W.G. and Lowenthal, J.W. (2002) Interleukin-2 directly induces activation and proliferation of chicken T cells in vivo. Journal of Interferon and Cytokine Research 22: 755-763.

Hilton, L.S., Bean, A.G.D. and Lowenthal, J.W. (2002) The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants. Veterinary Immunology and Immunopathology 85: 119-128.

Jones, D.C. and Black, D.B. (1999) Poultry vaccines currently available in Australia. Proceedings of the 11th Australian Poultry Science Symposium 11: 222-225.

Smith, A.B., Jones, D.C., Brown, E. and Black, D.B. (1997) Viruses in the meat chicken industry in south eastern Australia. Virology, 234: 667-676.


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Project Title:

Molecular epidemiology of Newcastle disease in Australia

RIRDC Project No.: CSA-11J Researcher: Dr. Allan Gould, Dr. Matthew Stevens, Mr. Paul Selleck and

Ms. Jacqueline Kattenbelt Organisation: CSIRO Livestock Industries



• To characterise Australian Newcastle disease viruses (NDVs) at the gene

sequence level in order to understand the types of viruses present and their potential to cause a virulent Newcastle disease outbreak.

Background Very little was understood as to the range and inter-relationships of Newcastle

disease viruses (NDVs) in Australia at the molecular level prior to the commencement of this research. It was assumed that only avirulent NDVs were present in Australia and that these viruses did not cause disease in poultry. The emergence of virulent NDV in NSW in 1998 demonstrated this lack of knowledge and the causal reasons for the initial and subsequent outbreaks of disease.

Research Phylogenetic studies were done on outbreak NDV isolates and previously isolated NDVs in Australia. The complete genomes of ten isolates of virulent and avirulent viruses associated with the ND outbreaks that occurred between 1998 and 2002 were determined to provide a database for further research. The genetic variability of the virulent locus within the virulent NDV isolates was compared to other sites and genes within the NDV genome. These results were used to understand the process whereby virulent viruses could emerge from an avirulent background and were used to test several hypotheses regarding their emergence. The ability of other avian viruses to alter the sequelae of an NDV infection was also investigated.

Outcomes The source of ND outbreaks from 1998-2002 was identified as being caused by the natural mutation of a pre-existing avirulent Australian NDV strain. The ability of this ‘progenitor’ virus and similar viruses to generate virulent virus was quantified at the quasispecies level. A Real-Time PCR was developed to detect virulent virus in a population of avirulent viruses. The complete genome sequence of ten NDV isolates (associated with the 1998-2000 outbreaks of virulent NDV in NSW) have been determined. This has enabled the genetic drift of the virus to be accurately determined and the confirmation that there is an association of the progenitor virus with virulent NDV outbreaks. Analysis of the individual viral sequences that constitute a field isolate (quasi-species analysis) has shown that buried within the avirulent progenitor virus population, virulent viruses may be hidden. Analysis of other Australian NDV isolates revealed the presence of a clade of viruses with the potential to cause virulent NDV outbreaks similar to the Mangrove Mountain outbreak.

Implications The cause of virulent NDV in Australia has been firmly linked to the presence of viruses that are only a few nucleotides away from virulence. The source(s) of this virus remain endemic within Australia and have the potential to continue to mutate and act as a source of virulent virus outbreaks within the poultry industry. Eradication of these viruses could be possible by the use of a survey and vaccination campaign.

Publications Gould, A.R., Kattenbelt, J.A. Selleck, P., Hansson, E., Della-Porta, A.J. and Westbury, H.A. (2001) Virulent Newcastle disease in Australia: molecular


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epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000. Virus Research 77: 51-60.


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Project Title:

Diagnostic tools for differentiation of vvIBDV and characterisation of local IBDV strains

RIRDC Project No.:

CSA-15J

Researcher: Dr. Jagoda Ignjatovic, Dr. Sandra Sapats and Mrs. Gaylene Gould Organisation: CSIRO Livestock Industries



• To develop ELISA and RFLP for differentiation of vvIBDV strains. • To demonstrate that changes in local IBDV field isolates remain such that they

can be clearly differentiated from all overseas strains.

Background Very virulent infectious bursal disease virus (vvIBDV) is exotic to Australia. A simple and rapid diagnostic test is needed in the event of an exotic incursion. In an earlier RIRDC project, a potential reagent specific for vvIBDV in ELISA was developed (CRAb88). Further evaluation of this reagent was needed. Furthermore, the technique of restriction fragment length polymorphism (RFLP) was developed in the USA for the differentiation of IBDV strains and work undertaken by the US researchers who developed this technique suggested that Australian strains were similar to vvIBDV strains. The usefulness of this method for differentiation of Australian strains therefore also required evaluation, and the US result for Australian strains needed to be further investigated. It was also possible that vvIBDV-like strains could emerge from local endemic strains and that monitoring of strains circulating in commercial poultry flocks would provide an early warning of the emergence of such strains.

Research The specificity of CRAb88 was tested in two overseas laboratories against a range of IBDV strains. The RFLP method was introduced into the research team’s laboratory and tested with all Australian IBDV strains. IBDV field isolates were obtained from various commercial poultry sites and their antigenic and genetic properties compared to other strains.

Outcomes CRAb88 was shown to react only with vvIBDV, detecting all vvIBDV regardless of their country of origin. An ELISA was subsequently developed and transferred to the diagnostic section of CSIRO Livestock Industries’ laboratory. The RFLP method was introduced, however by this method Australian IBDV strains were shown to belong to 12 different groups which differed from the 47 other groups identified amongst overseas strains. Thus, RFLP is not suitable for simple and meaningful strain differentiation in Australia. No similarity between Australian and vvIBDV was detected by this method. Seven new IBDV isolates collected from broiler sites between 2001 and 2003 were found to be antigenically and genetically similar to other earlier Australian IBDV strains.

Implications A simple and fast diagnostic test (ELISA) is now available in Australia for the differential diagnosis of exotic vvIBDV incursion. Monitoring of local IBDV strains showed no major changes have occurred in either their antigenicity or virulence.

Publications Sapats, S. and Ignjatovic, J. (2002) Restriction fragment length polymorphism analysis of the VP2 gene of Australian strains of infectious bursal disease virus. Avian Pathology 31: 559-566.

Ignjatovic, J. and Sapats, S. (2002) Characterisation of additional infectious bursal disease virus field isolates confirms existence of two distinct genetic groups in Australia. Australian Veterinary Journal 80: 689-694.


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Sapats, S.I., Heine, H.G., Trinidad, L., Gould, G.J., Foord, A.J., Doolan, S.G., Prowse, S. and Ignjatovic, J. (2003) Generation of chicken monoclonal antibody fragments that differentiate and neutralise infectious bursal disease virus (IBDV). Archives of Virology 148: 497-515.


15

Project Title:

Biological control of necrotic enteritis in meat chickens

RIRDC Project No.:

CSA-12A

Researcher: Dr. Robert Moore Organisation: CSIRO Livestock Industries



• To control necrotic enteritis in broiler chickens using a cost effective, user-

friendly, environmentally sustainable biological control strategy.

Background There is increasing pressure on the intensive animal production industries to decrease the use of antibiotics. Therefore, within the broiler chicken industry there is a need to develop alternative methods to control disease and maintain productivity. RIRDC have identified necrotic enteritis as a specific threat that needs to be addressed.

Research The research was aimed at developing antimicrobial peptides (bacteriocins), with activity against Clostridium perfringens, as products that could be used to protect chickens from the clinical manifestations and production losses caused by necrotic enteritis. The strategy was to clone and express synthetic bacteriocin genes and deliver the encoded recombinant proteins to the site of infection (the gut) in chickens. To test the products, a necrotic enteritis disease model needed to be established.

Outcomes Eight bacteriocin and two defensin genes were designed, synthesised, cloned, and expressed. Five of the recombinant bacteriocins were shown to have biological activity with three of them having activity against C. perfringens. The expressed bacteriocins were demonstrated to be safe in chickens and mice. A wide range of conditions was investigated in order to establish a necrotic enteritis model and eventually disease induction was possible. Unfortunately the model was developed too late in the project to test the products. In the meantime, the products were tested in other disease models and were shown to be effective in controlling a Listeria monocytogenes infection in the gut of chickens.

Implications The bacteriocins showed promise in controlling an infection in chickens and now must be tested and further developed for application against necrotic enteritis.

Publications Ingham, A., Ford, M., Moore, R.J. and Tizard, M. (2003) The bacteriocin piscicolin 126 retains antilisterial activity in vivo. J. Antimicrob. Chemother. 51: 1365-1371.

Ingham, A., Sproat, K., Tizard, M. and Moore, R.J. (2003) An optimised method and plasmid vectors for expression of non-modified bacteriocins in Escherichia coli. Submitted to J. Appl. Microbiol.

Ingham, A., Sproat, K., Tizard, M. and Moore, R. (2001) Recombinant antimicrobial peptides active against veterinary pathogens. Microbiology Australia 22: 25.5.

Ingham, A., Sproat, K., Tizard, M., and Moore, R. (2002) Recombinant antimicrobial peptides – bacterial missiles. Microbiology Australia 23: 3.3.


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Bird Nutrition and Feed Supply

Project Title:

Canola meal and cottonseed meal in broiler and layer diets

RIRDC Project No.:

DAQ-264J

Researcher: Dr. Rider Perez-Maldonado Organisation: Department of Primary Industries (Qld)

QPRDC PO Box 327 CLEVELAND QLD 4163


• To provide information on the chemical composition and variability with

processing of Australian canola meals and cottonseed meals at practical inclusion levels for use in broiler and layer diets.

• To provide recommendations to the poultry industries on the nutritional value of both canola and cottonseed meals when included in least-cost diets at levels close to the upper limit.

Background Although canola meal (CM) and cottonseed meal (CSM) offer great potential in the

poultry industry they are used at low levels (4-10%) in diets because of the presence of anti-nutritive factors (ANF) and variation in nutritional value due to location, environmental factors, cultivars, and processing. In Australia, the content of some ANF in CSM and CM have been reduced by genetic selection and by pre-press solvent extraction. Addition of soluble iron salts to diets reduces the negative effects of gossypol in CSM. New strains of laying birds and broiler chickens, improved canola and cotton varieties and better procedures for oil extraction from these crops provide new requirements and opportunities for the investigation of the suitability and nutritional value of these ingredients for poultry diets. In particular, there is potential for higher inclusion levels (>10%) above those normally used in practice for CSM and CM in poultry diets.

Research Four broiler trials and six layer trials were conducted using CSM and CM from various processors in Australia that were sampled over three years at the end of each processing cycle. These experiments evaluated CM and CSM obtained from processing plants located in NSW, Queensland, Victoria and WA. Broiler experiments were conducted over starter (0-21 days old) and finisher (21-42 days old) periods and evaluated chicken performance and health status. Layer trials lasted over 14 to 16 weeks and evaluated bird production performance and egg quality.

Outcomes Amino acid (AA) digestibility between CSM sources varied little and was consistent across samples. When compared with extruded meals, solvent extracted CSM showed an overall improved AME in both broiler and layer diets and contained low gossypol, condensed tannins and fibre levels, which are mostly removed during processing. The chemical composition within CM varied little across evaluations. However, between CM sources, chemical profile variation was common, particularly between solvent extracted and extruded meals. The overall AA digestibility across samples from different sources was satisfactory. Variations between solvent and extruded meals, particularly on lysine digestibility, were apparent. Glucosinolate levels among Australians CM were approximately one third of those found in Canadian “double zero” varieties. Sinapine content ranged from 11-15 g/kg, with extruded meals having the highest values. Seasonal, environmental and plant processing conditions accounted for most of the variation found in CSM and CM


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from different processors. For broilers, the overall results indicated that during the starter period, 200 g CSM/kg supported satisfactory performance. However, during the finisher period up to 300 g CSM/kg gave satisfactory performance, reducing abdominal fat pad without affecting chicken health. During the starter period, chicks fed on CM did well at 200 and 300 g CM/kg, depending upon CM source. During the finisher period, up to 300 g CM/kg gave the best results, reducing abdominal fat pad and intestinal viscosity without affecting chicken health status. In layer experiments, CM and CSM fed at various levels (100, 120, 150 or 200 g/kg) resulted in satisfactory performance in both brown and white strains of birds. Isabrown birds were more efficient when compared with White Supertint, birds, which gave higher egg weights and thus similar egg mass. The effect of gossypol or cyclopropenoid fatty acids (CPFA) levels in the CSM diets did not affect egg production. The addition of ferrous sulphate at the ratio 2:1 (ferrous sulphate to gossypol) did not affect egg production in this study. Egg quality studies on fresh eggs from brown layers fed on CM (100, 150, and 200 g/kg) indicated the production of ‘fishy’ tainted eggs at 150 and 200 g/kg level without any effect on yolk colour. ‘Fishy’ odour was not detected from eggs derived from White Supertint birds. On CSM, brown discolouration on the yolk was not observed in all evaluated eggs indicating that CSM from Narrabri is derived from low gossypol varieties. Mottling effect on eggs was only observed from brown and white layers fed CSM at 200 g CSM/kg. Cooked eggs from brown hens had a significantly higher level of ‘prawny’ odour than eggs from white birds.

Implications It is recommended that each feed manufacturer determine the chemical composition of CM and CSM ingredients before formulating poultry diets. Satisfactory bird performance is possible when feeding high levels of CSM and CM provided diets are formulated on a digestible AA basis. Coefficient tables provided in this report could be used for formulating these types of diets. An important consideration in using CSM in poultry diets is its low lysine (range 0.45-0.56) and threonine (0.57-0.68) digestibility coefficient values which can be overcome by adding synthetic amino acids to diets. Addition of iron salts is highly recommended when formulating diets with upper levels of CSM to overcome any residual gossypol effect. Therefore, with broilers 200 g CSM/kg or 200 g CM/kg can be used during the starter period and 300 g/kg of either CSM or CM can be used during the finisher period in diets formulated on a digestible amino acid basis. In layer hens, CM supported satisfactory egg production and egg quality in White Supertint birds when fed at 100, 150 and 200 g/kg levels. In brown birds, not more than 100 g CM/kg could be added without risking ‘fishy’ taint in eggs. Because the lipid content of CSM tended to produce mottling, it is advised to use only solvent extracted, low residual oil CSM at 150 g/kg maximum in white or brown laying hen diets.

Publications Trappett, P.C., Barram, K.M., Kemsley, M.K. and Perez-Maldonado, R.A. (2001) Evaluation of low glucosinolates canola meals on production and performance of laying hens. Proceed. Aust. Poult. Sc. Symp. 13: 247.

Perez-Maldonado, R.A., Blight, G.W. and Pos, J. (2001) Upper limits of inclusion of canola meal and cottonseed meal in diets formulated on a digestible amino acid basis for broiler chickens. Proceed. Aust. Poult. Sc. Symp. 13: 156.

Perez-Maldonado, R.A. (2001) Upper limits of inclusion of canola meal and cottonseed meal formulated on a digestible amino acid basis for chicken meat production. Proceed. Nut. Soc. Aust. 25: S33.

Bell, G.E., Nottingham, S.M. and Perez-Maldonado, R.A. (2002) Sensory evaluation of eggs from hens fed on canola meal and cottonseed meal based diets. Proceed. Aust. Poult. Sc. Symp. 14: 100.

Perez-Maldonado, R.A., Barram, K.M. and Kemsley, M.F. (2002) Maximum inclusion of canola meal in broiler starter diets formulated on a digestible and total amino acid basis. Proceed. Aust. Poult. Sc. Symp. 14: 180.

Perez-Maldonado, R.A., Barram, K.M., Trappett, P.C. and Kerven, G.L. (2002)


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Maximum inclusion of canola meal in broiler finisher diets formulated on a digestible and total amino acid basis. Proceed. Aust. Poult. Sc. Symp. 14: 181.

Perez-Maldonado, R.A. and Barram, K.M. (2003) Broiler performance in a semi-commercial environment using diets containing upper levels of canola or cottonseed meals. Proceed. Aust. Poult. Sc. Symp. 15: 177.


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Food Safety Project Title:

Salmonella typing and colonisation of chickens by characterised Salmonella Sofia

RIRDC Project No.:

IMV-3A

Researcher: Dr. Michael Heuzenroeder Organisation: Institute of Medical and Veterinary Science

Infectious Diseases Laboratories PO Box 14 Rundle Mall ADELAIDE SA 5000


• To continue to provide industry with a conventional and, where appropriate,

molecular based, Salmonella typing service. • To test the feasibility of AFLP typing as an epidemiological tool to replace PFGE

as the preferred molecular typing method. • To characterise by DNA sequence analysis the temperate (lysogenic) phage found

in S. Typhimurium phage type 64, which is a common phage type found in chickens and humans.

• To investigate Sofia MH76 colonisation of chickens using different methods of inoculation and S. Typhimurium challenge.

Background In previous projects molecular typing methods for Salmonella were instituted in

conjunction with classical typing methods (serotyping and phage typing) since they can offer greater discriminating power for determining distribution and location of potential pathogens. There are newer techniques such as AFLP that have the potential to be superior to the classical techniques and current molecular methods (such as PFGE) that need to be investigated as they may be faster and provide more discriminatory power. One of the reasons why certain strains predominate may be the carriage of bacterial viruses, also known as bacteriophages or phages. ST64T and ST64B are two phages found in S. Typhimurium DT64. It is possible that these phages can carry genes that influence disease potential and strain distribution in Salmonella and therefore knowledge of the DNA sequences of the phages may be helpful in determining whether virulence or other genes influence strain distribution. For over two decades, S. Sofia has been the major Salmonella isolated from chickens but it is almost never isolated from humans, therefore it has been suggested that it could be acting as a natural competitive exclusion agent, excluding pathogenic Salmonella strains such as S. Typhimurium DT64. Further investigation of this possibility may help to explain why Sofia is the predominant serotype found in chickens but doesn’t appear to be pathogenic for humans and may provide new opportunities for reducing the carriage of more pathogenic strains of Salmonella on chicken products.

Research AFLP was established in the laboratory using commercial bacterial fingerprinting kits. This technique was tested on S. Sofia isolates from over a 30 year period to establish whether unique Australian clones exist in order to assess the discriminatory power of the technique in comparison with PFGE. The DNA sequence was determined for the genomes of the ST64T and ST64B phages. The number of genes


20

contained within the phage genomes was determined. The putative gene products (proteins) produced by the phages were compared to the Genbank database to assign functions to the proteins. This was done to determine whether any of these proteins might be involved in virulence or influence current typing methods such as phage typing and serotyping. Research was also carried out to determine whether S. Sofia mutants (in the agf fimbrial gene) were disadvantaged in colonisation of chickens, however, no differences were observed. As previous experiments had failed to demonstrate that prior infection with S. Sofia had any significant effect on subsequent colonisation by S. Typhimurium, earlier exposure time of the chickens to S. Sofia was also investigated to determine whether this might influence co-colonisation with S. Typhimurium.

Outcomes AFLP typing was established to complement existing typing methods available in this laboratory for the monitoring of Salmonella in chicken flocks. It showed that no unique Australian clone of S. Sofia exists and thereby demonstrated that there are no obvious genetic differences that could explain the Australian situation with respect to S. Sofia. The DNA sequences of ST64T and ST64B were determined. It was found that ST64T could mediate genetic exchange between bacteria. ST64B, which was widespread in many S. Typhimurium strains, was defective and carried incomplete virulence factor genes. Both phages could potentially influence typing results and have the ability to acquire virulence factor genes. This data indicated a potential to influence S. Typhimurium virulence and strain distribution. Studies undertaken confirmed that S. Sofia MH76 did not prevent co-colonisation with S. Typhimurium in chickens.

Implications AFLP was found to be a useful typing tool but will not replace other established methods for typing, although in some cases it is clearly superior to established methods and should be retained. A molecular phage-typing scheme can be developed using genomic data gathered in this project. It is felt that additional work on S. Sofia was unlikely to demonstrate a significant potential for its use in displacing more pathogenic strains from the chicken production environment, and it is therefore felt that no further work in this is warranted.

Publications Mmolawa, P.T., Schmieger, H., Tucker, C.P. and Heuzenroeder, M.W. (2003) Genomic Structure of the Salmonella enterica Serovar Typhimurium DT 64 Bacteriophage ST64T: Evidence for Modular Genetic Architecture. J. Bacteriol. 85: 3473-3475.

Mmolawa, P.T., Willmore, R., Thomas, C.J. and Heuzenroeder, M.W. (2002) Temperate phages in Salmonella enterica serovar Typhimurium: implications for epidemiology. Int. J. Med. Microbiol. 291: 633-644.


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Project Title:

Development of campylobacter bio-replacement program and establishment of campylobacter reference laboratory

RIRDC Project No.:

UG-3A

Researcher: Dr. Victoria Korolik and Prof. Peter Coloe Organisation: Griffith University

Microbial Glycobiology, Institute for Glycomics PO Box 50 Gold Coast Mail Centre GOLD COAST QLD 9726


• To evaluate and refine the model system for colonisation of chickens with

selected Campylobacter strains. • To evaluate highly colonising and potentially non-virulent strains as potential

‘bio-replacement’ strains for application in the chicken industry. • To provide a Campylobacter reference laboratory based on molecular technology

for identification of virulent and non-virulent campylobacters, which can be used for epidemiological tracing of specific strains.

Background Campylobacter spp are now the most common cause of human enteritis worldwide

and it is well established that chicken meat is one of the major sources of human infection. It is therefore essential that the risk of transfer of Campylobacter spp to humans via chicken meat is minimised. Since Campylobacter spp can infect a high proportion of chicken flocks and intensive biosecurity programs around the world have failed to stop colonisation of commercial flocks by campylobacters, an alternative approach to control of Campylobacter spp in chicken flocks is required. A possible alternative approach is to use an avirulent strain of C. jejuni, which has been fully characterised, to establish continuing colonisation in chickens and to displace other strains which may be of more significance in a food safety context.

Research C. jejuni strains were assessed in terms of their colonisation type by inoculating two day old chicks with various strains of C. jejuni and assessing maximal colonisation levels by quantitating the bacterial numbers in the caeca of each chicken. Strains were also tested for their ability to out-compete other colonising strains by inoculating different strains into chickens already colonised with a different C. jejuni strain. An alternative methodology for control of pathogenic campylobacters in poultry by ‘bio-replacement’ was investigated and a potential bio-replacement strain was characterised for its ability to colonise chickens already colonised with other C. jejuni strains. This strain was also evaluated for its ability to sustain colonisation in the face of challenge with other strains, using the two day old chicken infection model developed previously. This strain was also tested for carriage of possible virulence factors using toxicity assays, invasion assays in tissue culture and a new mouse infection model. In addition, a PRC molecular detection and characterisation methodology for campylobacters was developed using DNA sequences from ribosomal RNA and surface antigen genes.

Outcomes Five different colonisation types were identified among C. jejuni strains. These were (1) immediate colonisation with prolonged excretion of viable C. jejuni bacteria; (2) delayed colonisation with prolonged excretion of viable C. jejuni; (3) immediate colonisation and slowly clearing excretion; (4) delayed colonisation and slowly


22

clearing excretion; and (5) no colonisation of the intestines with C. jejuni bacteria. Infection with strains of colonisation types 1 and 2 led to sustained colonisation of the intestines of chickens. Small experimental chicken flocks were exposed to different C. jejuni strains and within three weeks one or two strains had replaced all others in the gastrointestinal tract of the chickens. One such strain, C. jejuni strain 331, was shown to be a super-efficient coloniser of chickens, non-invasive in tissue culture, able to displace other strains present in the chicken intestine and not displaceable by other Campylobacter strains tested. This strain could potentially be used in a ‘bio-replacement’ program aimed at preventing the colonisation by pathogenic campylobacters of chickens destined for human consumption. A multiplex PCR of C. jejuni genomic DNA was developed to yield a PCR product with a unique polymorphic site that could be used to quickly and accurately identify and group C. jejuni isolates from purified DNA, cell culture, skin washings and faecal samples. This formed the basis for campylobacter reference laboratories now established at Griffith and RMIT Universities.

Implications One particular strain of C. jejuni has been identified and characterised as being a highly efficient coloniser of chickens yet lacking the ability to invade human cells in tissue culture or to produce toxins. A ‘bio-replacement’ program for control of Campylobacter in chicken flocks, based on this strain, will be evaluated in planned future research. Campylobacter reference centres have been established at Griffith and RMIT Universities and are available for identifying and typing isolates of Campylobacter submitted by industry or in future epidemiological studies.

Publications Ringoir, D.D. and Korolik, V. (2003) Differences in colonisation patterns of Campylobacter jejuni strains and changes in colonisation potential after passage in vivo. Veterinary Microbiology 92: 225-235.

Korolik, V., Friendship, D.T., Peduru Hewa, T., Alfredson, D., Fry, B.N. and Coloe P.J. (2001) Specific identification, grouping and differentiation of Campylobacter jejuni among thermophilic campylobacters using multiplex PCR. Epidemiology and Infection 127: 1-5.

Fry, B.N., Yuen-Yuen Chen, S.F., Newell, D.G., Coloe, P.J. and Korolik, V. (2000) The galE gene of Campylobacter jejuni is involved in LPS synthesis and virulence. Infection and Immunity 68: 2594-2601.


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Project Title:

Isolation of genes responsible for Campylobacter jejuni colonisation of the chicken intestinal tract

RIRDC Project No.:

UG-4A

Researcher: Dr. Victoria Korolik and Ms. Danielle Ringoir Organisation: Griffith University

Microbial Glycobiology, Institute for Glycomics PO Box 50 Gold Coast Mail Centre GOLD COAST QLD 9726


• To isolate and analyse specific genes which are involved in colonisation of the

chicken intestinal tract by C. jejuni. • To develop a strain-specific C. jejuni probe for molecular identification of

specific strains in culture and field samples.

Background Campylobacter species are recognised as the most common cause of gastroenteritis in humans world-wide. Campylobacter jejuni is responsible for 80% of these infections. A significant source of infection is consumption of undercooked and contaminated chicken meat. Birds and other animals are frequently colonised with C. jejuni as part of their normal flora. Ability to colonise is important in order for bacteria to maintain themselves and propagate in the host. C. jejuni strain 331 was previously shown to be highly colonising in chickens and non-invasive in human tissue culture cells, able to displace other strains from chicken intestinal tract and, once established, not able to be replaced by other Campylobacter strains. Genes that are present in a colonising strain such as strain 331, but not in a non-colonising strain, might be important for colonisation of the chicken intestinal tract and thus need to identified and characterised.

Research Subtractive hybridisation methods, designed for identifying minor genetic differences in closely related organisms, were used to genetically compare a non-colonising C. jejuni strain and a colonising C. jejuni strain. Unique genes that are present in the latter strain but are absent from the former strain were isolated and cloned to generate a small, subtracted genomic library of candidate genes. The genes isolated by this method were identified by sequencing the cloned DNA fragments and then by comparing the sequences with known gene databases. In addition, every strain carries several genomic DNA sequences unique to that strain. The subtracted genomic library was screened in order to identify genes unique to C. jejuni strain 331 by genomic hybridisation with C. jejuni 331 and 16 other randomly chosen strains. A unique sequence would hybridise to strain331 and none other.

Outcomes The genomic comparison between colonising and non-colonising strains identified genes from the lipooligosacharide locus, capsule locus, restriction modification enzymes, methyl accepting chemotaxis signal transduction proteins, lipoproteins and transport proteins. Genes in other organisms with similar functions have been implicated in having a role in colonisation. The methyl accepting chemotaxis signal transduction protein found in this procedure is specific for colonising strains, which indicates that this protein is important for colonisation of the chicken intestinal tract. First attempts are currently underway to disrupt this gene in order to elucidate its role in colonisation. In addition, four fragments which are specific for the colonising C. jejuni strain 331were identified and their specificity confirmed by screening against other C. jejuni strains. These fragments can be used for future design of a PCR protocol to identify C. jejuni strain 331.


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Implications Identification of genes related to colonisation will lead to a better understanding of the colonisation process of C .jejuni and to the identification of strain specific sequences which will permit the development of tests for the specific identification of C. jejuni strain 331, the strain which is being developed as a candidate ‘bio-replacement’ agent in project UG-3A.


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Environmental Management

Project Title:

Reduction of dust emissions from broiler and caged layer sheds

RIRDC Project No.:

SAR-33J

Researcher: Mr. Thomas Banhazi Organisation: South Australian Research and Development Institute

GPO Box 397 ADELAIDE SA 5001


• To determine the major risk factors associated with increased concentrations of

dust within, and dust emissions from, naturally and mechanically ventilated poultry houses.

• To develop strategies that will reduce both interior dust levels and dust emissions from buildings, resulting in more sustainable housing systems for egg and broiler production in semi-urban and more densely settled areas and reduced OH&S risks for staff.

Background Dust in poultry buildings is composed of a mixture of material, such as skin cells,

dried droppings, feathers, bedding debris, feed particles and a diverse range of micro-organisms. Dust particles can transport other pollutants like odour molecules, bacteria, endotoxins and viruses, and promote the distribution of these agents within and between livestock buildings. In order to reduce dust emissions from poultry buildings, factors influencing internal dust concentrations need first to be studied and understood, and dust concentration and emission reduction methods reported in the literature evaluated. A range of environmental factors, such as lighting, humidity, temperature and ventilation, have been demonstrated to have an influence on dust concentrations in some studies, but there are often interactions between these factors and animal-dependant variables. Different methods for reducing airborne pollutants, such as the negative electrostatic space charge system, ozonisation and water sprays, have been tested by various research groups. However, overseas research suggests the most useful technique currently available is the impregnation of bedding material with an oil and water mixture.

Research The concentrations of a range of airborne pollutants (total airborne bacteria, inhalable and respirable dust) present in 26 poultry housing facilities in South Australia were measured. Data was also collected on housing features used in the facilities. The measurements of airborne pollutants were made using standardised methods and instruments, which allowed reliable comparisons to be made between different buildings and classes of animals. The data collected identified the key housing factors associated with elevated concentrations of the measured pollutants. Two separate experiments were conducted to assess a dust abatement technique for use within poultry buildings and an emission reduction method utilising an innovative and low-cost air-scraping system.

Outcomes The results obtained in this study will assist development of strategies for reducing the effects of poor air quality on poultry production, health and welfare and meeting OH&S guidelines for the poultry industry. Specifically, a number of key housing and management factors have been identified as having a significant effect on air quality.


26

Oil impregnation of bedding material was identified as a promising technology that can be effectively used for dust reduction in broiler sheds. The innovative and low-cost air-scraping system developed by the research group is also a promising technology and could potentially be used by producers to reduce the concentration of emitted airborne particles from livestock buildings. However, the technology still needs refinement before it can be commercialised. Shed cleaning practices and the type of the shedding used were also identified to be important factors (amongst other factors) influencing the concentration of dust in poultry buildings.

Implications The adoption of routine dust concentration and emission reduction strategies in poultry buildings is an important objective because of the increasing environmental and occupational health and safety requirements associated with these industries. Oil impregnation of bedding and utilisation of air-scrapers appear to be potentially useful methods for reducing the concentrations and emissions of airborne particles. The implementation by industry of appropriate shed cleaning practices and adoption of tunnel ventilated shedding can also be expected to have significant benefits in terms of reducing dust in and emitted from poultry sheds.

CHICKEN MEAT PROGRAM – RESEARCH IN PROGRESS 27

RESEARCH IN PROGRESS

Flock Health

Project Title

Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates

RIRDC Project No.:

CSA-24J

Start Date: 01/07/02 Finish Date: 30/11/04 Researcher: Dr. Hans Heine Organisation: CSIRO Livestock Industries



• To provide fast, sensitive and specific molecular diagnostic tests for the

identification of very virulent isolates of infectious bursal disease virus (IBDV), highly pathogenic Newcastle disease virus (NDV) and avian influenza virus (AIV) by developing and implementing new real-time PCR assays (Sequence Detection System based on TaqMan) that will give reliable results in less than five hours.

• To develop a common assay format enabling simultaneous testing for IBDV, NDV and AI, as well as enabling differentiation between very virulent/highly pathogenic strains and circulating endemic non/low-pathogenicity strains.

• To implement the new tests at AAHL to ISO17025 standards by June 2004.

Current Progress Highly sensitive and specific real-time reverse-transcription PCR assays utilising the ABI PRISM 7700 Sequence Detection System coupled with TaqMan probe chemistry were developed to detect all serotype 1 strains of IBDV and to differentiate between virus groups. The design of real-time PCR primers and probes was based on phylogenetic and nucleotide sequence alignment analysis of over 100 geographically and pathogenically diverse IBDV strains. The intrinsic specificity of the probes allowed differentiation between very virulent and classical strains of IBDV without the need for sequencing. Four different TaqMan real-time PCR assays running under common reaction conditions were developed to detect all serotype 1 strains of IBDV or specifically Australian strains, overseas classical (including antigenic variant) strains or very virulent stains on the same microtiter plate on the ABI PRISM 7700. The specificity of the assays has been evaluated with a range of different IBDV strains including very virulent IBDV from Europe and Asia, classical IBDV from Europe, antigenic variants from the US, and endemic Australian strains. The duration of the assay from RNA extraction to analysis of results was less than four hours, far shorter than current molecular diagnostic tests. The development of TaqMan assays for NDV pathotyping is in progress.


Project Title

The effect of Newcastle disease vaccination with strain V4 on the course of infections with the Peats Ridge strain of Newcastle disease virus

RIRDC Project No.:

CSA-18J

Start Date: 01/10/01 Finish Date: 30/11/03 Researcher: Dr. Peter Daniels Organisation: CSIRO Livestock Industries

PO Bag 24 GEELONG VIC 3220


• To investigate whether prior vaccination with V4 can reduce subsequent

colonisation with Peats Ridge strain NDV and/or reduce the amount (and duration) of shedding of Peats Ridge strain NDV.

• To investigate whether V4 vaccination during a Peats Ridge infection can modify the course of the Peats Ridge infection..

• To provode data on the serological response of birds to infection with the virus Peats Ridge alone, and to the Peats Ridge strain following prior 'priming' with V4 vaccine.

Current Progress In recent years the Australian poultry industry has had to develop strategies not

only to protect itself from the effects of virulent strains of NDV, but also to manage the issue of endemic strains of NDV that might be capable of progressive mutation to virulence. Within this general context, two major objectives of this project were, firstly, to determine whether the pattern of colonisation of poultry with the Peats Ridge (non-virulent) strain of NDV is influenced by prior vaccination with V4 and, secondly, to assess whether an established Peats Ridge infection of poultry might be modified by subsequent vaccination with V4. To date, it has been shown that, although prior vaccination with V4 substantially reduces cloacal shedding of NDV following subsequent challenge with Peats Ridge strain, V4 vaccination in the face of established Peats Ridge infection does not clearly influence shedding of NDV by these birds. Currently, work is underway to determine the relative proportions of Peats Ridge strain and V4 virus within the NDV being shed via tracheal and cloacal routes of these birds using Taqman PCR technology. Initial validation and determination of limits of sensitivity for this test have been achieved.


Project Title

Postgraduate scholarship - Jacqueline Kattenbelt: Analysis of virulence determinants of Newcastle disease virus

RIRDC Project No.:

CSA-13J

Start Date: 15/07/00 Finish Date: 30/10/03 Researcher: Dr. Allan Gould Organisation: CSIRO Livestock Industries



• To investigate the molecular epidemiology of Australian Newcastle disease

virus (NDV) isolates and to establish a reverse genetics system for the virus.

Current Progress

Nucleocapsid, phosphoprotein, L (polymerase) and full-length genomic NDV clones have been inserted into specialised expression shuttle vectors. These constructs constitute the central components of the reverse genetics system for Newcastle disease virus used to generate ND virus by transfection of these plasmids into tissue culture cells. These constructs have been completely sequenced and defects in their construction corrected by standard molecular biological techniques. The viability of the constructs has been tested in a mini-genome using an extracellular SEAP reporter gene to replace the full-length NDV genome construct. Similarly, each construct was tested individually in a coupled transcription/translation system to detect the synthesis of the correct protein transcript. This was successful, as each construct produced a protein of the correct molecular weight. At present, attempts are being made to transfect these constructs into BHK cells constitutively producing T7 polymerase to produce viable ND virus. First attempts have been unsuccessful. However other procedures are being investigated for this transfection procedure as well as the use of a fowl pox virus expressing T7 polymerase in other cell types. A quasi-species analysis of the original Dean Park NDV outbreak virus from 1998 was undertaken to determine parameters associated with this outbreak in relation to subsequent outbreaks of the disease. Arrangements have been made with CLI (CSIRO Livestock Industries) in Brisbane to transfer several NDV constructs into “yeast-2-hybrid” vectors held in these laboratories in a collaborate effort to study protein-protein NDV interactions.


Project Title

Avian leukosis-J (ALV-J) in Australia: laboratory technologies and research needs

RIRDC Project No.:

UM-49A

Start Date: 01/10/00 Finish Date: 31/07/03 Researcher: Dr. Trevor Bagust Organisation: The University of Melbourne

Faculty of Veterinary Science Cnr Park Drive and Flemington Road PARKVILLE VIC 3052


• To develop the most appropriate laboratory technologies and reagents for

detection of avian leukosis virus sub-group J (ALV-J) and its associated disease effects for Australia's chicken meat industry.

Current Progress Avian leukosis sub-group J virus (ALV-J) causes the formation of tumours

while lowering the productivity and liveability of broiler breeders. ALV-J has entered Australia in contaminated imported genetic stocks. This project has established techniques for the detection of ALV-J, and successfully applied these to blood, meconium, albumen, feathers and tumour tissues. Polymerase chain reaction (PCR) suitable for specific detection of ALV-J using these tissues has been developed and is routinely being applied, with virus propagation in chicken fibroblast cell cultures, to screen for ALV-J infection in Australian flocks. Application of these techniques by the International Avian Health Laboratory at the University of Melbourne, in collaboration with the poultry industry, enabled some 40 strains of ALV-J to be obtained from flocks located in Queensland, NSW, Victoria and SA. In November 2002, 12 representative isolates were forwarded to the AFRC Institute for Animal Health, Compton (UK) and their identity has now been confirmed by that laboratory as ALV-J. Most recently, laboratory methodologies for ALV-J detection have been refined to increase the speed and efficiency of surveillance by using serums, and which permit screening of larger numbers of samples per flock. This advance will directly enhance the effectiveness of detection of ALV-J infections when present at low prevalence in Australian great grandparent and grandparent flocks.


Project Title

Typing of Pasteurella multocida

RIRDC Project No.:

UQ-100J

Start Date: 01/01/02 Finish Date: 31/12/04 Researcher: Dr. Linda Blackall Organisation: The University of Queensland

Department of Microbiology and Parasitology ST LUCIA QLD 4072


• 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.

Current Progress A total of 30 out of 110 isolates of Pasteurella multocida in the study have been

revived and obtained in pure culture. Their identity as Pasteurella multocida was confirmed by biochemical tests. A suitable DNA extraction kit was obtained and DNA was extracted from these 30 isolates. Some of the DNA sample concentrations were quantified using Fluorescent DNA Quantitation kit to determine the quality of the DNA extractions. The DNA extractions were confirmed as suitable for PCR amplification by successfully conducting a Pasteurella multocida species specific PCR on all samples. A total of seven house keeping enzymes were chosen for this Multi-Locus Sequence Typing study. The selection was based on the genetic diversity of these enzymes as shown in a prior multi-locus enzyme electrophoresis study. Two sets of primers were designed to amplify the genes associated with two of the seven selected enzymes. Once the PCR products are obtained and sequenced, the sequence data will be analysed and the sequence types of the isolates will be established. Finally, the data will be placed on the world wide accessible web site.


Project Title

Control of intestinal spirochaete infections in chickens

RIRDC Project No.:

UMU-29J

Start Date: 01/09/01 Finish Date: 30/08/03 Researcher: Prof. David Hampson Organisation: Murdoch University

Division of Veterinary and Biomedical Sciences MURDOCH WA 6150


• To develop improved methods to control infection by Brachyspira

intermedia and Brachyspira pilosicoli - bacterial pathogens causing significant economic loss in Australian layer and broiler breeder flocks.

Current Progress Studies have been conducted on a broiler breeder and a layer farm to determine

the source and dynamics of infection with intestinal spirochaete species. No colonisation was found in the breeder farm. On the layer farm approximately 20% of faeces were positive for spirochaetes. Of these, 36% were B. intermedia and 6% B. pilosicoli, whilst 58% were of unknown species. The isolates are being typed. No environmental sources of infection were identified. Neither species survived more than a few days in faeces, and both were rapidly inactivated by disinfectants. In an experimental infection trial, layers were fed diets based on one or other of two Western Australian wheat varieties, then infected with B. intermedia. Significant differences in colonisation were detected in the two groups, but this was not related to the viscosity of the ileal contents. A diet-related effect was not seen when the experiment was repeated using birds infected with B. pilosicoli. An experiment is planned in which birds will be fed diets based on wheat, barley, or barley plus sorghum, each fed with or without addition of exogenous enzymes. Birds will be experimentally infected with B. intermedia and the outcomes related to the NSP contents of the diets.


Project Title

Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis

RIRDC Project No.:

UTS-4J

Start Date: 01/05/02 Finish Date: 30/04/04 Researcher: A/Prof. Nicholas Smith Organisation: University of Technology, Sydney

Institute for the Biotechnology of Infectious Diseases Westbourne Street GORE HILL NSW 2065


• To test the efficacy of recombinant versions of Eimeria maxima gametocyte

antigens as a subunit, maternally-delivered vaccine against coccidiosis.

Current Progress

The immunogenicity of recombinant gametocyte antigens of Eimeria maxima was evaluated. Groups of eight to nine chickens of two breeds (Australorps and Cobb 500) were injected with various doses and combinations of recombinant versions of the 56 kDa and 82 kDa E. maxima gametocyte antigens at 8, 12 and 16 weeks of age, and antibody production in response to these antigens was determined. Immunisation with the native vaccine formulation (positive control) stimulated strong antibody responses and negative control chickens did not produce antibodies. Immunisation with either the recombinant 56 kDa protein or the recombinant 82 kDa protein resulted in the production of antibodies that recognised strongly both the native and recombinant versions of the respective antigens. The effect was dose-dependent. Furthermore, the purified recombinant proteins were well recognised by protective antibodies generated by immunisation with the native proteins and by protective antibodies produced in response to actual infection with E. maxima. Both Australorps and Cobb 500 birds responded well to the immunisation. These results provide evidence that the recombinant proteins share sufficient similarity with the native antigens to suggest that they may be valuable in a recombinant subunit vaccine to control coccidiosis. Efficacy trials of the antigens will commence shortly.


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

Start Date: 1/03/03 Finish Date: 28/02/06 Researcher: A/Prof. Nicholas Smith Organisation: University of Technology, Sydney

Institute for the Biotechnology of Infectious Diseases Westbourne Street GORE HILL NSW 2065


• To identify and characterise structural proteins and enzymes involved in

oocyst wall formation in Eimeria maxima.

Current Progress It has been shown previously that tyrosine-rich gametocyte proteins play an important role in oocyst wall formation in the avian parasite E. maxima. They are proteolytically processed during development to become the oocyst wall, through a peroxidase-coupled, sclerotisation-type reaction. These proteins and enzymes are localised to the wall forming bodies of macrogametocytes. The research that will be undertaken within this project will commence with a database search in order to design primers for enzymes that catalyse tyrosine cross-linking eg phenol oxidase and peroxidase. This work will be completed within the coming six months. Wall-forming bodies from gametocytes of E. maxima will then be purified and enzyme activities in these and in whole gametocyte extracts identified. This will include isoenzyme characterisation using two-dimensional electrophoresis. Primers to the proteins and enzymes involved in oocyst wall formation will be designed and the genes that encode them amplified by PCR. These genes will be sequenced and cloned and recombinant proteins expressed. These recombinant proteins will then be used to immunise animals to produce antibodies to enable the localisation of the proteins within the parasite by immuno-microscopy. The function of these proteins will be studied using gene transfection and knockout technologies.


Project Title

Systematic pathotyping of Australian Marek's disease (MDV) isolates

RIRDC Project No.:

UNE-83J

Start Date: 01/07/02 Finish Date: 30/11/05 Researcher: Dr. Stephen Walkden-Brown Organisation: University of New England

Animal Science School of Rural Science and Natural Resources ARMIDALE NSW 2351


• To pathotype nine current and three older Australian isolates of Marek's

disease virus (MDV) using internationally recognised protocols. • To undertake molecular characterisation of these isolates to facilitate

identification and relationships between isolates. • To test the pathogenicity of two isolates in current Australian strains of meat

and layer bird. • To determine the extent of protection provided by HVT and bivalent

serotype 2/HVT vaccines against recent isolates. • To determine whether recent isolates are more pathogenic than older

isolates. • To develop an improved knowledge base for rational decision making

regarding MDV vaccines and vaccination procedures.

Current Progress In October 2002, a new 24 isolator facility at UNE was opened. It has been the subject of ongoing modification and improvement and is currently occupied with a second large experiment on another project. The facility is now operating ideally. At the collaborating organisation (RMIT) there has been a very active process of isolating current Marek’s disease virus (MDV) field strains with good industry support. From July to December 2002, 16 field submissions involving over 200 individual birds were made. To date in 2003 there have been an additional five submissions involving 23 birds. Submissions have come from Victoria, NSW, Queensland, SA and WA. Seven isolates appear promising and are at various stages of being tested for ability to grow to high titre in cell culture, and being screened for freedom from contamination with vaccine strains of MDV and other pathogens, including CAV, ALV(J), REV and IBDV. The first pathotyping experiment in which four new MDV isolates will be evaluated for pathogenicity against reference strains of MDV in specific pathogen free chickens is scheduled to start on 4 August 2003 in the UNE isolator facility. Primers to excise the Meq gene of these isolates for sequencing and comparison, have been designed and ordered.


Project Title

Molecular techniques for monitoring Marek's viraemias in broilers and layers

RIRDC Project No.:

UJC-10J

Start Date: 01/07/02 Finish Date: 31/07/05 Researcher: Dr. Graham Burgess Organisation: James Cook University

Department of Microbiology and Immunology TOWNSVILLE QLD 4811


• To develop and transfer to the industry molecular techniques for monitoring

blood samples for the presence of Marek's disease virus. • To use a panel of simple, cost-effective molecular diagnostic techniques to

describe the effects of various management strategies on Marek's infection. • To develop and validate quantitative assays for Marek's disease viral DNA

based on real-time PCR. • To assemble a comprehensive collection of DNA for Australian wild type

Marek's disease viruses.

Current Progress A collection of DNA samples from 15 Australian serotype 1 Marek’s disease virus (MDV) isolates has been assembled. Negotiations to obtain DNA from an extensive panel of strains from the USA have been completed. The sequence of two areas of the MDV 1 genome that could be useful for strain differentiation has been identified, including the UL 36 gene and the retroviral insertion in the ICP 4 gene. Clinical trials using specific pathogen free (SPF) birds are now underway to ensure that the ICP 4 marker is detectable following in vivo passage. A third area of the serotype 1 genome of interest is a regulatory site that may be a marker of pathotype. New collection media that has the potential to improve the sensitivity and repeatability of the collection technique is being tested in a clinical trial with collaborators. A quantitative real-time PCR assay for serotype 1 MDV has been optimised and the controls required for standard curve generation have been produced. Titration of viral vaccine stocks to determine vaccination dose can therefore now proceed. This will provide a new quality assurance assay for vaccine manufacturers and users of MDV vaccines. In a clinical trial, SPF birds are being challenged and feather pulp is being used to develop a procedure for isolation of vial genome using pulsed field gel electrophoresis.


Project Title

Molecular evaluation of responses to vaccination and challenge by Marek's disease viruses

RIRDC Project No.:

RMI-12J

Start Date: 19/12/02 Finish Date: 31/12/05 Researcher: Prof. Greg Tannock Organisation: Royal Melbourne Institute of Technology

Department of Biotechnology and Environmental Biology PO Box 71 BUNDOORA VIC 3083


• 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 into chick embryos under conditions used for in ovo vaccination.

• To carry out protection experiments after in ovo and day old vaccination in which the endpoints will be measured from the challenge virus load in critical target organs.

Current Progress This project commenced in December 2002 following the arrival of Dr

Jianming Tan from the University of Guelph in Canada to fill the Post-Doctoral position on the project. Activities undertaken since then have been concerned with (a) the installation of four chicken isolators which were obtained from Intervet Pty. Ltd. in Newcastle for the conduct of challenge experiments, and (b) the optimisation of conditions for the quantitative polymerase chain reaction (PCR) which will allow the measurement of viral loads in chickens infected with Marek's disease virus (MDV), which will form the basis of a challenge model for the assessment of vaccine strategies. Preliminary experiments were carried out by conventional PCR to determine the specificity of five pairs of primers with specificities for the three MDV serotypes, some of which were different to those described in the literature. The most suitable of these primers will be used in tests to measure viral loads by quantitative PCR (QPCR). Two sets of primers were selected as being suitable for virulent serotype 1 viruses, one for serotype 2 viruses and a further two for serotype 3 vaccine strains. PCR products from single serotype 1 and 3-specific PCR products were cloned into vectors which will be used as internal standards in estimating MDV genome copy number in the QPCR. The standardised QPCR has been used to measure increases in genome copy number in cells infected with serotype 1 and 3 viruses. Results obtained have shown that the test has good reproducibility and will now be applied to the measurement of viral loads in vaccinated and challenged chickens.


Project Title

Use of cytokines to enhance vaccine efficacy in poultry

RIRDC Project No.:

CSA-26J

Start Date: 01/02/03 Finish Date: 31/01/06 Researcher: Dr. Andrew Bean Organisation: CSIRO Livestock Industries



• 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.

Current Progress Marek’s disease (MD) is an economically important disease in chickens and

since the initial use of Marek’s Disease vaccines the disease has been substantially modified in its expression and prevalence. Of crucial importance to the poultry industry is the continuous emergence of hyper-virulent viruses, which are less well controlled by current vaccines. With this in mind, understanding the interactions of this herpes virus and the host immune system is fundamental to understanding the disease mechanisms and the design of new vaccination strategies. The specific objective of the project is to develop novel vaccine formulations that will enhance the ability of current MD vaccines to control emerging hyper-virulent Marek’s disease in poultry. This involves the evaluation of various cytokines and an assessment of their ability to enhance vaccine efficacy and immune competence. By manipulation of the immune system via the co-delivery of cytokines it is hoped that the immune response to vaccination can be enhanced. Several chicken cytokines (IFN-γ, IL-2, IL-6, IL-18) have been cloned and expressed using E. coli expression systems. Furthermore, their in vitro ability to stimulate chicken immune cells has been tested. The in vivo activity of these cytokines is currently being assessed to determine their potential as adjuvants for co-delivey with MDV-related vaccine strains.


Project Title

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

RIRDC Project No.:

CSA-21J




• To reduce disease by enhancing mucosal immunity in chickens with the aid

of cytokine therapy.

Current Progress Most diseases of poultry are initiated at mucosal surfaces, such as the lung, as these surfaces are in intimate contact with the external environment. There is an increasing need for enhanced protection provided by vaccines that are capable of promoting effective mucosal and systemic immunity to bacterial and viral pathogens. Oral vaccination with protein antigen alone generally induces poor immunity, therefore the development of potent mucosal immune enhancers is essential. Newly discovered chicken cytokines ChIL-6 and B cell activating factor (BAFF) have the potential to direct and control the outcome of mucosal responses. This highlights their potential importance in vaccination strategies as adjuvants to augment mucosal immunity. To date, ChIL-6 has been expressed in both prokaryotic and eukaryotic expression systems and the recombinant protein has been shown to be biologically active. To investigate the immunological role of IL-6 in chickens, anti-ChIL-6 antibodies have been prepared towards the development of an ELISA to measure ChIL-6 levels. Additionally, initial in vivo trials involving administration of recombinant ChIL-6 protein in chickens have been carried out to elucidate the role of IL-6 in avian immune responses. Similarly, the cytokine BAFF has been cloned and expressed in a eukaryotic system. A bioassay has been designed based on mammalian studies to determine the biological activity of BAFF.


Project Title

Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus infection

RIRDC Project No.:

CSA-25J




• To examine the early immune response to Marek’s disease virus infection

and apply this information to enhance the development of therapeutic strategies.

Current Progress Marek’s disease virus (MDV) is considered to pose a major future threat to the

Australian poultry industries. Understanding the interactions of this herpes virus and the host immune system is fundamental to understanding the disease mechanisms. Additionally, utilising and augmenting the immune response may provide protection to this economically significant disease. This project focuses on assessing the early responses to MDV, concentrating on the early events associated with the immune response to the virus. Preliminary work has been carried out to identify innate immune molecules in chickens that are of importance and will be examined during MDV infection. To enable the dissection of these early events, an appropriate disease model must be established. With this in mind, a field strain of MDV has been isolated from a commercial farm and was identified as a serotype 1 virus via PCR. This may be useful in establishing a model of infection. In vitro techniques have been developed and the production of cell free virus is under way. This cell free virus will enable the initial interaction between pathogen and host to be studied, particularly with respect to respiratory infection. Methods are currently being developed to examine viral infection in the air sacs and lungs of chickens, as a possible site of initial interaction between pathogen and host.


Project Title

Postgraduate scholarship - Scott Sheedy: Live vectoring of therapeutic and prophylactic proteins and pathogenesis in necrotic enteritis

RIRDC Project No.:

CSA-20A

Start Date: 01/03/02 Finish Date: 28/02/05 Researcher: Dr. Robert Moore Organisation: CSIRO Livestock Industries



• To use live bacterial vectors to deliver therapeutic proteins (eg. cytokines;

vaccine antigens) to the gut of chickens. Such delivery may have applications in the treatment of gut diseases such as necrotic enteritis.

• To investigate the importance of the alpha toxin produced by Clostridium perfringens in the pathogenesis of necrotic enteritis.

Current Progress A series of E. coli isolates recovered from chicken guts have been tested for

their ability to colonise and persist in the gut of chickens when re-inoculated. Three persistent isolates have been identified. To investigate these isolates as potential live vectors for the delivery of therapeutic and prophylactic proteins, recombinant plasmid molecules must be able to be introduced. Thus, the transformability of the strains has been confirmed using two different plasmid replicons. These isolates are now ready for the introduction of plasmids expressing the proteins that are to be delivered using the live vectors; for example cytokines, bacteriocins, and vaccine antigens. Such proteins may have therapeutic applications against necrotic enteritis. The role of the alpha toxin from Clostridium perfringens in the development of necrotic enteritis remains unclear. Recently, work was published that demonstrated that the sequence of an alpha toxin encoding gene from an avian isolate of C. perfringens was quite different to the sequences found in mammalian derived isolates of C. perfringens. This issue has been investigated by sequencing the alpha toxin gene from 27 chicken isolates of C. perfringens. All the encoded proteins had very high homology with the mammalian isolates, so it therefore appears that the published avian sequence is very unusual and not typical of other avian isolates. The next step in the analysis of alpha toxin involvement in pathogenesis is to make knockout mutants and test their disease causing potential.


Project Title

The development of vaccination strategies to control necrotic enteritis in poultry

RIRDC Project No.:

RMI-11A

Start Date: 01/01/00 Finish Date: 31/12/03 Researcher: Prof. Peter Coloe Organisation: Royal Melbourne Institute of Technology



• To develop an effective vaccine against necrotic enteritis and to evaluate the

vaccine against a challenge model of the disease. This vaccine will be orally deliverable, cost effective to manufacture and deliverable within established farming practices.

Current Progress The main focus of the work during this period was to develop a reliable

challenge model that would reproduce necrotic enteritis (NE) in chickens. A number of challenge models were evaluated but none yielded a high percentage of NE in challenged birds, with obvious NE only occurring in only around 5% of birds, to a maximum of 16% of the birds in one trial. However, mild NE was detected, ranging from 20-32% of birds across all groups, but this level of NE was considered inadequate for a reliable assessment of vaccine efficacy. Not withstanding this, a study was undertaken to assess the efficacy of a commercial clostridial vaccine delivered intradermally to protect against C. perfringens (the organism that causes NE) challenge. No significant differences were seen between the vaccinated and non-vaccinated groups. The role of the intestinal contents in inhibition of clostridial toxin was evaluated and in one trial 15/18 challenged birds contained fully or partially neutralising agents against C. perfringens alphatoxin that could be destroyed by heating. For the remainder of 2003 additional work will be undertaken towards the development of an alternative challenge model and alternative ways of delivering potential vaccine antigens. The natural inhibitors of C. perfringens alpha toxin will be further assessed also.


Project Title

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

RIRDC Project No.:

UNE-75A

Start Date: 1/10/00 Finish Date: 30/09/03 Researcher: A/Prof. Mingan Choct and Dr. Andreas Kocker Organisation: University of New England

School of Rural Science and Agriculture ARMIDALE NSW 2351

Phone: (02) 6773 5121 and (02) 6773 2778 Fax: (02) 6773 3275 and (02) 6773 3922 Email: [email protected] and [email protected] Objectives

• To establish a model in which necrotic enteritis (NE) can be successfully

induced in 2-4 week old broiler chickens. • To test the efficacy of various feed supplements, including enzymes, organic

acids and prebiotics, in controlling the occurrence of NE, maintaining and improving bird performance and as replacement strategies for currently used antibiotic growth promotants.

Current Progress In four experiments, the efficacy of feed enzymes, organic acids, a prebiotic and

a probiotic as alternative feed supplements to the currently used antimicrobial growth promotants (AGP) in broiler feed was tested in a previously establish challenge model. Alternative supplements were added directly to the experimental feed according to the supplier’s recommendations and all diets were cold pelleted. In all four experiments conducted, birds challenged with Clostridium perfringens (CP) without any supplement (CP control) had lower bodyweights and increased feed conversion ratios (FCR) at 32 days of age compared to birds fed the control diet with AGP. These differences would suggest that in these experiments, birds were successfully infected with sub-clinical clostridial enteritis. There were no significant differences between the unsupplemented control group (CP control) and diets containing alternatives to AGP. However, birds fed diets supplemented with organic acid or glycanase enzyme had numerically improved bodyweights and reduced FCR compared to the CP control. Similarly, the addition of a fruit extract (representative of a prebiotic) or a probiotic product (based on B. subtilus) numerically improved bodyweights and FCR compared to the CP control. The results of these four experiments indicate that some of the tested products can potentially maintain performance of birds in the face of challenge with CP. However, at this point further studies are necessary to clearly establish the ability of these products to significantly reduce the severity of dysbacteriousis and subclinical or clinical necrotic enteritis.


Bird Nutrition and Feed Supply

Project Title

Evaluation of new millet varieties as a poultry feed ingredient

RIRDC Project No.:

DAQ-302A

Start Date: 21/10/02 Finish Date: 30/04/04 Researcher: Mr. Danny Singh Organisation: Department of Primary Industries (Qld)

Animal Research Institute Locked Mail Bag No 4 MOOROOKA QLD 4105


• An evaluation of the nutritional value of Australian pearl millet varieties as a

high quality feed for poultry. • Recommendations to the poultry industries on the nutritional value of pearl

millet in least-cost diet formulation. • Promotion of the benefits of pearl millet varieties as a feed grain to both

grain growers and poultry producers.

Current Progress Fifty three lines of pearl millet were analysed for protein and amino acid content. The protein content ranged from 9.0% to 17.9%. Seventy five percent of the samples contain greater than 14.0% protein. Only two samples had less than 10% crude protein, 5.5% had between 10.1% and 12.0% crude protein, and 16.6% had between 12.1% and 14.0% crude protein. The amino acid profile of the lines showed similar trends. Based on the protein content and amino acid profile, as well as other favorable agronomic characteristics, three combinations of the parent lines were crossed. Seeds from these crosses were planted at Biloela Research Station, Biloela, Queesland. These will be harvested in late June and the first series of broiler feeding experiments are scheduled to start in July.


Project Title

Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets

RIRDC Project No.:

UWA-76J

Start Date: 01/05/03 Finish Date: 21/01/05 Researcher: Dr. Ian Williams Organisation: University of Western Australia

Animal Science CRAWLEY WA 6009


• 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.

• To destroy the thick cell walls and their main antinutritional factor, pectic substances, by expansion and enzymatic treatment.

• 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.

Current Progress At the time of preparing this report the project had been underway for one

month. An appointment to the position of research assistant on the project was made on the 24th April, 2003. All incubation equipment has been purchased and assembled. Buffers and reagents have been prepared and all necessary chemicals purchased or ordered. AB Enzymes GmbH, Darmstadt, Germany has agreed to supply the pure enzymes, polygalacturonase and pectin methyl esterase, free of charge. Two applications to the Animal Ethics and Experimentation Committee have been prepared and submitted for experiments to commence in the coming six months. The objectives and approach of these experiments are as follows. The first will investigate whether a more complete breakdown of the pectins in the cell walls of lupins can be achieved by incubating them with two enzymes, pectin methyl esterase and polygalacturonase, instead of polygalacturonase alone. It is believed that these two enzymes might act synergistically. An in vitro system has been established to test this hypothesis. This study will require about four months to run the assay and complete the analysis of viscosity, water-holding capacity, filtration rate, cell walls, pectin, methyl ester, length of pectin and molecular weight of pectin. The second experiment is aimed at defining the optimal dose of pectinase in lupin-based diets for egg layers. The benefits and optimal dose of pectinase for broiler chickens fed dehulled, lupin-based diets has previously been established. This layer experiment will compare two levels of lupins (10% and 20%), two types of lupins (whole and dehulled) and four doses of pectinase (0, 0.6, 0.8 and 1.0 g/kg diet). Food and water intake, weight gain, metabolisable energy, faecal moisture and egg quality (Haugh unit, shell thickness, yolk colour and soiled eggs) will be measured. It is anticipated that this experiment will be commenced in September 2003 and be completed by April 2004.


Project Title

The net energy values of the Australian feed ingredients for poultry

RIRDC Project No.:

UNE-82J

Start Date: 01/07/02 Finish Date: 30/11/03 Researcher: A/Prof. Mingan Choct Organisation: University of New England



• To measure the net energy (NE) value of the common Australian feed

ingredients for broilers and layers. • To compare performance of birds fed diets formulated on the basis of NE

and apparent metabolisable energy (AME).

Current Progress The project will investigate whether formulation of poultry diets using the net energy (NE) value will offer advantages over using the apparent metabolisable energy (AME) value, which is the current default system of energy measurement. Therefore the commonly used feed ingredients such as wheat, sorghum, barley, millrun, soybean meal, canola meal, and meat and bone meal will be tested for their net energy values in layers and broilers using a closed-circuit calorimetric system. Then two commercial-type diets will be formulated, one using the NE values and the other the AME values, to compare feed efficiency and growth rate of broilers in a semi-commercial environment. All preparatory work, including sourcing of ingredients, construction of new closed circuit calorimeters in addition to the four existing ones, and obtaining animal ethics approval and rearing layers for the trials, has been completed. Trials will commence before the end of June. It is estimated that the work will all be completed by November 2003.


Project Title

Estimating lysine availability by slope-ratio chick assay

RIRDC Project No.:

DAQ-277A

Start Date: 01/04/02 Finish Date: 31/03/03 Researcher: Dr. Rider Perez-Maldonado Organisation: Department of Primary Industries (Qld)

QPRDC PO Box 327 CLEVELAND QLD 4163


• To establish and validate a slope-ratio chick assay to determine the

availability of lysine in selected samples of canola meal and cottonseed meal.

• To compare lysine availability values with ileal apparent digestibility values determined in the same samples of canola meal and cottonseed meal.

Current Progress During December 2002, following the completion of a pilot study, a

comprehensive trial was conducted including a basal diet based on wheat, sorghum, gluten wheat, starch, dextrose, rice hulls and vitamins/minerals to produce a diet containing 0.4% lysine. Synthetic lysine was added to this basal diet to obtain iso-energetic mash diets with lysine contents of 0.4, 0.47, 0.54, 0.61 and 0.68%. The trial evaluated the lysine availability of solvent extracted canola meals (CM) from Melbourne, Numurkah (Victoria), New South Wales, and an expeller-extracted CM from Boree (NSW). A commercial (Riverina) solvent extracted cottonseed meal (CSM) from Narrabri was evaluated as well. Each diet was offered to a group of six birds replicated four times in a randomised block design in metabolisable cages within a controlled environment house. Due to some difficulties experienced with the conduct of this initial trial, it was decided that the trial would be repeated, with some modifications made to amino acid specifications and feed preparation methods. Therefore, during April-May 2003 a new growth trial was carried out which included similar test ingredients as in 2002, but diets were prepared as steam pelleted crumbles and specifications were modified to supply higher amino acid contents. The results of this new trail were satisfactory. Preliminary statistical analysis of lysine bioavailability (LBA) estimates indicate that CSM has a LBA coefficient of 0.56 and CM from Melbourne, Numurkah and Boree have LBAs of 0.83, 0.91, and 1.1 respectively. This indicates that the bioavailability of lysine in canola meal is superior to that of cottonseed meal and that the bioavailability of lysine in extruded meals is higher than for solvent extracted meals. It is also significant that, for CSM, the bioavailability of lysine demonstrated in this trial is similar to lysine digestibility values obtained in previous trials. These low lysine digestibility and availability values (0.56) are almost certainly due to anti nutritional factors present in the meal, such as condensed tannins and residual gossypol. Heat during processing may also have a negative effect. Further statistical analyses are being conducted to include a multiple regression model and more regression analysis for feed conversion ratio versus % lysine.


Project Title

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

RIRDC Project No.:

UNE-86A

Start Date: 1/02/03 Finish Date: 31/01/06 Researcher: A/Prof. Mingan Choct Organisation: University of New England



• To elucidate mechanisms by which grain processing and feed constituents

affect the gut physiology of birds from early life, which in turn affects life-long productivity.

Current Progress It is widely accepted that the nutrient digestibility of a feedstuff gives a direct

indication of the performance of the bird it is fed to. There are however instances where highly digestible feedstuffs are poorly digested by the broiler chicken, passing through to the posterior digestive tract, with little of the feed being digested and absorbed on the way through. This may allow fermentative digestion, and hence proliferation of pathogenic bacterial species, leading to reduced performance and morbidity. In recent studies it has been shown that the presence of coarse particles (coarse indigestible fibre) in the feed of chickens improves the development of anterior digestive tract organs, helps regulate digesta flow through the tract and improves starch digestibility. This project aims to elucidate mechanisms by which the improvement in the development of the digestive tract of broilers post-hatch may be affected through feed texture/structure manipulation, with the goal of improving life-long productivity. A trial commencing on 29/7/03 will investigate the effect of sorghum particle size on gut physiology, digestive enzyme secretion and performance of male and female broilers, from 1 to 45 days of age.


Project Title

Use of dietary fatty acids to increase protein accretion in broilers

RIRDC Project No.:

US-104A

Start Date: 01/02/01 Finish Date: 30/04/04 Researcher: Dr. Ron Newman Organisation: The University of Sydney

Faculty of Veterinary Science 405 Werombi Rd CAMDEN NSW 2570


• To develop a simple feed technology to reduce fat deposition and increase

muscle protein accretion in broilers. This will be achieved by manipulating dietary fatty acid intake to alter tissue sensitivity to the metabolic hormones involved in lipid and protein metabolism.

• To improve nutrient utilisation and reduce in feed costs through the adoption of this technology.

Current Progress Previous research undertaken by this research group has shown that feeding

broilers diets containing 50g/kg of n-3 polyunsaturated fatty acids (PUFAs) for six weeks alters body composition while feeding n-6 PUFAs improves broiler performance when compared to feeding a more saturated fat. Recent studies have investigated feeding broilers varying ratios of n-3 and n-6 PUFAs to determine the fatty acid ratio required to optimise both carcass composition and broiler performance. Although this study did not identify a definitive n-3:n-6 PUFA dietary ratio, the results confirmed a positive correlation for n-6 PUFAs and performance and a positive correlation with n-3 PUFAs and body composition. A more recent study that used these same dietary n-3:n-6 PUFA ratios but in a pelleted form demonstrated an improvement for all dietary groups in both feed efficiency and growth. In addition, these same pelleted diets resulted in a significant reduction in abdominal fat deposition at 30g/kg n-3 PUFA inclusion. Therefore, feeding broilers 30g/kg of n-3 PUFAs and 30g/kg of n-6 PUFAs will optimise performance and significantly improve body composition. This finding is currently being confirmed.


Project Title

Digestible amino acids and improved broiler performance

RIRDC Project No.:

UQ-107A

Start Date: 01/04/03 Finish Date: 31/03/06 Researcher: Prof. Wayne Bryden Organisation: The University of Queensland

School of Animal Studies GATTON QLD 4343


• To improving broiler performance by • 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.

Current Progress The focus of this project, which has just commenced, is on the contribution of cereal grains to the dietary supply of amino acids for broiler growth and production. The initial studies of the project will concentrate on the amino acid supply from wheat and sorghum. The digestible amino acid content of both cereals will be assessed using standard methodology and will examine both cereal grains across a range of protein contents. Samples are presently being obtained that give a wide range of different protein contents for both cereal grains.


Project Title

Premium grains for livestock program (stage 2)

RIRDC Project No.:

GRD-3J

Start Date: 01/07/00 Finish Date: 30/06/04 Researcher: Dr. John Black Organisation: John L Black Consulting

Locked Bag 21 WARRIMOO NSW 2774


• To develop rapid and objective analytical tests for assessing the quality of

feed grains. • To enhance grain nutritional value through breeding and processing. • To develop a model(s) to predict feed grain quality for certain livestock

species. • To provide an integrated and coordinated framework in which the above

objectives can be achieved.

Current Progress This project is the second stage of a major research program for improving feed grains quality and marketing that has been negotiated in response to identified industry needs. Several experiments have been conducted over the past year to further quantify the characteristics of cereal grains that determine their energy value for broilers, laying hens, pigs, cattle and sheep. Rice was included for the first time and produced the highest apparent metabolisable energy (AME) values for broilers yet recorded in the Program of around 17.5 MJ/kg dry matter. Another experiment showed that germination substantially increased (≅ 1 MJ/kg) the AME of a barley sample for broilers, but had relatively minor affects on the AME of wheat, sorghum and triticale. Comparisons of the energy value of the same grains when offered to broiler chickens and laying hens are continuing and there are substantial differences for some grains. Although statistical analyses are to be completed, the AME of a low amylose, naked barley (Merlin), was almost 1.5 MJ/kg higher in layers than in broilers. A difference of similar magnitude was observed for naked, high lipid oats. However, the differences between broilers and laying hens were small for some other grains. The Premium Grains for Livestock Program has been extended for a year to allow completion of experiments using grains with specially selected characteristics, full statistical analyses, evaluation of concepts and development of NIR assays for industry.


Food Safety

Project Title

Development and validation of Campylobacter microarrays for virulence detection and strain differentiation in poultry products

RIRDC Project No.:

RMI-14A

Start Date: 01/06/02 Finish Date: 31/05/05 Researcher: Prof. Peter Coloe Organisation: Royal Melbourne Institute of Technology



• To utilise existing knowledge on Campylobacter spp. to select the most

appropriate virulence factors for use in the development of a Campylobacter microarray. The development will involve the design and selection of appropriate oligonucleotides through the use of bacterial genomic techniques and alignment of gene sequences from known virulence factors to the Campylobacter genome.

• To also look into the design of microchips for the immobilisation of oligonucleotides and hybridsation conditions for simultaneous detection of a variety of selected virulence factors.

Current Progress A comprehensive literature review and a genomic database review on the

virulence determinants of Campylobacter spp. have been undertaken. The objective of this review was to identify genes that play a role in virulence, and especially those genes that may function differently in the chicken compared to how they function in mammals. In addition, a large collection of Campylobacter spp and a collection of individual strains of C jejuni from a variety of sources has been established and new strains will be added as needed The work, on-going and planned for the next six months, involves alignment of campylobacter gene sequences and the synthesis of a selection of specific primers for campylobacter specific genes which could be associated with virulence. These will be used to screen, using conventional PCR methods, a selection of the Campylobacter spp isolates for the presence or absence of these potential virulence factors. Once the methodology is established it will be extended to detecting mRNA to show the level of expression of the particular genes.


Project Title

On-farm reduction strategies for Campylobacter spp.

RIRDC Project No.:

DAQ-282A

Start Date: 01/01/02 Finish Date: 31/12/04 Researcher: Ms. Jillian Templeton Organisation: Department of Primary Industries (Qld)

Agency for Food and Fibre Sciences Locked Mail Bag No 4 MOOROOKA QLD 4105


• To develop targeted control strategies, based on knowledge of sources of the

organism, to prevent the entry of Campylobacter spp. into broiler flocks.

Current Progress A cross-sectional study to determine the on-farm prevalence of Campylobacter spp. has been completed. The study examined 64 randomly selected meat chicken farms prior to partial depopulation. The study also examined potential on-farm sources of Campylobacter spp. including water supply, re-used litter, darkling beetles/larvae, warm-blooded animals, wild birds and flies. Only 17 of the 64 farms tested were Campylobacter-positive (26.6%). This is a much lower prevalence than was obtained in our 1999 cross-sectional study where 26 of 56 farms tested were Campylobacter-positive (46.4%). Of the potential sources examined, flies collected around pick-up crates were the most frequently colonised. From August-December 2002 longitudinal studies were conducted to clarify the role that flies play in the transmission of Campylobacter spp. The results suggest that flies are not an important source of introduction of Campylobacter spp. to the flocks. During 2003, our focus has been to investigate if darkling beetles (Alphitobius diaperinus) play a role in the transmission of Campylobacter spp. On-farm longitudinal studies and artificial infection experiments are being performed.


Project Title

Tannins to control microbial pathogen colonisation of broiler chickens

RIRDC Project No.:

UA-61A

Start Date: 01/04/03 Finish Date: 31/03/04 Researcher: A/Prof. John Brooker Organisation: The University of Adelaide

Animal Science Department Roseworthy Campus ROSEWORTHY SA 5371


• To establish the optimum inclusion levels of grape seed tannins in broiler

chicken diets without compromising growth rates or having other anti-nutritional effects on the chickens.

Current Progress This is a small feeding trial to establish the correct inclusion rate of grape seed

tannins in a broiler chicken diet. This information will then be used for future research into the effectiveness of this treatment as a means of limiting colonisation of broiler chickens by microbial pathogens. The experimental protocol for this work has been established, but because of space and staff considerations, the trial will not be conducted until approximately September, 2003.


Environmental Management

Project Title

Meat chicken EMS: transfer to industry

RIRDC Project No.:

FSE-2A

Start Date: 01/03/03 Finish Date: 30/11/03 Researcher: Mr. Eugene McGahan Organisation: FSA Environmental

PO Box 2175 TOOWOOMBA QLD 4350


• To develop a practical, competency based training and assessment workshop

package in "Environmental Management Planning and Training for Meat Chicken Producers".

• To recommend methods for facilitating on-site audit processes for meat chicken farms.

Current Progress Early work on the project has involved the development of the materials

required for an environmental management training package. These materials include a participant’s training manual, a facilitator’s manual and a series of overheads to assist facilitators conducting the training course. The first draft of these documents will be available for comment by30 June 2003. Once the first drafts of these documents have been edited, pilot training will be conducted with industry representatives. The training package will be designed to assist meat chicken farmers in developing and implementing a site-specific Environmental Management System (EMS). The meat chicken farm EMS identifies and evaluates potential environmental risks, it includes the implementation and maintenance of an environmental management plan (EMP), and it requires independent auditing to verify that the system is working. The training package will take the form of a risk-based assessment for individual farmers. Participants will leave the training course with an understanding of how their operation may impact on the environment and a good, draft environmental management plan for their enterprise.


Project Title

Sustainability improvements in the Victorian chicken meat industry (Phase 2)

RIRDC Project No.:

JSC-2A

Start Date: 01/02/03 Finish Date: 31/05/04 Researcher: Mr. Jim Smith Organisation: James Smith Consulting

55 Sims Street SANDRINGHAM VIC 3191


• To achieve broader uptake of the Chicken Care best practices by Victorian

broiler growers, and more specifically: • To identify the extent of implementation of Chicken Care by growers and

address any barriers to its further adoption. • To identify and provide improvements to Chicken Care information transfer,

processes and tools as required by the community, regulators and growers. • To incorporate the RIRDC National Environment Management System

(EMS) and the industry biosecurity and animal welfare audit programs into the Chicken Care best practices model.

Current Progress Chicken Care was launched by the Victorian chicken meat industry in August

2000 to improve the industry’s environmental performance and to address broad community concerns in this area. The steering committee for this project reviewed the status and effect of the Chicken Care initiative as at May 2003 and concluded that, overall, (a) grower operations and implementation of best practices have improved and that progress was being made by the twenty farms previously the subjects of community complaints, (b) local councils and the EPA have publicly acknowledged that growers have become more cooperative with neighbours and agencies and are addressing issues raised, (c) effective information tools and training are being used by 40% of Victorian growers, and (d) the industry continues to need the Chicken Care program and needs to find ways as provided in this project for its broader uptake by growers. A survey to measure adoption level of the best practices model was sent to the 120 Chicken Care trained growers in late 2002. Follow-up assistance from processor company servicemen has now been arranged to achieve the response rates necessary for a reliable analysis of adoption to be undertaken by end-June. Work has commenced to update the Chicken Care best practices model. This will incorporate the recently published national animal welfare, biosecurity and environmental management standards. It will reduce the complexity of grower paperwork and implement recent suggestions from growers, the community advisory panel (CCAP) and the program steering committee (CCSC). A process for external verification of grower self-auditing of their environmental management has been developed and trialled at four farms. Processor auditors and members of CCAP and CCSC carried out the verification which was concluded to be a useful process and confirmed that 95% of grower self-assessments were accurate or understated. One productive meeting for each of CCAP and CCSC has been convened so far in 2003. Four new local councils have joined CCAP, and the CCSC has begun a process to realign its grower members to those responsible for Chicken Care


participation in each branch. Training workshops planned for April have been rescheduled to August/September so they can focus on the revised best practices model. A processor/grower workgroup has developed the draft framework for a guidance note covering Broiler Farm OHS Hazards – an area of weakness identified in previous surveys. The topics for three other guidance notes to be developed (Shed Clean-Out, Typical Maintenance Requirements and Landscaping) have been agreed by the project CCSC.


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SCHOLARSHIPS

CSA-13J Postgraduate scholarship - Jacqueline Kattenbelt: Analysis of virulence determinants of Newcastle

disease virus Refer to report page 29 CSA-20A Postgraduate scholarship - Scott Sheedy: Live vectoring of therapeutic and prophylactic proteins and

pathogenesis in necrotic enteritis Refer to report page 41 CSA-21J Postgraduate scholarship - Manija Asif: Cytokines and innate molecules for enhanced mucosal

immunity in the chicken Refer to report page 39 CSA-25J Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus infection

Refer to report page 40 MS023-03 Masters in avian health - Dr Reza Fadavi Firooz UTS-6A Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the

apicomplexan parasite, Eimeria maxima Refer to report page 34 UNE-86A Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal

integrity, and performance of broiler chickens Refer to report page 48 PROGRAM REVIEW AND DEVELOPMENT MS001-55 Environmental Management Sub-Program Steering Committee MS001-58 Collaborative necrotic enteritis steering group MS023-28 Steering committee meetings with Marek's disease researchers WS023-22 Strategic Planning Workshop, Sydney, 10 July 2003 TRAVEL/CONFERENCE/WORKSHOPS TA023-19 2003 Australian Poultry Science Symposium, February 2003 - funding for two invited speakers TA023-22 12th International Conference Negative Strand Viruses 2003, Italy, June 2003 - Ms Jacqueline

Kattenbelt TA023-24 XIII World Veterinary Poultry Congress, USA, July 2003 - Dr Trevor Bagust TA023-25 XIII World Veterinary Poultry Congress, USA and IBDV/CAV meeting, Italy, June 2003 - Dr Jagoda

Ignjatovic TA023-26 XIII WVPA Congress, Denver, July 2003 - Dr Graham Burgess WS001-07 Support for the 12th Australian Poultry and Feed Convention/7th WPSA Asian Pacific Federation

Conference, Gold Coast, October 2002 WS023-09 National Biosecurity Workshops

RESEARCH TO BE SUPPORTED IN 2003/2004

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Flock Health Project Title Project No Principal Investigator Organisation Phone No Biological control of necrotic enteritis in meat chickens CME03-01 Dr Robert Moore CSIRO Livestock Industries (03) 5227 5760 New diagnostic assays to improve control of coccidiosis in poultry CME03-13J Dr Glenn Anderson Department of Primary Industries

(Qld) (07) 3362 9494

Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics CME03-15J Dr Jagoda Ignjatovic CSIRO Livestock Industries (03) 5227 5769 Diagnostic tools for differentiation of vvIBDV and characterisation of Australian strains CSA-15J Dr Jagoda Ignjatovic CSIRO Livestock Industries (03) 5227 5769 The effect of Newcastle disease vaccination with strain V4 on the course of infections with the Peats Ridge strain of Newcastle disease virus

CSA-18J Dr Peter Daniels CSIRO Livestock Industries (03) 5227 5272

Postgraduate scholarship - Scott Sheedy: Live vectoring of therapeutic and prophylactic proteins and pathogenesis in necrotic enteritis

CSA-20A Dr Robert Moore CSIRO Livestock Industries (03) 5227 5760

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

CSA-21J Dr Andrew Bean CSIRO Livestock Industries (03) 5227 5792

Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates CSA-24J Dr Hans Heine CSIRO Livestock Industries (03) 5227 5278 Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus infection

CSA-25J Dr Andrew Bean CSIRO Livestock Industries (03) 5227 5792

Use of cytokines to enhance vaccine efficacy in poultry CSA-26J Dr Andrew Bean CSIRO Livestock Industries (03) 5227 5792 Investigations into the development of a sustainable management strategy for the darkling beetle, Alphitobius diaperinus (Panzer) in broilers

DAQ-273A Dr Trevor Lambkin Dept of Primary Industries (Qld) (07) 3896 9434

Advanced clinical diagnostics: use of real-time immuno-PCR and LightUp probes MUL03-36 Dr Debby Cousins Department of Agriculture (WA) (08) 9368 2429 The development of vaccination strategies to control necrotic enteritis in poultry RMI-11A Prof Peter Coloe Royal Melbourne Institute of

Technology (03) 9925 2481

Molecular evaluation of responses to vaccination and challenge by Marek's disease virus RMI-12J Prof Greg Tannock Royal Melbourne Institute of Technology

(03) 9925 3088

Marek's disease research in Australia - a review RMI-15J Prof Greg Tannock Royal Melbourne Institute of Technology

(03) 9925 3088

Molecular techniques for monitoring Marek's viraemias in broilers and layers UJC-10J Dr Graham Burgess James Cook University (07) 4781 5472 Avian Leukosis-J (ALV-J) in Australia: laboratory technologies and research needs UM-49A Dr Trevor Bagust University of Melbourne (03) 9344 9676 Avian leukosis subgroup-J (ALV-J): epidemiological studies for control of ALV-J in Australian broiler breeder flocks

UM-68A Dr Trevor Bagust University of Melbourne (03) 9344 9676

Control of intestinal spirochaete infections in chickens UMU-29J Prof David Hampson Murdoch University (08) 9360 2287 Effects of organic acids, prebiotics, and enzymes on control of necrotic enteritis and performance of broiler chickens

UNE-75A A/Prof Mingan Choct University of New England (02) 6773 5121

Systematic pathotyping of Australian Marek's disease (MDV) isolates UNE-83J A/Prof Stephen Walkden-Brown

University of New England (02) 6773 5152


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Typing of Pasteurella multocida UQ-100J A/Prof Linda Blackall University of Queensland (07) 3365 4645 Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis UTS-4J A/Prof Nicholas Smith University of Technology, Sydney (02) 9514 4013 Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the apicomplexan parasite, Eimeria maxima

UTS-6A A/Prof Nicholas Smith University of Technology, Sydney (02) 9514 4013

Investigations into control strategies for the darkling beetle Mr Trevor Lambkin Dept of Primary Industries (Qld) (07) 3896 9434 Bird Nutrition and Feed Supply Project Title Project No Principal Investigator Organisation Phone No Early feeding of prebiotics on development of the digestive system and gut microflora of broilers

CME03-22 A/Prof Mingan Choct University of New England (02) 6773 5121

Premium grains for livestock program extension CME03-57 Dr John Black Grains Research & Development Corporation

(02) 4753 6231

Evaluation of new millet varieties as a poultry feed ingredient DAQ-302A Mr Danny Singh Dept of Primary Industries (Qld) (07) 3824 3081 The net energy values of the Australian feed ingredients for poultry UNE-82J A/Prof Mingan Choct University of New England (02) 6773 5121 Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal integrity, and performance of broiler chickens

UNE-86A A/Prof Mingan Choct University of New England (02) 6773 5121

Digestible amino acids and improved broiler performance UQ-107A Prof Wayne Bryden The University of Queensland (07) 5460 1253 Use of dietary fatty acids to increase protein accretion in broilers US-104A Prof Wayne Bryden University of Sydney (02) 4655 0658 Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets UWA-76J Dr Ian Williams University of Western Australia (08) 9380 3780 Support for Chair in Poultry Science, University of Sydney Dr Tom Scott University of Sydney (02) 4655 0612 Food Safety Project Title Project No Principal Investigator Organisation Phone No Campylobacter bio-replacement program to control food poisoning organisms in poultry CME03-29 Dr Victoria Korolik Griffith University (07) 5552 8321 On-farm reduction strategies for Campylobacter spp. DAQ-282A Ms Jillian Templeton Department of Primary Industries

(Qld) (07) 3362 9520

Salmonella typing and colonisation of chickens by characterised S. Sofia IMV-3A Dr Michael Heuzenroeder Institute of Medical & Veterinary Science

(08) 8222 3275

Development of a sequence-based bacteriophage typing system for Salmonella IMV-5A Dr Michael Heuzenroeder Institute of Medical & Veterinary Science

(08) 8222 3275

Development and validation of Campylobacter microarrays for virulence detection and strain differentiation in poultry products

RMI-14A Prof Peter J Coloe Royal Melbourne Institute of Technology

(03) 9925 7104

Tannins to control microbial pathogen colonisation of broiler chickens UA-61A A/Prof John Brooker The University of Adelaide (08) 8303 7638 Development of campylobacter bio-replacement program and establishment of campylobacter reference centre

UG-3A Dr Victoria Korolik Griffith University (07) 5552 8321

Environmental Management Project Title Project No Principal Investigator Organisation Phone No Evaluating risks posed by pathogen emissions from meat chicken sheds CME03-44 Dr Pat Blackall Department of Primary Industries

(Qld) (07) 3362 9498


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Efficacy of windbreak walls for odour reduction CME03-56 Mr Geordie Galvin Department of Primary Industries (Qld)

(07) 4688 1118

Meat chicken EMS: transfer to industry FSE-2A Mr Eugene McGahan FSA Environmental (07) 4632 8230 Sustainability improvements in the Victorian chicken meat industry (Phase 2) JSC-2A Mr Jim Smith James Smith Consulting (03) 9598 8717 Trials of odour control technologies for broiler farms Dr Julie Simons Department of Primary Industries

(Vic) (03) 9217 4358

Risk assessment of the use of litter products as fertilizer/soil conditioner and development of guidelines for its safe and sustainable use

Mr Peter Nicholas FSA Environmental (07) 4632 8230

Other Project Title Project No Principal Investigator Organisation Phone No CRC for the Australian Poultry Industries CPO-1A A/Prof Mingan Choct CRC for the Australian Poultry

Industries (02) 6773 5121

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