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RIRDC Completed Projects in 2004-2005 & Research in Progress as at June 2005

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CHICKEN MEAT PROGRAM - COMPLETED PROJECTS

Flock Health CSA-20A Postgraduate scholarship - Scott Sheedy: Live vectoring of therapeutic and prophylactic proteins and pathogenesis in necrotic enteritis DAW-113A Advanced Clinical Diagnostics: the use of real-time immuno-PCR and LightUp probes Food Safety DAQ-282A On-farm reduction strategies for Campylobacter spp. Environmental Management FSE-3A Risk Assessment on the Use of Chicken Litter and Guidelines for its Safe Use

Flock Health
 

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:	01/03/2002
Finish Date:	28/02/2005
Researcher:	Dr. Robert Moore
Organisation:	CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220
Phone:	(03) 5227 5760
Fax:	(03) 5227 5555
Email:	rob.moore@csiro.au
Objectives	·1 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. ·2 To investigate the importance of the alpha toxin produced by Clostridium perfringens in the pathogenesis of necrotic enteritis.
Background	The Australian poultry industries are seeking to minimize the use of antibiotics and other chemicals to control disease. Necrotic enteritis is one disease that is likely to cause significant problems to poultry producers following the removal of antibiotics. It would therefore be desirable to have other treatment strategies available for this disease. There are a number of different classes of protein that could be used as natural therapeutics to control disease. Such proteins include cytokines, antimicrobial peptides, and vaccine antigens. One problem in using these proteins is to effectively deliver them to the sites were action is required. The focus of this study was to investigate the potential of live bacteria as vectors for the delivery of therapeutic proteins. The bacterium C. perfringens is the causative agent of necrotic enteritis. To understand the genesis of the disease it is important to understand the pathogenic mechanisms used by the bacterium. The alpha toxin produced by C. perfringens is believed to be an important virulence factor so we studied the primary sequence of the toxin from a range of local field isolates.
Research	Our aim was to identify non-pathogenic, commensal bacteria which were able to colonise and persist in the gut of chickens. We had to be able to manipulate the persistent strains so all candidate strain s were tested for their ability to be transformed with plasmids and to express a model recombinant protein. Following the identification of suitable strains they were transformed with plasmids expressing either a cytokine gene or a gene encoding an antimicrobial peptide and introduced into birds to determine if they exerted a biological effect in vivo. To investigate the alpha toxin we sequenced the gene from 27 field isolates and compared the sequences with published sequences from unrelated strains.
Outcomes	A collection of 100 Escherichia coli isolates from local chickens were characterized by pulse field gel electrophoresis of their genomic DNA to identify 25 distinct types. Representative isolates of each type were tested in a number of bird trials to determine which strains were able to colonise and persist throughout the typical 42 day life span of a broiler chicken. Three isolates had the required characteristics and all could be efficiently transformed and expressed the model recombinant protein, green fluorescent protein. Transformation of the strains with genes expressing chicken interleukin-6 (IL-6) and the antimicrobial peptide piscicolin 126 (P126) showed that the isolates could express these protein in vitro and the expressed proteins had the appropriate biological activity in in vitro assays. When isolates expressing these proteins were used to colonise the guts of chickens it was clear that IL-6 was produced and exerted a local biological effect in priming lymphocytes for higher levels of proliferation following stimulation. High levels of P126 activity could be recovered from the caecal contents of chickens colonized with the expressing construct. In one trial birds treated with a P126 appeared to be completely protected from necrotic enteritis however this could not be repeated in subsequent trials. We suspect that the local gut conditions may be reversibly denaturing the P126 and it may be possible to use a more appropriate antimicrobial peptide to provide reliable protection against disease. It is clear that the live bacterial vectors can be used to deliver biologically active proteins. The survey of alpha toxin genes from local isolates of C. perfringens showed that the sequences were very different from the only other previously published sequence from an avian isolate. The chicken isolates carry genes closely related to those found in C. perfringens from most other sources, including mammals.
Implications	This project has demonstrated the feasibility of using live bacteria vectors to deliver proteins to the site at which action is required, e.g. the gut. Unlike most previously reported work on live bacterial vectoring the strains that we have identified are able to persist in the host for long periods of time and hence can provide sustained release over the life of the birds. Further research is now required to define the most appropriate proteins to deliver to the birds to ameliorate the effects of C. perfringens in causing necrotic enteritis. With further development the basic concept outlined here will provide alternative treatment strategies for the Australian poultry industry.
Publications	Sheedy, S.A., Ingham, A.B., Rood, J.I., and Moore, R.J. (2004) Highly conserved alpha-toxin sequences of avian isolates of Clostridium perfringens. J. Clin. Microbiol. 42:1345-47.


 

Project Title:	Advanced Clinical Diagnostics: the use of real-time immuno-PCR and LightUp probes
RIRDC Project No.:	DAW-113A
Researcher:	Dr. Debby Cousins
Organisation:	Department of Agriculture Locked Bag 4 Bentley Delivery Centre BENTLEY WA 6983
Phone:	(08) 9368 3451
Fax:	(08) 9474 1881
Email:	dcousins@agric.wa.gov.au
Objectives	·1 To gain an understanding of the science and technology involved in the production and use of LightUp probes and the use of real-time immuno-PCR. ·2 To conduct initial studies into the production of LightUp probes and their use in routine diagnostics, focussing on the detection of Campylobacter subspecies as a model. ·3 To evaluate the feasibility of adopting this technology in Australia and the need for research partnerships.
Background	There is a need for rapid and accurate detection of pathogens, particularly those that affect safe food production or human and animal health. The focus is currently on molecular methods to do so. The use of peptide nucleic acids and real-time immuno-PCR are new advances that could provide significant benefits in routine diagnostics.
Research	Funding from this project facilitated the training of a young Australian scientist in two new technologies in the Swedish laboratory where the technologies were developed and are most advanced. A pilot project applying the new LightUp probe was undertaken in Sweden comparing this system with conventional PCR systems. Real-time PCR conditions were optimised for the detection of Campylobacter jejuni and Campylobacter coli. LightUp probes specific for each organism were then studied for their specificity and sensitivity using these conditions. A survey was conducted to ascertain the expectation of the Australian poultry industry in relation to the use of diagnostic technologies in their businesses.
Outcomes	A young Australian scientist was trained in real-time PCR, the use of LightUp probes and immuno-PCR technologies using both formal coursework and in-house methods. LightUp probes were shown to be more specific than standard real-time PCR detection systems in the pilot project. Although a decrease in sensitivity was noted in the pilot project using LightUp probes this is unusual for such systems and may have been due to sample storage issues. Good networks were established between researchers and supervisors in Australia and Sweden which will have benefits in establishing the technologies in the Department of Agriculture Western Australia, in facilitating development of future research projects and in technology transfer to other Australian laboratories. Increased understanding of the expectations of the poultry industry in terms of the use of new technologies.
Implications	This project has shown that there are benefits associated with real-time LightUp probes and in further assessment of immuno-PCR as a technique for identifying small amounts of antigen or protein. The establishment of these methods in routine diagnostics should be further developed.

Food Safety

Project Title	On-farm reduction strategies for Campylobacter spp.
RIRDC Project No.:	DAQ-282A
Start Date:	01/01/2002
Finish Date:	31/12/2004
Researcher:	Ms. Jillian Templeton
Organisation:	Department of Primary Industries (Qld) Agency for Food and Fibre Sciences Locked Mail Bag No 4 MOOROOKA QLD 4105
Phone:	(07) 3362 9520
Fax:	(07) 3362 9429
Email:	Jillian.Templeton@dpi.qld.gov.au
Objectives	· To develop targeted control strategies, based on knowledge of sources of the organism, to prevent the entry of Campylobacter spp. into broiler flocks.
Background	Food-borne illness caused by Campylobacter spp. is the most frequently reported notifiable disease in humans in Australia. Epidemiological evidence continues to implicate poultry as the most important source of human Campylobacter infection. In order to control the level of contamination of poultry products, the industry needs to understand when and how campylobacters are introduced to poultry flocks.
Research	Cross-sectional and longitudinal studies of Campylobacter spp. were conducted on broiler farms in South East Queensland. Investigations were conducted on potential sources and effective control strategies.
Outcomes	Only 26.6% of farms sampled were Campylobacter-positive at partial depopulation. Reservoirs of Campylobacter were found around poultry sheds (sheep, wallabies, wild birds). Drinking water, litter, flies and darkling beetles did not appear to be sources. Well-maintained footbaths, properly used, were shown to prevent transmission of the organism to the flock. Partial depopulation is the other point of introduction; transport crates were shown to be frequently contaminated on arrival at the farm.
Implications	The most effective control strategy prior to partial depopulation is a footbath at the shed entrance. Crate cleaning is difficult and expensive but improvements in control of farm to farm transmission could be achieved by preventing crates recently used for the transport of older birds being used for partial depopulation.
Publications	Results have been presented to the industry at seven meetings including: Miflin, JK and Templeton, JM 2002, ‘Studies in the epidemiology of Campylobacter spp. in poultry’, Scientific Meeting, Australian Veterinary Poultry Association, Gold Coast, Queensland. Templeton, JM and Miflin, JK 2002, ‘Flagellin gene typing is a powerful tool for studying the source and spread of Campylobacter jejuni in broiler flocks’, WPSA Asian Pacific Federation Conference, Gold Coast, Queensland. Templeton, JM and Miflin, JK 2003 ‘Update on research into Campylobacter spp. in broilers’, Queensland Poultry Science Symposium, Gatton, Queensland. Templeton, JM and De Jong, AJ 2004, ‘An evaluation of the role the darkling beetle (Alphitobiusdiaperinus) plays in the transmission of Campylobacter spp. in broiler flocks’, 5th Asia Pacific Poultry Health Conference, Gold Coast, Queensland.


 
 
 
 

Environmental Management
 

Project Title	Risk Assessment on the Use of Chicken Litter and Guidelines for its Safe Use
RIRDC Project No.:	FSE-3A
Start Date:	14/04/2004
Finish Date:	31/05/2005
Researcher:	Mr Peter Nicholas
Organisation:	Food Science Australia FSA Environmental PO Box 2175 TOOWOOMBA QLD 4350
Phone:	(07) 4632 8230
Fax:	(07) 4632 8057
Email:	peter.nicholas@fsaconsulting.net
Objectives	·1 To undertake a risk assessment on the use of raw and treated litter products for a range of different end-use practices. ·2 To develop a set of guidelines for the safe and sustainable use of litter and treated or composted litter products based on this risk assessment.
Background	Spent litter (litter that has been cleaned out of a shed following use) and spent litter products can add value to a range of production systems. Direct application of untreated spent litter is becoming more difficult due to increasingly stringent regulations governing the use of spent litter, increased competition from other organic fertilisers and increasing urbanisation of traditional poultry farming areas. A sound understanding of the properties of spent litter combined with a properly managed process for utilising spent litter will maximise the benefits and minimise potential negative impacts of spent litter to human/animal health and the environment.
Research	This project will build on previous research identifying the environmental risks associated with meat chicken litter management by quantifying any significant human health and environmental risks from chicken litter use. Practices to reduce risks will then be identified and validated. These will be developed into practical, user-friendly guidelines for the safe use of chicken litter. These will allow those spreading the litter to minimise human health, animal health and environmental risks from the spreading of chicken litter.
Outcomes	Adopting safe reuse guidelines will: ·3 Have clear social benefits for those living near chicken meat farms through a reduced risk of human health impacts.  ·4 Significantly reduce the potential for adverse environmental impacts to water and soil.  ·5 Allow the nutrients in litter to be spread at fertiliser rates, thereby providing total-farm economic gains. The draft risk assessment and guidelines are currently being finalised prior to circulation for comment.
Implications	These guidelines have identified a number of key areas requiring further research – these recommendations will be detailed within the final report.
Publications	No publications have arisen from this project as at 30 June 2005.

RESEARCH IN PROGRESS

Flock Health
CSA-24J Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates
CSA-25J Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus  infection
CSA-30J Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics
DAQ-316A New diagnostic assays to improve control of coccidiosis in poultry
DAQ-319A Preliminary investigations into the efficacy of novel treatments and application methods for darkling beetle (Alphitobius diaperinus [Panzer]) control in Australian broiler houses
UNE-83J Systematic pathotyping of Australian Marek's disease (MDV) isolates
UQ-100j Typing of Pasteurella multocida

Bird Nutrition and Feed Supply
UNE-86A Postgraduate scholarship - Mr Nicholas Rodgers: Relationships between grain quality, intestinal integrity, and performance of broiler chickens
UQ-107A Digestible amino acids and improved broiler performance
US-127A Establishment of a Chair in Poultry Sciences at The University of Sydney
US-133A Assessment of the anti-nutritive effects of phytate by dephytinisation
US-134A Early dietary and management intervention on broiler breast meat yield

Food Safety
IMV-5A Development of a sequence-based bacteriophage typing system for Salmonella
RMI-14A Development and validation of Campylobacter microarrays for virulence detection and strain differentiation in poultry products
UG-7A Campylobacter bio-replacement program to control food poisoning organisms in poultry

Environmental Management
DAQ-318A Evaluating risks posed by pathogen emissions from meat chicken sheds
DAQ-321A Efficacy of windbreak walls for odour reduction
DAV-213A Trials of odour control technologies for broiler farms
DAQ-323A Managing litter re-use for minimal nutrient runoff to surface water

Flock Health

Project Title	Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates
RIRDC Project No.:	CSA-24J
Start Date:	01/07/2002
Finish Date:	30/11/2005
Researcher:	Dr. Hans Heine
Organisation:	CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220
Phone:	(03) 5227 5278
Fax:	(03) 5227 5555
Email:	Hans.Heine@csiro.au
Objectives	·1 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. ·2 To develop a common assay format, enabling simultaneous testing for IBDV, NDV and AI as well as the differentiation of very virulent/highly pathogenic strains from circulating endemic non/low-pathogenic strains. ·3 To implement the new tests at the Australian Animal Health Laboratory (AAHL) to ISO17025 standards by June 2004.
Current Progress	The project has been extended as extra funding was provided by the Australian Biosecurity CRC for the development of diagnostic capabilities for influenza H5N1 isolates in view of the widespread occurrence of highly pathogenic avian influenza (HPAI) in Asia. The development of rapid diagnostic capabilities for H5N1complemented the RIRDC project goals that were focused on developing and improving the capabilities for diagnosis of Australian HP H7 isolates. All previously documented outbreaks of HPAI in domestic poultry in Australia have been of the H7 subtype. During the last year we have developed TaqMan real-time PCR tests that enabled the rapid and sensitive detection of influenza type A and the differentiation between subtypes H5 and H7 in diagnostic samples including cloacal swabs and tissue homogenates. Other published TaqMan PCR tests were biased towards the North American lineage of AI and had low analytical sensitivity or failed with the Eurasian lineage and Australian strains. We have optimized tests for the sensitive detection of the Eurasian lineage, including the predominant H5N1 isolates, and for the Australian HP H7 isolates that have been identified in previous outbreaks in poultry.


 
 
 
 

Project Title	Postgraduate scholarship - Kristie Jenkins: Improved therapeutics for Marek's disease virus infection
RIRDC Project No.:	CSA-25J
Start Date:	01/03/2003
Finish Date:	01/01/2006
Researcher:	Dr. Andrew Bean
Organisation:	CSIRO Livestock Industries Private Bag 24 GEELONG VIC 3220
Phone:	(03) 5227 5792
Fax:	(03) 5227 5531
Email:	Andrew.Bean@csiro.au
Objectives	·1 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 future threat to the Australian poultry industry. However, understanding the interactions of this virus and the host immune system may allow us to produce novel ways to combat this economically significant virus. This project centers on assessing the early response to MDV, particularly focusing on the innate immune response. Receptors involved in the innate immune response have been cloned in the chicken. The immune molecules produced when these receptors are stimulated have been partially characterized to determine their therapeutic potential. Understanding these receptors and the molecules they stimulate may allow the development of strategies to target them, resulting in enhanced response. Trials are underway to assess the therapeutic potential of molecules.


 
 

Project Title	Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics
RIRDC Project No.:	CSA-30J
Start Date:	01-Jul-03
Finish Date:	31-Jul-06
Researcher:	Dr. Sandra Sapats
Organisation:	CSIRO Livestock Industries Private Bag No 24 GEELONG VIC 3220
Phone:	(03) 5227 5769
Fax:	(03) 5227 5555
Email:	Sandra.Sapats@csiro.au
Objectives	·1 To monitor genetic changes in circulating IBDV strains (Molecular Epidemiology) in order to determine if field strains are changing significantly. ·2 To optimise the already developed Reverse Genetic system for IBDV for the genetic modification and the molecular characterisation of biological properties of IBDV. ·3 To apply the Reverse Genetics system for the construction of recombinant IBDV to ascertain the importance of specific mutations in VP2 gene related to virulence and antigenicity and in particular how these effect virulence of endemic strains.
Current Progress	Samples collected in 2004 from broiler farms located in Queensland, NSW and Tasmania were tested for the presence of IBDV. Virus was detected in two farms from Queensland and three farms from NSW with broilers ranging in age from 28-35 days of age. Both farms from Tasmania tested negative for IBDV. Sequence analysis of isolated virus revealed that the majority of isolates were genetically similar to Australian IBDV vaccine strains. One isolate from Queensland was a re-isolation of vaccine virus. One NSW isolate had four unique amino acid changes, three of which had been previously detected in a NSW isolate in 2003. None of these amino acid changes have been associated with increased virulence of IBDV.  The genetic material of an Australian IBDV (002/73) has been cloned. We have optimized a system that allows us to generate infectious virus from this genetic material. The viral VP2 protein is the major protein recognized by the chicken’s immune system and is believed to play a major role determining the virulence of the virus. We have made several amino acid changes within the VP2 protein and are determining the impact of these changes on disease severity in the chicken.


 

Project Title	New diagnostic assays to improve control of coccidiosis in poultry
RIRDC Project No.:	DAQ-316A
Start Date:	01/01/2004
Finish Date:	31/12/2006
Researcher:	Dr. Glenn Anderson
Organisation:	Department of Primary Industries & Fisheries (Qld) Animal Research Institute Locked Mail Bag No. 4 MOOROOKA QLD 4105
Phone:	(07) 3362 9494
Fax:	(07) 3362 9429
Email:	glenn.anderson@dpi.qld.gov.au
Objectives	·1 To develop and validate new DNA-based quantitative assays for the diagnosis of poultry coccidiosis. To develop and validate new serological assays for the diagnosis of poultry coccidiosis.
Current Progress	Real time PCR probes specific for each of the seven Eimeria species have been designed using sequence data generated in the first year of the project and are now undergoing evaluation. Considerable progress has been made toward understanding the antibody response to infection in chickens and developing specific serological tests. Immunoblot tests specific for E. tenella and E. necatrix have been developed and have undergone initial evaluation. Immunoblots with parasite antigens prepared from the gut of infected birds at varying times after infection suggested that the antibody response recognized exclusively gametocyte antigens. This was confirmed using immunoperoxidase and fluorescent antibody tests on sections of gut that demonstrated strong antibody binding to gametocytes but not to merozoites or oocysts. Gametocytes of E. tenella vaccine strain have now been purified from the gut of infected birds in useful quantities. The gametocyte antigen has been used in immunoperoxidase and fluorescent antibody tests to demonstrate the specificity of monoclonal antibodies developed previously. At least three monoclonal antibodies have been shown to bind to gametocytes. The purified gametocyte antigen is also being evaluated for development of a generic ELISA and species-specific ELISAs. Initial results suggest that the generic ELISA using gametocyte antigen could be much more sensitive and specific than an ELISA developed previously using oocyst antigen.


 
 

Project Title	Preliminary investigations into the efficacy of novel treatments and application methods for darkling beetle (Alphitobius diaperinus [Panzer]) control in Australian broiler houses
RIRDC Project No.:	DAQ-319A
Start Date:	01/07/2003
Finish Date:	31/12/2004
Researcher:	Mr. Trevor Lambkin
Organisation:	Department of Primary Industries & Fisheries (Qld) Entomology Building, Indooroopilly Research Centre 80 Meiers Road INDOOROOPILLY QLD 4068
Phone:	(07) 3896 9434
Fax:	(07) 3896 9446
Email:	trevor.lambkin@dpi.qld.gov.au
Objectives	·1 To undertake preliminary, mainly laboratory based investigations into the efficacy of a number of novel litter treatments in controlling darkling beetle (Alphitobius diaperinus).
Current Progress	Research by QDPI & F has shown that the largest numbers of adults and larvae of darkling beetle occur in the litter during the first three weeks of a batch. In response to these findings, the efficacy of five compounds applied as litter treatments for beetle control were laboratory trialed. Three Bayer compounds (imidacloprid SC and granule, beta-cyfluthrin SC) were all highly effective in suppressing progeny. The granule formulation was the most effective, also in terms of adult mortality and suppression of subsequent fecundity, but Bayer has concerns about residues. Beta-cyfluthrin is the safest compound to use but potential beetle insecticide cross resistance is an issue. Trialing of a diatomaceous earth indicated that a dose rate of 5kg/m3 of litter suppressed progeny by 75%. Strategic application of this dose along broiler house edges and under feed pan lines would augment other control agents. Issues surrounding the OHS of the DE require clarification. The laboratory testing of spinosad SC (Elanco) as a litter treatment was discontinued due its unexpected poor performance. Instead, the field trialing of spinosad as a floor and litter treatment over three batches was commenced in September 2004. Completion date for this trial is early September 2005.


 
 
 
 

Project Title	Systematic pathotyping of Australian Marek's disease (MDV) isolates
RIRDC Project No.:	UNE-83J
Start Date:	01/07/2002
Finish Date:	30/11/2005
Researcher:	A/Prof. Stephen Walkden-Brown
Organisation:	University of New England Animal Science School of Rural Science and Natural Resources ARMIDALE NSW 2351
Phone:	(02) 6773 5152
Fax:	(02) 6773 3922
Email:	swalkden@pobox.une.edu.au
Objectives	·1 To pathotype nine current and three older Australian isolates of Marek's disease virus (MDV) using internationally recognised protocols. ·2 To undertake molecular characterisation of these isolates to facilitate identification and relationships between isolates. ·3 To test the pathogenicity of two isolates in current Australian strains of meat and layer bird. ·4 To determine the extent of protection provided by HVT and bivalent serotype 2/HVT vaccines against recent isolates. ·5 To determine whether recent isolates are more pathogenic than older isolates. ·6 To develop an improved knowledge base for rational decision making regarding MDV vaccines and vaccination procedures.
Current Progress	The last 12 months has seen important breakthroughs on the project with improved isolation and growth of Marek’s disease virus (MDV) in cell culture at RMIT due to a change from culturing on chicken embryo fibroblasts to chick kidney cells. Successful isolation of MDV from field poultry dust samples and feather tips were also achieved. An initial screening experiment in SPF chickens at UNE during mid-2004 revealed a number of prospective pathogenic new isolates and showed that chicks could be infected with MDV from infective poultry dust from the field without transmission of other poultry pathogens. In late 2004/early 2005 four pathogenic new isolates were formally pathotyped in sham- and HVT-vaccinated SPF chickens at UNE against the standard reference MDV strain MPF57 and another isolate from the early 1990s, Woodlands1. There was evidence of induction of early mortality between weeks 1 and 2 post-challenge in the more virulent isolates. There was also a strong positive relationship between immune organ weight (thymus and bursa of Fabricius) at days 13 and 56 post-challenge and the level of protection against MD provided by HVT vaccination (vaccination at hatch, MDV challenge at day 5). Protective index ranged from 38% for the most virulent new isolate to 100% for the reference strain MPF57. The two most virulent isolates were recent, one isolated from dust and the other from feather tip material. These two isolates are currently being pathotyped in commercial broiler and layer chickens in an Australian Poultry CRC project.


 

Project Title	Typing of Pasteurella multocida
RIRDC Project No.:	UQ-100J
Start Date:	01/01/2002
Finish Date:	31/12/2004
Researcher:	Prof. Linda Blackall
Organisation:	The University of Queensland Department of Microbiology & Parasitology ST LUCIA QLD 4072
Phone:	(07) 3365 4645
Fax:	(07) 3365 4620
Email:	blackall@awmc.uq.edu.au
Objectives	·1 To establish a Multi-locus Sequence Typing (MLST) system for Pasteurella multocida. ·2 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. ·3 To provide typing tools which will support improved prevention and control programs for fowl cholera.
Current Progress	The study has focused on 50 isolates of Pasteurella multocida, including three taxonomic reference strains and 16 serovar reference strains. The remainder are Australian avian isolates that have been deliberately selected to represent the known diversity of this species (established by earlier multi-locus enzyme electrophoresis and DNA finger-printing studies). Purified DNA was prepared from all the cultures. Using specifically designed primers, PCR products of at least 450-500 base pairs were generated for all 50 isolates for all seven house-keeping enzymes selected for the study. These 350 PCR products have then been sequenced. These 350 sequences are now being edited and subjected to a final "data clean up". A few isolates appear to have sequence variation at the primer site for one enzyme and alternative PCR products using a slightly altered primer set are currently being prepared. Preliminary analysis of the isolates shows promising sequence variation that indicates the potential to create an effective typing scheme. Current work is focused on preparing the sequences for analysis to create the dendrogram that will show the relationships amongst the Multi-Locus Sequence Types (STs) that form the heart of this project. The data will be placed on the world wide accessible web site.


 
 

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:	01/02/2003
Finish Date:	31/01/2006
Researcher:	Prof. Mingan Choct
Organisation:	University of New England School of Rural Science and Agriculture ARMIDALE NSW 2351
Phone:	(02) 6773 5121
Fax:	(02) 6773 3050
Email:	mchoct@une.edu.au
Objectives	·1 To elucidate the mechanisms by which grain processing and feed constituents affect gut physiology of birds from early life, which in turn affects life-long productivity.
Current Progress	An experiment conducted in May-June 2004 at the Agricultural University of Norway (NLH), studied the effects of sorghum particle size and processing method on performance, AMEn, gut development, and digesta pH of male broilers. Whole (WS), hammer-milled [3mm screen] (HM3), roller-milled [0.15mm roller spacings] (RM0.15), and hammer-milled [1mm screen] (HM1) sorghum were used as 75% of the respective experimental diets. The results from this experiment have shown that feeding roller-milled sorghum (RM0.15) with an equivalent volume weighted mean (~740µm) as hammer-milled sorghum (HM3), enables lowered (P<0.0001) feed conversion ratio (FCR) (1.46 vs. 1.52) at 35d of age (Table 1). Feeding WS as part of a pelleted diet increased (P<0.005) AMEn, increased (P<0.001) relative gizzard weight and decreased (P<0.01) relative proventriculus weight with respect to the HM1 (446.8µm) diet at 21d and 35d. Feeding WS lowered (P<0.0001) gizzard pH, and raised (P<0.01) duodenum pH at 35d relative to the HM1 diet. Feeding RM0.15 enables lower (P<0.0001) FCR at 21 and 35d, compared to the HM3 and HM1 diets, which were more (P<0.0001) efficient that the WS treatments (Table 1). Feeding whole sorghum improves gut development and possibly gut function, but feeding roller-milled sorghum improves efficiency. Table 1. Performance of male broilers from 11 to 35d of age1 Performance parameter WS HM3 RM0.15 HM1 Bodyweight gain (g) 1823.3a 1905.2b 1916.4b 1865.7ab Cumulative feed intake (g) 2853.8 2902.2 2795.9 2804.5 FCR (g/g) 1.57c 1.52 b 1.46a 1.50b 1Values in rows with unlike superscripts differ significantly (P<0.05)


 
 
 
 

Project Title	Digestible amino acids and improved broiler performance
RIRDC Project No.:	UQ-107A
Start Date:	01/04/2003
Finish Date:	31/03/2006
Researcher:	Prof. Wayne Bryden
Organisation:	The University of Queensland School of Animal Studies GATTON QLD 4343
Phone:	(07) 5460 1257
Fax:	(07) 5460 1236
Email:	hos@sas.uq.edu.au
Objectives	·1 To delineate the digestible amino acid supply from the grain portion of the diet. ·2 To estimate the ileal digestible amino acid requirements of broilers fed wheat/sorghum based diets. ·3 To estimate the availability and requirements of digestible amino acids such as lysine and methionine for lean tissue deposition. ·4 To improve broiler performance by integration of the above information into feed formulation practices.
Current Progress	Extensive studies have been conducted to determine the digestible amino acid supply from wheat (16 samples) and sorghum (7 samples) of varying protein content. The results indicate that the ileal amino acid digestibility of high protein wheat cultivars are greater than those of lower protein cultivars. The amino acid digestibility in wheat appears to be largely influenced by crude protein content. On the other hand, no relationship was found between AME and protein digestibility in wheat. In the studies with sorghum a positive relationship was observed between average amino acid digestibility and crude protein content indicates that as grain protein of content increases so does amino acid digestibility. In a separate study it was demonstrated that the dietary lysine requirement for maximum growth and minimal feed conversion ratio is 1.21% for 1-18 day old broilers or chicks in the starter phase. It was also demonstrated in another starter phase study that the digestible methionine requirement for immune competence is greater than that for maximum growth.


 
 
 
 

Project Title	Establishment of a Chair in Poultry Sciences at The University of Sydney
RIRDC Project No.:	US-127A
Start Date:	01/01/2004
Finish Date:	30/12/2007
Researcher:	Prof. Tom Scott
Organisation:	The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570
Phone:	(02) 4655 0612
Fax:	(02) 4655 0693
Email:	toms@camden.usyd.edu.au
Objectives	·1 To establish a Chair in Poultry Science at the University of Sydney in order to maintain a high level research capability for the industry at that facility. ·2 To assist in the development of a centre of excellence in poultry science at the University of Sydney and to develop a targeted research program in poultry nutrition, health and safety focused on the needs of the Australian poultry industries.
Current Progress	The Poultry Science Chair has started two RIRDC supported research projects and one CRC research supported project; applications for support from ARC (Australian Research Council) and AECL have also been submitted. The Australian Poultry Science Symposium for 2005 was hosted February 7-9, 2005 and highlighted issues pertaining to sorghum and starch utilisation and factors associated with feed intake. The APSS organising committee is currently finalising plans for the 2006 meeting and will highlight feed processing and factors affecting egg and chick quality. Four fourth year honours thesis projects have been developed and currently underway, these include areas of nutrition (germinated grains, calcium solubility), embryology (defining true fertility of hatching eggs ), and health (oral delivery of vaccines). A MSc student (course work) has also been enrolled and will conduct research in the area of broiler breeder male fertility. The Chair of Poultry Science has also been invited to present information at the International Broiler Nutrition Conference (New Zealand, April 2005), the Coolum Feed Workshop (Queensland, May 2005), and the Recent Advances in Animal Nutrition in Australia (Armidale, July 2005).


 
 

Project Title	Assessment of the anti-nutritive effects of phytate by dephytinisation
RIRDC Project No.:	US-133A
Start Date:	01/01/2005
Finish Date:	31/01/2008
Researcher:	Dr Peter Selle
Organisation:	The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570
Phone:	(02) 4655 0612
Fax:	(02) 4655 0693
Email:	toms@camden.usyd.edu.au
Objectives	·1 Yield of breast meat is determined by muscle fibre number and subsequent growth. Muscle fibre numbers can be manipulated by early dietary intervention directly or as we hypothesise by increasing early gut development to facilitate nutrient intake and absorption to meet these requirements.
Current Progress	An extensive literature search of ‘dephytinisation’ is nearing completion. Preliminary dephytinisation procedures have been satisfactorily completed with sorghum and soyabean meal. Validation of suitable methods to determine phytate in samples where other sources of phosphorus may be a confounding factor (eg, complete diets, ileal digesta, excreta) is continuing. We are working with a colourimetric method (Latta and Eskin, 1980) in association with Dr Bob Caldwell (University of Sydney). We are also working with an enzymatic hydrolysis method (Shen et al. 2005) with Dr Alan Richardson (CSIRO, Canberra). The Animal Ethics Committee has specifically approved the project.  Intrinsic phytase activity of wheat complicates dephytinisation via hydrothermal treatment with exogenous phytase and the intention was to eliminate phytase by steam-pelleting. As this could impact on phytate in wheat, experimental broiler diets based on the same wheat, raw or steam-pelleted, without and with exogenous phytase, were evaluated. Broilers offered steam-pelleted wheat were less responsive to phytase (AME, N retention, growth performance). It is likely that steam-pelleting reduces phytate and protein solubility, rendering phytate less susceptible to enzymatic hydrolysis and the formation of protein-phytate complexes may be reduced. Consequently, alternative methods to eliminate phytase in wheat prior to dephytinisation need to be investigated.


 
 

Project Title	Early dietary and management intervention on broiler breast meat yield
RIRDC Project No.:	US-134A
Start Date:	01/07/2004
Finish Date:	31/12/2007
Researcher:	Prof. Tom Scott
Organisation:	The University of Sydney Chair of Poultry Science Faculty of Veterinary Science PMB 3 University of Sydney Poultry Research Unit CAMDEN NSW 2570
Phone:	(02) 4655 0612
Fax:	(02) 4655 0693
Email:	toms@camden.usyd.edu.au
Objectives	·2 Yield of breast meat is determined by muscle fibre number and subsequent growth. Muscle fibre numbers can be manipulated by early dietary intervention directly or as we hypothesise by increasing early gut development to facilitate nutrient intake and absorption to meet these requirements.
Current Progress	There is significant variation in yield of breast meat of broilers, but the consensus is that it accounts for approximately 20% of the weight of a broiler, however, provides about 60% of the value. The project will evaluate the impact of parental, in ovo (in egg during incubation) and post-hatch nutrition on the development of breast muscle cell numbers and size of broiler chickens. An extensive review of the literature has facilitated the planning and development of research protocols to define treatment effects on early bird growth, efficiency and ultimately on development of breast meat yield.  One preliminary study has been completed to evaluate in ovo injections of nutrients into the developing egg. A total of 600 hatching eggs (500 broiler and 100 layer-type eggs) were incubated for 16 days, at this time either 1.0 or 2.0 ml of four saline-based nutrients were injected into eggs containing viable embryos; the eggs resealed and incubation continued. A total of eight treatments were evaluated (including a non-injected control). Hatchability of injected eggs, except for one treatment, was equal to that of non-injected control egg hatchability. A second in ovo injection trial is underway; and a preliminary broiler breeder nutrition study has been initiated.


 
 

Project Title	Development of a sequence-based bacteriophage typing system for Salmonella
RIRDC Project No.:	IMV-5A
Start Date:	01/07/2003
Finish Date:	01/07/2006
Researcher:	Dr. Michael Heuzenroeder
Organisation:	Institute of Medical & Veterinary Science Infectious Diseases Laboratories PO Box 14 Rundle Mall PO ADELAIDE SA 5000
Phone:	(08) 8222 3275
Fax:	(08) 8222 3543
Email:	heuzenroeder@imvs.sa.gov.au
Objectives	·1 To continue to provide conventional typing (serotyping and phage typing) to the industry on an ongoing basis for the purposes of organism tracing and the monitoring of Salmonella serovars for public health and research objectives. ·2 To use AFLP and PFGE typing (where appropriate) for typing industry Salmonella strains when conventional typing methods fail to offer sufficient discriminatory power. ·3 To establish an objective sequence-based molecular typing system that would enhance and extend the current classical phage typing system. ·4 To characterise genes in endogenous phages in Salmonella to determine the predicted impact of these genes on current typing methods.
Current Progress	Industry specimens were received greater numbers in comparison to previous years. In 2003/4, 3,883 specimens were received (a 72% increase). To May in 2004/5 5,794 specimens were received. In 2004, Salmonella Sofia was the most common isolate (52%) followed by serovars Infantis (13.3%) and Typhimurium (12.1%) from chickens. In humans, S. Typhimurium was the most common isolate (29.5%). Amplification of phage genes provided greater typing ability than PFGE. This novel typing method is called Multiple Amplification of Phage Loci Typing (MAPLT). Further separation could be achieved by sequencing the amplified products (MLST). MAPLT and MLST of phage loci proved superior to MLST using housekeeping genes, which are used for MLST typing of Salmonella by others. A publication and a provisional patent have been major outcomes from this work. An evaluation study of Variable Number of Tandem Repeats (VNTR) of a subgroup of S. Typhimurium strains previously typed by MAPLT and MLST has been undertaken. Five primer sets were used in the study. It was concluded that a combination of approximately seven MAPLT and VNTR primer sets could provide maximum discrimination of S. Typhimurium isolates. We are currently testing both MAPLT and VNTR on common phage types of S. Enteritidis.


 
 

Project Title	Development and validation of Campylobacter microarrays for virulence detection and strain differentiation in poultry products
RIRDC Project No.:	RMI-14A
Start Date:	01/06/2002
Finish Date:	31/05/2005
Researcher:	Prof. Peter Coloe
Organisation:	Royal Melbourne Institute of Technology Department of Biotechnology & Environmental Biology PO Box 71 BUNDOORA VIC 3083
Phone:	(03) 9925 7104
Fax:	(03) 9925 7110
Email:	pcoloe@rmit.edu.au
Objectives	·1 To utilise existing knowledge on Campylobacter spp. to select the most appropriate virulence factors for use in the development of a Campylobacter microarray.  ·2 To design and select appropriate oligonucleotides for use in a Campylobacter array through the use of bacterial genomic techniques and alignment of gene sequences from known virulence factors to the Campylobacter genome.  ·3 To investigate the design of microchips for the immobilisation of oligonucleotides and hybridsation conditions for simultaneous detection of a variety of selected virulence factors.
Current Progress	Bioinformatics has been used to assess Campylobacter jejuni virulence and twenty-six genes representing potential virulence factors have been identified.  These genes have been amplified from C. jejuni strains from a variety of sources, and each product sequenced and compared. The sequence information shows that the genes are highly conserved across the different strains. Primers have also been designed based on the newly published C. coli genome information, and PCR products that will differentiate between C. coli and C. ejuni have been identified. Nine genes involved in lipo-oligosaccharide (LOS) modification have been identified and specific LOS primers have been prepared and evaluated on control strains All PCR products have been used to prepare a PCR based microarray to identify campylobacter isolates and to differentiate between C. jejuni and related species and this PCR array is now being used to evaluate specificity and selectivity using a variety of control and field isolates Isolates representing a wide collection of serotype strains of C. jejuni have also been screened for NeuB2, a gene involved in flagella modification  In addition to using the PCR array for identification, it has been applied as an expression array to analyse human and chicken isolates grown at 37°C and 42°C . However so far there has been no detectable difference in expression


 
 

Project Title	Campylobacter bio-replacement program to control food poisoning organisms in poultry
RIRDC Project No.:	UG-7A
Start Date:	01/07/2003
Finish Date:	31/07/2007
Researcher:	Dr. Victoria Korolik
Organisation:	Griffith University School of Health Sciences, Gold Coast Campus PMB 50 GOLD COAST MAIL CENTRE QLD 9726
Phone:	(07) 5552 8321
Fax:	(07) 5552 8908
Email:	v.korolik@griffith.edu.au
Objectives	·1 To develop methods for controlling Campylobacter spp in poultry by utilising a collection of non pathogenic Campylobacter strains as bio-replacement organisms to exclude pathogenic Campylobacter spp from commercial chicken flocks.  ·2 To determine the overall effect of colonisation by super-colonising Campylobacter strains on the general health of chickens.
Current Progress	Consumption of poultry, contaminated with C. jejuni and C. coli, which are found as normal flora in all birds, is considered to be one of the major causes of campylobacter enteritis worldwide.  This project aims at minimising risk of human exposure to pathogenic campylobacters and involves "bio-replacement" of potentially virulent Campylobacter strains by a non-virulent C. jejuni strain.  Persistence of "bio-replacement" C. jejuni strain 331 in GI tract of the chickens in experimental flocks of 100 birds, in simulated farm environment, was previously established. The ability of the "bio-replacement" strain to out-compete other highly colonising strains was tested by superinfecting chicks, colonised by "bio-replacement" strain 331, at days 14, 28 and 35. Secretion of campylobacteria was monitored by cloacal swabs from 15 randomly chosen birds on days 12, 17, 31, 38, 49 and 56. The identity of resident C. jejuni strains was established by PCR/FlaA typing. The results of this trial showed that all 4 strains were present and were continuously circulating in the flocks with none able to maintain dominance. The ability of the "bio-replacement" strain 331, to out-compete other, highly colonising challenge strains, following second introduction of 331 had been conducted and preliminary PCR/FlaA analysis indicate that, with one exception, on day 56 of life, all chicken trial groups secrete "bio-replacement" strain 331 only.


 

Environmental Management

Project Title	Evaluating risks posed by pathogen emissions from meat chicken sheds
RIRDC Project No.:	DAQ-318A
Start Date:	01/01/2004
Finish Date:	31/12/2006
Researcher:	Dr. Pat Blackall
Organisation:	Department of Primary Industries & Fisheries (Qld) Animal Research Institute Locked Mail Bag No 4 MOOROOKA QLD 4105
Phone:	(07) 3362 9498
Fax:	(07) 3362 9429
Email:	pat.blackall@dpi.qld.gov.au
Objectives	·6 To quantify emission rates for pathogenic bacteria (Salmonella spp, Escherichia coli, Campylobacter jejuni/coli, Staphylococcus) from typical tunnel ventilated meat chicken sheds. ·7 To evaluate the potential for human health effects by these emissions evaluated by combining measured emissions, gaussian air dispersion models, bacterial survival models and applying quantitative risk assessment methodology.
Current Progress	The initial work on this study has been focused on the development and validation of methods for the detection and enumeration of both dust particles and the bacteria of interest.  The laboratory-based microbiological work in a dust generator was completed and media for the enumeration of the key target bacteria Escherichia coli, Salmonella, Campylobacter jejuni and Staphylococcus spp. were selected. Similar validation work was undertaken for the equipment used to capture and enumerate dust particles.  Next, a range of methods for dust and pathogen capture in the field were considered.  This was a difficult task as the physics of air flow both within a tunnel shed and close to the exhaust fan create many problems for efficient and representative particle capture.  The outcome was a novel method that allows airborne particle capture directly from the fan exhaust to be performed using a duct that leads from the fan and a specially developed and validated isokinetic capture port.  This approach has been validated in a field trial.  As well, additional on-farm microbiological sampling to develop suitable experimental designs has also been performed. This sampling has covered the full range of bacteria of interest and has allowed field validation of the microbiological methods.


 
 

Project Title	Efficacy of windbreak walls for odour reduction
RIRDC Project No.:	DAQ-321A