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RIRDC Completed Projects in 2005-2006 & Research in Progress as at June 2006
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COMPLETED PROJECTS (click here for Research in Progress)

Flock Health

Bird Nutrition and Feed Supply Efficiency of Production Product Quality and Safety .
COMPLETED PROJECTS

Flock Health
 
Project Title: Use of cytokines to enhance vaccine efficacy in poultry
RIRDC Project No.: CSA-26J
Researcher: Dr. Andrew Bean
Organisation: CSIRO Livestock Industries
Private Bag 24
GEELONG VIC 3220
Phone:  (03) 5227 5792
Fax:  (03) 5227 5531
Email:  Andrew.Bean@csiro.au
Objectives  ·1 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.

·2 To enhance disease resistance and vaccine efficacy in poultry by administering therapeutic doses of chicken interferon-gamma to commercial broilers and layers
Background  Marek’s disease is caused by a highly contagious oncogenic herpes virus and has significant economic impacts for the world poultry industry. Even in the face of the current successful vaccine approaches, Marek’s disease (MD) remains a concern, particularly with the development of more virulent strains of Marek’s disease virus (MDV). If this situation continues it may lead to the recurrence of MDV as a serious economic threat. Therefore, in order to maintain the highest possible levels of animal health, welfare and productivity it is vital to be proactive in the development of safe alternative vaccination strategies and therapeutics.
Research  Previous studies have identified the potential for cytokines to act as therapeutics and vaccine adjuvants. In this project, chicken cytokines were evaluated for their immunoenhancing capacity during MDV infection.
Outcomes  The final report on this project outlines the analysis of the immune response, with particular emphasis on the cytokine response, during MDV infection. Further to this, biologically active recombinant chicken cytokines were produced from an E. coli expression system and used to assess their potential as therapeutics and vaccine adjuvants during MDV infection. It was found that the cytokines IL-2 and IL-6 show immunostimulatory activity and with this in mind this evaluation of cytokines activity provides evidence for the rational use of chicken cytokines as therapeutics for disease.
Implications  The further development of this technology will allow the Australian poultry industry to alleviate some of the concerns associated with vaccination against MDV infection. The results from this work test the feasibility of using cytokines as natural therapeutics and adjuvants and these cytokines are now available for a similar type of assessment as treatments for other immunosuppressive viral infections such as infectious bursal disease virus, chicken infectious anaemia virus and infectious bronchitis.

 
 
Publications Asif, M., Jenkins, K.A., Hilton, L.S., Kimpton, W.G., Bean, A.G.D. and Lowenthal, J.W. (2004). Cytokines as adjuvants for avian vaccines. Immunol. Cell. Biol, 82: 638-643.

Moore, R.J., Doran, T.J., Wise, T.G., Riddell, S., Granger, K., Crowley, T.M., Jenkins, K.A., Karpala, A.J., Bean, A.G.D. and Lowenthal, J.W. (2005). Chicken functional genomics. Australian Journal of Experimental Agriculture, 2005.

Invited presentations (at meetings): Lowenthal, J.W., Bean, A.G.D., Tyack, S.G., Hilton, L.S., Asif, M., Jenkins,KA and Johnson, M.A.(2004). Oral delivery of novel therapeutics. World’s Poultry Congress, Istanbul, Turkey. 2004

Schat, K.A., Bean, A.G.D., Broadway, M., Asif, M., Thomas, J. and Lowenthal, J.W. (2004). In vitro propagation of dendritic-like cells from chicken spleens. Avian Immunology Research Meeting, Munich, 2004 

Bean, A.G.D., Thomas, J.D., Bruce, M.P., Asif, M., Broadway, M.M., O’Neil, T.E., Jenkins, K.A., and Lowenthal, J.W. (2004). Immune system enhancement following in ovo adminstration of chicken cytokines, AIRG, Munich, 2004 

Bean, A.G.D., Jenkins, K.A., Asif, M., Thomas, J., Hilton, L.S., O’Neil, T.E., Lowenthal, J.W. (2003). Innate Immunity: recognition and response, APSS, Sydney, Australia, 2003.

Bean, A. G. D., Asif, M., Jenkins, K. A., Hilton, L. S., O’Neil, T. E., Lowenthal, J. W. (2003). Evaluation of the immunoenhancing capability of innate cell associated cytokines, MVADS, Dublin, Ireland, 2003.

Asif, M., Thomas, J.D., Jenkins, K.A., Kimpton, W.G., Bean, A.G.D., Lowenthal, J.W. (2003) Enhancement of Mucosal Immunity by Cytokines, ASI, Perth, Australia, 2003.

Jenkins, K.A., Bruce, M.P., Thomas, J.D., Kimpton, W.G., Schat, K.A., Lowenthal, J.W. and Bean, A.G.D. (2003). The Innate Immune Response to an Alpha Herpes Virus, ASI, Perth, Australia, 2003.

Thomas, J.D., Bruce, M.P., Asif, M., Broadway, M., O’Neil, T.E., Jenkins, K.A., Muralitharan, M., Lowenthal, J.W. and Bean, A.G.D. (2003). In ovo Administration of Cytokines to Chickens, ASI, Perth, Australia, 2003.

 
 
Project Title: Postgraduate scholarship – Kristie Jenkins: Improved therapeutics for Marek’s disease virus infection
RIRDC Project No CSA-25J
Researcher: Dr. Andrew Bean
Ms. Kristie Jenkins
Organisation: CSIRO Livestock Industries
Private Bag 24
GEELONG VIC 3220
Phone:  (03) 5227 5792
Fax:  (03) 5227 5531
Email:  Andrew.Bean@csiro.au

Kristie.Jenkins@csiro.au
Objectives  ·3 To examine the early immune response to Marek's disease virus infection and apply this information to enhance the development of therapeutic strategies.
Background  Prevention of viral infections is an important issue for the poultry industry. Understanding the innate immune system in chickens may help identify novel and effective ways to control viral infections in chickens such as Marek’s Disease Virus (MDV). An important group of receptors involved in the innate immune response are Toll-like receptors (TLRs). When this project began, chicken TLRs were relatively uncharacterised. With this is mind, the aim of this study was to identify and characterise TLRs that are involved in viral recognition in chickens. Examination of anti-viral TLRs in chickens revealed that chickens appear to contain only one functional receptor to represent the mammalian TLR9 subfamily. This receptor was subsequently cloned, characterised and shown to have the highest similarity to mammalian TLR7. Although the TLR9 subfamily gene was found to have the highest similarity to TLR7 it did display characteristics of TLR9, leading to the speculation that this receptor also recognises mammalian TLR9 ligands in chickens. 
Research  In vitro characterisation of the response of chicken splenocytes to TLR9 subfamily ligands found TLR7 and TLR9 ligands stimulate a response similar to that elicited by the homologous mammalian receptors. In contrast, a lack of response was induced by a huTLR8 ligand. In mammals, TLR7 and TLR9 ligands have been shown to have adjuvant potential. The analogous in vitro responses observed may also suggest TLR7 and TLR9 ligands may display adjuvant activity in chickens. To assess this, the in vivo response to these ligands had to first be determined. The avian system possesses the unique opportunity to administer substances in ovo and currently a number of poultry vaccines such as those for MDV are administered in this manner. Thus the ability of the TLR ligands to be administered in ovo was characterised to determine their potential use as adjuvants to these vaccines. 
Outcomes  The in ovo administration of the ligands was shown to be successful with no detrimental effects on hatchability. Administration of TLR9 ligands resulted in increased levels of IL6 mRNA detectable at 7 days post hatch. Subsequent examination of the prophylactic and adjuvant potential of TLR7 and TLR9 ligands in the face of Marek’s disease virus (MDV) revealed their ability to reduce MDV induced impact on CD4+ cells. 
Implications  The experiments performed in the current study revealed that chickens contain a smaller repertoire of viral recognising TLRs than mammals, with only one functional receptor identified to represent the mammalian TLR9 subfamily. Although an orthologue for TLR9 was not identified, chicken cells were found to respond to both TLR7 and TLR9 ligands in a manner analogous to mammalian cells. The current knowledge of the action of TLR ligands in mammals, in conjunction with their immunomodulating ability shown in this study, draws attention to their potential use as therapeutic agents for the poultry industry. 
Publications To date no publications have been produced from this work.

 
 
 
 
Project Title: Postgraduate scholarship – Manija Asif: Cytokines and innate molecules for enhanced mucosal immunity in the chicken
RIRDC Project No CSA-25J
Researcher: Dr. Andrew Bean
Ms. Manija Asif
Organisation: CSIRO Livestock Industries
Private Bag 24
GEELONG VIC 3220
Phone:  (03) 5227 5792
Fax:  (03) 5227 5531
Email:  Andrew.Bean@csiro.au
Objectives  ·4 To reduce disease by enhancing mucosal immunity in chickens with the aid of cytokine therapy.
Background  To maintain high standards of poultry health and production there is a need for the continuing development of novel strategies directed towards improving health and welfare. With this in mind, there is a greater emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently, existing vaccine adjuvants may have deleterious side effects, such as inflammation, which can lead to down grading of meat quality and in turn to lower profits. Therefore, to enhance vaccination strategies, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants has attracted considerable attention in this respect. Moreover, their potential role as adjuvants has been addressed by several recent studies. As cytokines are regulatory proteins secreted by a variety of cell types that play a crucial role in controlling the immune response, they provide a useful candidate for adjuvant studies. Furthermore, the recent identification of a number of chicken cytokine genes has provided the opportunity to study their effectiveness in enhancing the immune response during infection or vaccination. One such cytokine, interleukin-6 (IL-6) has been shown in mammals to play a major role in controlling the immune response. The aim of this project was to characterise the biological functions of chicken IL-6 (ChIL-6) as a precursor to an assessment of its potential as a therapeutic or vaccine adjuvant.
Research  To examine the immunoenhancing properties of ChIL-6, recombinant ChIL-6 (rChIL-6) protein was expressed and its biological activity was determined. Antibodies and enzyme linked immunoassays specific for ChIL-6 were developed to characterise the in vivo function of this cytokine. In vivo and in ovo administration of rChIL-6 significantly enhanced the in vitro proliferation of lymphocytes in response to ConA. Furthermore, adjuvant potential assessment studies revealed that rChIL-6 administration with an antigen increases secondary IgY responses. Since IL-6 is a pro-inflammatory cytokine in mammals, the inflammatory role of ChIL-6 in a disease model, infectious bronchitis virus, was assessed using a susceptible (S-line) and resistant (HWL) line of SPF birds. Significant over expression of IL-6 was observed in S-line birds suggesting a possible exacerbation of the disease associated with IL-6 in this susceptible line.
Outcomes  The experiments performed in these studies show that ChIL-6 predisposes lymphocytes to enhanced proliferative potential when administered to the embryo or adult birds. Furthermore, ChIL-6 was found to act as an adjuvant by increasing antibody responses to an antigen. 
Implications  These functions of ChIL-6 suggest that it may have the potential to be used as an adjuvant or therapeutic agent for the poultry industry. This potential needs to be explored in further investigations. 

 
 
Project Title: Biological control of necrotic enteritis in meat chickens
RIRDC Project No.: CSA-29A
Researcher: Dr. Robert Moore
Organisation: CSIRO Livestock Industries
Private Bag 24
GEELONG VIC 3220
Phone:  (03) 5227 5760
Fax: (03) 5227 5555
Email:  rob.moore@csiro.au
Objectives  ·1 To complete the development of a reproducible necrotic enteritis (NE) disease model that can be implemented in PC2 containment conditions, thus allowing the testing of disease treatments, such as bacteriocin products and recombinant vaccines developed in previous projects.
Background  An earlier RIRDC project, CSA-12A, developed potential treatments for NE based on antimicrobial proteins called bacteriocins. To test the efficacy of these treatments an NE disease model was required but could not be completed in the time available. This project was aimed at completing the development of a suitable disease induction model.
Research  A series of chicken infection trials were undertaken in order to optimise the challenge procedure. A collection of recent field isolates of Clostridium perfringens, the bacterium responsible for the disease, was tested in chickens and a number were found to be capable of eliciting necrotic lesions in the gut. A range of factors, including room temperature, coccidia treatment, bacterial growth conditions, gut motility and feed changes, were assessed for their impact on the development of NE disease under experimental conditions. Final trials briefly looked at the impact of one bacteriocin based treatment.
Outcomes  Optimised procedures and conditions to induce reliable levels of mild to moderate disease were defined. The most important factors appear to be the growth conditions of the challenge bacteria, feed withdrawal over the challenge period, a reduction in housing temperature just prior to challenge, and the particular isolates of C. perfringens used. Methods for assessing and recording disease outcomes were also refined. 

A quantitative polymerase chain reaction (qPCR) method was established which is capable of high throughput analysis of bacterial numbers in gut samples. This provides another level of analysis beyond the simple scoring of gut lesions. In one trial, one of the groups treated with the bacteriocin was fully protected, showing no signs of disease. In a subsequent trial this finding was not repeated; despite this the initial observation makes us optimistic that the bacteriocin treatments can be successfully developed.
Implications  This project has developed the disease induction process to the point were it is now suitable to be used to assess the performance of intervention strategies being developed to combat NE. It can be used to test bacteriocin treatments, vaccines, and other possible therapeutics such as cytokines.
Publications To date no publications have been produced from this work.

 
 
 
 
Project Title: The development of vaccination strategies to control necrotic enteritis in poultry
RIRDC Project No.: RMI-11A
Researcher: Prof. Peter Coloe
Organisation: RMIT University 
Plenty Road 
BUNDOORA VIC 3083 
Phone:  (03) 9925 7104
Fax:  (03) 9925 7110
Email:  pcoloe@rmit.edu.au 
Objectives  ·1 To lay a foundation for understanding and controlling necrotic enteritis (NE) by developing a model for the reproduction of necrotic enteritis in chickens.

·1 To develop and evaluate a vaccine, based on toxoid, for the control of necrotic enteritis that can be delivered orally, that is cost effective to manufacture and that can be delivered within established farming practices.
Background  A major aim of the poultry industry is to produce high quality product without the need for supplementation with antimicrobial agents. To achieve this objective, over recent years there has been a strong move towards the use of vaccination strategies to replace long term chemical use for the control of disease. Effective vaccination of poultry against necrotic enteritis (NE) would not only illustrate the industry’s proactive stance with respect to replacing chemical control measures for disease, but should enhance the productivity of flocks. Since an outbreak of NE (which is caused by the pathogen, Clostridium perfringens) can result in between 2% to 10% mortality, as well as causing significant losses in flock productivity, effective vaccination will have a major cost benefit outcome for the industry. Effective vaccination to control C. perfringens alphatoxin will also ensure the development of a healthy, effective gut microflora. This microflora will limit the possibility of other undesirable organisms colonising the chicken and posing a threat to human health after processing.
Research  The approach taken in this project was to establish a reproducible model for NE, to produce and test a killed vaccine based on toxoid and, if this proved successful, to clone and deliver a live modified vaccine.

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

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

However, without an effective chicken model to test the purified toxin in this work did not proceed further. The availability of cloned alphatoxin will enable evaluation of this as a protective antigen when a model of NE in chickens becomes available.
Implications  The development of an efficacious vaccine for necrotic enteritis would be a significant step forward for the chicken industry. A vaccine that is cost effective and can be delivered orally would enhance the productivity of the industry and also enhance disease control and flock welfare.

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

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

 
 
Project Title: Preliminary investigations into the efficacy of novel treatments and application methods for darkling beetle (Alphitobius diaperinus [Panzer]) control in Australian broiler houses
RIRDC Project No.: DAQ-319A
Researcher: Mr. Trevor Lambkin
Organisation:  Department of Primary Industries & Fisheries (Qld)
Entomology Building, Indooroopilly Research Centre
80 Meiers Road
INDOOROOPILLY QLD 4068
Phone: (07) 3896 9434
Fax: (07) 3896 9446
Email:  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).
Background  A long history of applications of contact insecticides to the floors of broiler houses in Australia for the control of darkling beetle may have been largely ineffective and has resulted in the development of strong insecticide resistance in beetle populations, particularly in eastern Australia. Recent research undertaken in Australia has identified that control strategies should mostly be targeted to the treatment of litter for management of this pest. Therefore, in 2003/04, preliminary investigations commenced to laboratory trial a number of novel litter treatments and to field trial a new broiler house floor application, to determine their efficacy in controlling all stages of the beetle.
Research  Imidacloprid 200SC and granules, beta-cyfluthrin 25 g/L, spinosad and diatomaceous earth were laboratory tested as litter treatments, with spinosad also field trialled as a broiler house floor treatment. Efficacy in suppressing development of progeny (larvae) was measured for each treatment. 
Outcomes  The most effective litter treatment overall was the imidacloprid bait granule, but beta-cyfluthrin was also very effective in suppressing progeny. Diatomaceous earth reduced progeny significantly but at doses much higher than the other two compounds. Spinosad was found to be ineffective as a litter treatment but gave significant suppression of populations when applied to the floors of broiler houses.
Implications  The four formulations trialled for broiler house control of A. diaperinus offer the chicken meat industry an alternative for beetle control, but only if certain issues are addressed. These are the need for OH&S and bird safety implications to be resolved for diatomaceous earth and imidacloprid respectively, for all formulations be integrated into an integrated system of rotation (or use of two products concurrently), and for a cost effective method is found to apply treatments to litter. Each formulation, if used appropriately, is a realistic option for beetle control. If used together in the form of an integrated system, it is possible that a lower dose of each compound might be used. 
Publications To date no publications have been produced from this work.

 
 
Project Title: Molecular evaluation of responses to vaccination and challenge by Marek’s disease viruses
RIRDC Project No.: RMI-12J
Researcher: Prof. Greg Tannock
Organisation: Royal Melbourne Institute of Technology
Department of Biotechnology and Environmental Biology
PO Box 71
BUNDOORA VIC 3083
Phone:  (03) 9925 7142
Fax: (03) 9925 7110
Email:  gtan@rmit.edu.au
Objectives  ·1 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.

·2 To extend the use of QPCR to measure viral loads in various organs of infected chickens after infection with virulent and vaccine viruses.

·3 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.

·4 To measure the distribution of serotype 1 and 3 vaccine viruses after inoculation to chick embryos under conditions used for in ovo vaccination.

·5 To carry out protection experiments after in ovo and day 1 vaccination in which the endpoints will be measured from the challenge virus load in critical target organs.
Background  Despite the significance of Marek’s disease (MD) as a major cause of economic loss to the chicken industry, comparatively little is known about the mode of action of vaccines that are widely used. Most data have been obtained using cell culture to measure the presence of Marek’s disease virus (MDV) together with other traditional methods based on the capacity of the virus to cause death, morbidity and disease-specific lesions. All of these have limitations and the project attempted to overcome them by the application of newer molecular techniques for the enumeration of the MDV genome in infected tissues. The animal model so developed was then applied as a means of studying the disease and for an evaluation of optimal methods of vaccination.
Research  Early stages of the project concerned the optimisation of methods for estimating MDV DNA in cultures and chicken tissues by QPCR.

The technique was initially used to optimise methods for the isolation of field isolates of MDV in cell culture. In later studies, QPCR was used to estimate viral loads in various tissues of infected chickens and chicken embryos after inoculation with virulent strains of MDV and commonly used vaccine strains. The resulting animal challenge model was used to estimate the efficacy of two commonly used Australian vaccines; the Rispens strain and HVT.
Outcomes  Optimum methods for the isolation of field viruses, as defined by QPCR, include the use of chicken kidney (CK) culture and inoculation of freshly prepared CK cells in suspension and culture for six days. Highest viral DNA levels were observed in the spleen and thymus of birds inoculated with the virulent type 1 strain MPF57. Levels in the same organs of chickens inoculated with the Rispens and HVT vaccines were

1:10,000-1:100,000 lower. When day-old chickens were vaccinated with the Rispens vaccine and challenged with MPF57, reductions in MPF57 in the spleen occurred over nine weeks that were consistent with clinical measures of protection that were observed in parallel. A similar challenge experiment was conducted in hatched chickens that had been inoculated with HVT in ovo at days 11 and 17 of embryonic development. Overall responses to challenge were comparable. In all challenge experiments, vaccination was shown to reduce levels of challenge virus in the spleen but did not lead to the induction of sterilising immunity
Implications  The challenge model developed in this project provides a useful objective measure of immunity to MDV, which could be very useful in the evaluation of new vaccines. The model has shown quite unequivocally that vaccine evaluation based on clinical endpoints alone provides no indication of their incapacity to eliminate MDV from a flock. Accordingly, effective decontamination of litter, in conjunction with vaccination, will remain an essential strategy to reduce the exposure of young immunologically immature birds to challenge virus. 
Publications Tan, J., Cooke, J., Clarke, N., Crisp, F.and Tannock, G.A. (2004). Proceedings of the 7th International Symposium on Marek's Disease, Oxford, UK, July 2004.

 
Project Title: Avian leukosis subgroup-J (ALV-J): epidemiological studies for control of ALV-J in Australian broiler breeder flocks
RIRDC Project No.: UM-68A
Researcher: Dr. Trevor Bagust
Organisation: The University of Melbourne
Faculty of Veterinary Science, Building 404
Cnr Park Drive & Flemington Road
PARKVILLE VIC 3010
Phone:  (03) 8344 9676
Fax: (03) 8344 9675
Email:  trevorjb@unimelb.edu.au
Objectives  ·1 To undertake a structured epidemiological survey to determine the current prevalence and distribution of ALV-J in Australian broiler breeder stocks at Great Grandparent (GGP) and GP levels.

·2 To undertake molecular characterisation and comparative pathotyping of recent Australian strains of ALV-J isolated from large commercial poultry breeding organisations.

·3 To provide the tools that would allow industry to control ALV-J in Australian broiler breeder flocks.
Background  Avian leukosis virus subgroup J (ALV-J) causes tumours, mortalities and sub-optimal production in the broiler industry. The presence of this virus has previously been confirmed in Australia. In an earlier RIRDC project (UM-49A Avian leukosis virus subgroup J (ALV-J): Developing laboratory technologies for diagnosis in Australia), the molecular technology was developed to culture and detect ALV-J. This technology is now applied in this project to help industry control ALV-J.
Research  A detailed structural epidemiological survey was carried out to determine the prevalence of ALV-J in the Australian industry. Further, Australian isolates of ALV-J were characterised antigenically and by sequence analysis to determine their likely origin and the extent of antigenic drift. Investigation of isolation procedures for ALV-J from field and experimentally infected birds was undertaken to establish the optimal samples for detection of ALV-J. Selected isolates were also pathotyped in both major strains of birds used in the Australian industry.
Outcomes  The structured epidemiological survey of Australian flocks identified flocks positive for ALV-J and these were removed by industry. Over the course of this project a decline in the prevalence of ALV-J is reported with the likely eradication of ALV-J from the Australian industry. The presence of ALV-A was reported with instances of co-infection occurring with both ALVJ and ALV-A. Australian isolates of ALV-J showed significant antigenic drift from UK and US prototypes and sequence analysis indicated that Australian isolates of ALV-J were imported from both European and US sources. Pathotyping of Australian isolates of ALV-J demonstrated these isolates induced myeloid leukosis and caused significant decreases in body weight.
Implications  This project has demonstrated the control and likely eradication of ALV-J from the Australian poultry industry. Furthermore, it also revealed the presence of ALV-A. Iif steps are not taken to remove ALV-A from Australian stocks the risk of further ALV recombinations increases. Investigation of sampling procedures indicated the dangers in relying solely on the one sample type when screening for ALV-J. Australian isolates of ALV-J have been introduced to this country from both European and US sources, thus highlighting the need to screen imported stocks to ensure their freedom from ALV-J.
Publications Bagust, T., Fenton, S. and Reddy, M. (2004). Australian Veterinary Journal,82: 701-706.

Fenton, S., Reddy, M. and Bagust, T. (2005). Avian Pathology, 34: 48-54.

Bagust, T.J., Fenton, S.P. and Reddy, M.R. (2003). Proceedings of WVPA congress Denver USA 2003.

Fenton, S.P., Bagust, T.J. and Reddy, M.R. (2003). Proceedings of The Avian Veterinarian Poultry Association conference Melbourne, November 2003.

Fenton, S., Bagust, T. and Reddy, M. (2004). Proceedings of the Avian Poultry Assoc. Asia Pacific Conference #5. Gold Coast Qld 20-23 April 2004.

 
Project Title: Systematic pathotyping of Australian Marek’s disease (MDV) isolates
RIRDC Project No.: UNE-83J
Researcher: A/Prof. Stephen Walkden-Brown
Organisation: University of New England
Animal Science
School of Rural Science and Natural Resources
ARMIDALE NSW 2351
Phone:  (02) 6773 5152
Fax: (02) 6773 3922
Email:  swalkden@pobox.une.edu.au
Objectives  ·1 To evaluate current Australian strains of MDV for their ability to induce disease and overcome the effects of vaccination. 

·2 To do this in a way that facilitates comparisons with similar studies overseas.
Background  In the USA there is clear evidence of evolution of Marek’s disease virus (MDV) towards greater virulence (ability to induce disease) over time, possibly as a response to vaccination. A feature of this increased virulence is the successive failure of different vaccines against MDV to provide effective protection against the disease. Australia suffered a serious outbreak of MDV from 1991-1996 and part of the response to this has been to introduce routine in ovo vaccination of broiler chickens with potential to facilitate such an evolution in virulence. Previous Australian studies had shown that there were highly virulent strains of MDV in the country.
Research  Between 2002 and 2005, 533 field samples were submitted for isolation of MDV, with 655 isolations on cell culture attempted. Unfortunately isolation rates were low and only six isolates (four new and two old) grew sufficiently for inclusion in formal pathotyping experiments while a further 11 grew sufficiently to infect chickens in screening experiments. These isolates showed a wide range of virulence in SPF (specific pathogen free) chickens with the more virulent strains inducing pathology typical of very virulent isolates overseas. A new early mortality syndrome induced by MDV was observed and detailed for the first time in Australia. There was no clear evidence of a trend towards increased virulence over the last decade although the number of isolates tested was relatively small. In unvaccinated SPF chickens, virulence was well predicted by a range of measurements on chickens as soon as two weeks after challenge.
Outcomes  The research undertaken confirmed the presence of very virulent MDV in Australia. It did not confirm evolution in virulence of Australian MDV over time. Isolation of MDV in cell culture proved to be impractical, and isolation directly in chickens should be pursued instead. New methods for screening for virulence in unvaccinated SPF chickens were developed. The work also demonstrated that virulence in unvaccinated chickens is not necessarily related to the ability of MDV to overcome the effects of vaccination.
Implications  The work has allowed for an improved understanding of current status of Australian MDV. HVT, the main vaccine used in the broiler industry, induces low levels of protection against several isolates of MDV. This is suggestive of some evolution in virulence occurring in Australian MDVs, although this was not able to be confirmed in the current project. Ongoing monitoring for MDV virulence and vaccine efficacy is worthwhile. The project has provided good data on how such ongoing monitoring might be conducted.

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

 
 
Project Title: Typing of Pasteurella multocida
RIRDC Project No.: UQ-100J
Researcher: Prof. Linda Blackall
Organisation: The University of Queensland
Department of Microbiology & Parasitology
ST LUCIA QLD 4072
Phone:  (07) 3365 4645
Fax: (07) 3365 4620
Email:  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.
Background  Fowl cholera remains one of the major bacterial diseases of poultry. The importance of P. multocida as a pathogen of birds and many other species is such that the complete chromosome of this pathogen was recently sequenced, allowing the development of typing systems based on this sequence knowledge. MLST is a new generation typing method that has considerable advantages over all other typing methods. The technique is highly reproducible, highly discriminatory and is totally portable. This last feature means that once an MLST typing scheme has been established any laboratory can compare their isolate with any previously typed isolate by simply comparing the DNA sequences. All typed isolates stored in the central Web data-bank are accessible to anyone with Web access. The rapid progress in DNA sequencing technology means that this form of DNA-based typing is now feasible (and indeed already functioning for major human pathogens such as Neisseria meningitidis and Campylobacter jejuni.)
Research  A total of 63 isolates of P. multocida from Australian poultry – all associated with fowl cholera outbreaks - as well as three international reference strains, representing the three subspecies within P. multocida, were used. The complete sequence of seven different house-keeping genes was determined. The sequences were then analysed using a specialised suite of software. This analysis showed that 39 different MLST Sequence Types (STs), representing 39 distinct allelic profiles, were present in the study. One particular ST (ST-2) was found to be the predominant ST (18.18%) in these isolates.

A dendogram showing the genetic relatedness of the isolates was produced. This dendogram showed a strong correlation with the dendogram created using the earlier phenotypic-based technology of multi-locus enzyme electrophoresis (MLEE). In particular, both MLST and MLEE have shown that there are two major sub-divisions within the species P. multocida. In accordance with the prior MLEE and ribotyping data, MLST has confirmed that a diverse range of P. multocida is associated with fowl cholera in Australia. A retrospective analysis confirmed that MLST has the power to be a useful typing tool , allowing outbreaks of fowl cholera to be followed and understood.
Outcomes  The major outcome of this project was the first web based MLST scheme for P. multocida, with this scheme being constructed on Australian poultry isolates. This is a significant advance, both in the Australian and international context. The scheme will enhance the management and control of fowl cholera outbreaks, both in Australia and around the world. The data-base generated in this study has been provided to the major MLST Web site http://www.mlst.net/.
Implications  The development of the MLST database and its placement on the central website serviced by the Oxford University means that laboratories around Australia and around the world can now directly compare isolates and build a truly international picture of the diversity within this important pathogen of poultry and other commercial livestock.
Publications To date no publications have been produced from this work.

 
 
Project Title Efficacy trials of a maternally-delivered recombinant vaccine against coccidiosis
RIRDC Project No.: UTS-4J
Researcher:  A/Prof. Nicholas Smith
Organisation: University of Technology, Sydney
Institute for the Biotechnology of Infectious Diseases
Westbourne Street
GORE HILL NSW 2065
Phone: (02) 9514 4013
Fax: (02) 9514 4201
Email: Nick.Smith@uts.edu.au
Objectives ·1 To test the immunogenicity of recombinant versions of gametocyte antigens of Eimeria maxima.

·2 To test the efficacy of recombinant versions of Eimeria maxima gametocyte antigens as a subunit, maternally-delivered vaccine against coccidiosis.
Background  Coccidiosis caused by species of Eimeria costs the poultry industry an estimated $1 billion per year worldwide. The resistance of the parasite to currently used drugs, the high cost of research and development of new anticoccidial agents ($100 million for each new drug) and the demands by the public for chemical-free agricultural practices drive the quest for a vaccine to control this disease. A vaccine using native proteins from the gametocytes of Eimeria maxima is currently marketed in several countries as CoxAbic®. We have cloned and expressed recombinant versions of the gametocyte antigens to succeed the native antigens as a second generation anticoccidial vaccine. The immunogenicity and efficacy of the recombinant proteins, however, needs to be tested. A recombinant version of the vaccine may allow simplification of production and quality control because it will avoid cycling of parasites through chickens. The recombinant vaccine may potentially also be cheaper since the high costs of maintaining chickens pathogen free to ensure the safety and quality of the native vaccine can be avoided.
Research  The genes encoding two immunodominant antigens of Eimeria maxima, gam56 and gam82, have been cloned into a bacterial expression vector and the proteins expressed and purified. The testing of the recombinant gametocyte antigens involved a systematic sequence of evaluations. Different recombinant proteins were compared in a variety of different doses and combinations. Immunogenicity and efficacy were determined relative to negative controls and positive controls (birds vaccinated with the proven native gametocyte vaccine formulation).

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

The second stage of the evaluation involved immunisation of broiler breeder hens housed on floor litter, with evaluation of transfer of antibodies from hens to yolks to hatchlings and efficacy evaluated in hatchlings by counting oocysts excreted by individual birds.
Outcomes  Both recombinant proteins were recognised by protective chicken antibodies that were raised to the native 56 and 82 kDa gametocyte proteins, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of protective antibodies to the native proteins by 76%, which was comparable to the inhibition of 98% observed when the native antigens themselves were used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised the native proteins by ELISA, and gametocytes by immunoblotting. The antibodies induced by immunisation with the recombinant 56 kDa protein were efficiently transferred by Cobb500 hens to their egg yolks and absorbed by the hatchlings, resulting in reduced oocyst excretion.
Implications  A recombinant version of the 56 kDa gametocyte protein of E. maxima appears to be a very promising potential substitute for the native protein components of CoxAbic®. It induces a strong, and competitive, anti-native protein antibody response in hens that is efficiently transferred into egg yolks and, thence, to hatchlings, affording them a level of protection against challenge infection with Eimeria. This result can, however, only be regarded as preliminary and it is recommended that repeated trials via maternal immunisation be carried out to confirm the encouraging results reported here.
Publications Belli, S.I., Mai, K., Skene, C., Gleeson, M.G., Witcombe, D.M., Katrib, M., Finger, A., Wallach, M.G. and Smith, N.C. (2004). Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima. Vaccine, 22: 4316-4325.

 
 
Project Title: Postgraduate scholarship - Ms Kelly Mai: The molecular basis for oocyst wall formation in the apicomplexan parasite, Eimeria maxima
RIRDC Project No.: UTS-6A
Researcher: A/Prof. Nicholas Smith
Ms. Kelly Mai
Organisation: University of Technology, Sydney
Institute for the Biotechnology of Infectious Diseases
Westbourne Street
GORE HILL NSW 2065
Phone:  (02) 9514 4013
Fax: (02) 9514 4026
Email:  Nick.Smith@uts.edu.au

kelly.mai@uts.edu.au
Objectives  ·1 To identify and characterise structural proteins and enzymes involved in oocyst wall formation in Eimeria maxima.
Research and Outcomes Compositional analysis of oocyst walls (isolated from bleached unsporulated oocysts of E. maxima) using gas chromatography and mass spectrometry indicated that the oocyst wall is composed of peptides, carbohydrates (mainly glucose and ~ 1% novel carbohydrates) and, surprisingly, no lipid. These results are currently being confirmed. 

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

A recombinant version of gam56 - designated r56 – was constructed and produced. It was subsequently demonstrated that this recombinant protein can be crosslinked in vitro using exogenous peroxidase in the presence of peroxides. R56 proteins were crosslinked into high molecular weight oligomers including dimers, tetramers and polymers as detected by SDS-PAGE gel electrophoreses and Western Blotting. In addition, biochemical data (HPLC analysis) showed that high concentrations of dityrosines were detected in crosslinked protein samples. These results support the hypothesis that crosslinking of proteins originating in the wall forming bodies of the gametocytes of E. maxima is mediated by the formation of dityrosine bridges through oxidation of tyrosine residues in proteins. 
Implications These data establish a molecular model to explain a fundamental developmental phase in the Eimeria lifecycle, namely the formation of the oocyst and, most particularly, its distinctive protective wall, which is essential for transmission of this genus of parasites. 
Publications To date no publications have been produced from this work.

 
 
Project Title: Effects of organic acids, prebiotics, and enzymes on control of necrotic enteritis and performance of broiler chickens
RIRDC Project No.: UNE-75A
Researcher: Prof. Mingan Choct and Dr Andreas Kocher
Organisation: School of Rural Science and Agriculture 
The University of New England
ARMIDALE NSW 2351
Phone:  (02) 6773 5121
Fax: (02) 6773 3050
Email:  mchoct@une.edu.au
Objectives  ·4 To produce data and demonstrate the effect of using organic acids, prebiotics and enzymes in broilers as alternatives to antibiotic growth promotants to maintain feed efficiency and general bird health. 

·5 To evaluate the effect of these alternative products on the prevention of necrotic enteritis. 
Background  Most chicken meat producers in Australia use antibiotics, such as zinc bacitracin, to control outbreaks of necrotic enteritis. However, continued use of antibiotics in feed may encourage the development of resistance to antibiotics by some organisms. In order to examine the effectiveness of alternative products to antibiotics in controlling necrotic enteritis (NE) and other diseases, a reliable experimental model must first be established. 
Research  Thirteen experiments were conducted to establish a reliable necrotic enteritis model and to test organic acids, prebiotics and enzymes as alternatives to antibiotics in broilers. Some examples or probiotics were also tested. 
Outcomes  The first 24 months of the project focused on the establishment for a reliable necrotic enteritis model, whereas during the last 18 months’ experiments were conducted to test the efficacy of a number of organic acid products, xylanases, a probiotic (B. subtilus) and oligosaccharides using the previously established NE model. The NE model was reproduced consistently with 1-3% NE-related mortalities (lesion scores 1-4), and clear reductions in growth and feed conversion efficiencies of broilers. Enzymes worked most effectively in maintaining bird performance and reducing the number of Clostridium perfringens in the small intestine. Organic acids were intermediate in their effects on these parameters, whereas the effect of oligosaccharides was not consistent.
Implications  Under a subclinical infection, the effect of NE on bird performance can be alleviated by the use of appropriate enzymes or organic acids, but under the circumstances of a clinical outbreak of NE, it is doubtful that any of these alternatives can control or prevent the damage caused by it.
Publications Kocher, A., Anderson, G., Choct, M. and Carter, R.R. (2002). An Australian model for experimental reproduction of clostridial enteritis in broilers. Proceedings of the Australian Poultry Science Symposium, 14: 193. 

Kocher, A., Choct, M., Teo, A., Tan, H.M. and Carter, R.R. (2004). Effectiveness of alternative feed supplements to broiler diets using a necrotic enteritis challenge model. Proceedings of the Australian Poultry Science Symposium, 16: 84.

Kocher, A., Rodgers, N.J. and Choct, M. (2004). Efficacy of alternatives to AGP's in broilers challenged with C. perfringens. Proceedings of the Australian Poultry Science Symposium, 16: 130-133.

Bird Nutrition and Feed Supply
 
Project Title: Mechanical and enzymatic improvements of dehulled lupins for broiler and layer diets (AECL-managed project WAU-1A)
RIRDC Project No.: UWA-76J 
Researcher: Dr. Ian Williams
Organisation: The University of Western Australia
Faculty of Natural and Agricultural Sciences
CRAWLEY WA 6009
Phone: (08) 6488 3780
Fax: (08) 6488 1040
Email:  iwilliam@animals.uwa.edu.au
Objectives ·1 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.

·2 Breakdown of thick cell walls of whole and dehulled lupins by expansion and enzymatic treatments.

·3 To increase the inclusion rates of whole and dehulled lupins up to 20% in broiler and layer diets by the above treatments without significant losses in productivity.
Background Australia produces 87% of the world’s lupins which are an excellent source of protein and energy. While the world faces a shortage in plant protein meals, feed manufacturers and poultry producers cannot use more than about 5% lupins in broiler and 7% in layer diets. The main reason is because lupins contain complex cell-wall polysaccharides (33%) that are indigestible. The main component of cell walls is pectin which varies from 33 to 71%. Pectin increases the viscosity of digesta in the bird’s digestive tract, increases water intake and wet droppings and, consequently, reduces food intake and efficiency of feed utilisation. Poultry cannot digest pectin because they don’t secrete the appropriate enzymes so their use of lupins is limited. However, by treatment of lupins with exogenous enzymes and mechanical heat treatment (expansion), it might be possible to increase the nutritive value of lupins for poultry.
Research  Three experiments were carried out to investigate whether exogenous pectinases and expansion might increase the nutritive value of lupins for broilers and layers. The first involved the inclusion of 10 and 20% whole and dehulled lupins using pectinase (endo-polygalacturonase) for layers. The second experiment evaluated enhancement of pectinase activity through expansion of lupins before including them at 10 and 20% in the diets for broilers. The third experiment investigated the in vitro breakdown of pectin in dehulled lupins by two pectinases, endo-polygalacturonase (PG) and pectin methyl esterase (PME).
Outcomes  Low levels of PG (0.6 g/kg diet) improved the nutritive value of both whole and dehulled lupins for laying hens while higher doses appeared detrimental to performance. Higher levels (0.8 g/kg diet) of PG improved the nutritive value of whole and dehulled lupins for broilers. Expansion was effective in breaking down cell walls of lupins but this was not reflected in improved performance of birds.

PG on its own broke down about 20% of the pectin in cell walls but a combination of pectinases broke down 50% of the pectin. Initially, PME removed the methyl ester radicals to allow PG to break down more of the glycosidic bonds of pectin. 
Implications  Treatment of lupin-based diets with PG should allow feed manufacturers to incorporate at least 10% lupins in both broiler and layer diets and, depending on the level of wet droppings that can be tolerated, may allow up to 20% inclusion. Layers can tolerate higher levels of lupins than broilers. Although expansion breaks down the cell walls of lupins it cannot be recommended as a process for improving nutritional value because of negative effects. On the in vitro evidence, a combination of two pectinases, PG and PME, offers much greater potential to improve the nutritive value of lupins and, at the same time, control the problem of wet droppings than PG on its own.
Publications Ali, A., Williams, I.H., Martin, G.B. and Sipsas, S. (2005). Hydrolysis of lupin pectin by pectinases for broilers. Proceedings of the Australian Poultry Science Symposium, 17: 219 – 222.

Ali, A., Williams, I.H., Martin, G.B. and Sipsas, S. (2006). The optimal dose of pectinase in lupin-based diets for laying hens. Proceedings of the Australian Poultry Science Symposium. 18: 218.

Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. (2006). Expansion and pectinase treatment of lupins for broilers. Proceedings of the Australian Poultry Science Symposium. 18: 62.

 
Project Title: Digestible amino acids and improved broiler performance
RIRDC Project No.: UQ-107A
Researcher: Prof. Wayne Bryden
Organisation: The University of Queensland
School of Animal Studies
GATTON QLD 4343
Phone:  (07) 5460 1257
Fax: (07) 5460 1236
Email:  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.
Background  The supply of protein and amino acids in poultry diets is a significant cost of production. Digestible amino acid values are becoming important as the basis of poultry feed formulations. However, for industry to optimise the digestible amino acid approach to feed formulation, it must be confident in estimates of broiler requirements for digestible amino acids.
Research  Studies were conducted to determine ileal digestible lysine, methionine and threonine requirements in broiler chickens from 1 to 21 and 22 to 42 days of age. Responses of sex and genotype of broilers to diets formulated on total and digestible amino acid values were investigated along with the dietary supply of ileal digestible amino acids from Australian wheat and sorghum.
Outcomes  A series of studies that examined the lysine, methionine and threonine requirements of broiler chickens in both the starter and grower periods were conducted. The influence of the sex and strain of the broiler chickens on digestible amino acid requirements was also evaluated. It was also demonstrated that the supply of digestible amino acids could be estimated from the protein content of wheat and sorghum.
Implications  The data derived in this project for amino requirements matches nicely with previous data generated by the authors on ileal digestible amino acid supply from feed ingredients. Moreover, further refinement of the digestible amino acid system for both supply and requirements will be required if the industry is to minimise protein and amino costs in the future. To achieve this goal, ongoing research is required on amino acid digestion and utilisation.
Publications Bryden, W.L. and Li., X. (2003). Prediction of amino acid digestibility of complete broiler diets. Proc. Aust. Poult. Sci. Sym., 15: 67.

Li., X., Sun, Y., Huang, K., Hyde, M.L., Muir, W.I., Gill, R.J. and Bryden, W.L. (2003). Broiler performance and methionine and lysine concentrations in diets formulated using digestible amino acid values. Proc. Aust. Poult. Sci. Sym., 15: 71.

Bryden, W.L. and Li., X. (2003). Estimating amino acid availability from digestibility coefficients: application to poultry diets. Proc. Nutr. Soc. Aust., 27: 22.

Bryden, W.L., Li., X., Nigusti Ayu, M., Huang, K.H. and Pym, R.A.E. (2004). Growth performance of broilers and fed diets formulated using digestible amino acid values. WPC 2004 XXII World’s Poultry Congress. Turkey.

Lemme, A., Ravindran, V. and Bryden, W.L. (2004). Ideal digestibility of amino acids in feed ingredients for broilers. Wld’s Poul. Sci. J. 60: 421-435.

Shini, S., Li., X., and Bryden, W.L. (2005). Methionine requirement and cell-mediated immunity in chicks. Asia Pacific Journal of Clinical Nutrition (2005) 14 (suppl). s123.

Shini, S., Li., X., Mulyantini, N.G.A., Zhang, D., Shini, A., Kumar, A., Hosking, B.J. and Bryden, W.L. (2005). Methionine requirements and immune function in broiler chicks. Recent Advances in Animal Nutrition in Australia 15: 13a. 

Li., X., Mulyantini, N.G.A., Zhang, D., Gurney, K. and Bryden, W.L. (2006). Apparent ileal amino acid digestibility of Australian sorghum for broiler chickens. Aust. Poult. Sci. Symp. 18: 48.

Zhang, D., Li., X., Huang, K., Huynh, H.T, Mulyantini, N.G.A., Kumar, A. and Bryden, W.L. (2006). The response of broiler to dietary digestible lysine levels in the starter phase. Aust. Poult. Sci. Symp. 18: 66.

Bryden, W.L., Zhang, D. and Li., X. (2006). Relationship between total crude protein content and apparent ileal amino acid digestibility of wheat for broilers. XII European Poultry Conf. (in press).

Zhang, D., Li., X. and Bryden, W.L. (2006). Dietary digestible lysine contents and broilers performance during the starter period. XII European Poultry Conf. (in press).

Efficiency of Production
Project Title: Investigation of the cause of miliary hepatitis in laying chickens
RIRDC Project No.: DAV-226J
Researcher: Dr. W. Mike Forsyth
Organisation:  Department of Primary Industries (Vic)
Primary Industries Research Victoria, Attwood Site
475 Mickleham Road
ATTWOOD VIC 3049
Phone: (03) 9217 4200
Fax: (03) 9217 4399
Email:  mike.forsyth@dpi.vic.gov.au
Objectives  ·1 To determine whether the origin of the microorganism causing miliary hepatitis is the liver or the intestine. This would be a starting point for further work to use more advanced cultural techniques such as strict anaerobiasis, egg inoculation and tissue culture. 

·2 To find a model of miliary hepatitis in laying chickens and to test two bacterial isolates in this model to see if they are a cause of miliary hepatitis.
Background  This was a pilot trial to establish a model so that laboratory manipulation can establish the cause of the disease.
Research  Laying hens at their peak of production were inoculated by mouth with extracts of intestines and liver, and by two bacteria previously collected from affected birds. They were then observed for ten days to establish whether the disease, miliary hepatitis, could be reproduced.
Outcomes  The disease was not reproduced in this trial by the tissues or by the two bacterial inoculums given by mouth.
Implications  Understanding of the cause of miliary hepatitis has been advanced but the disease remains an enigma to the industry. The evidence from these studies indicates that two bacteria isolated in the course of investigation of natural disease appear not to be primary pathogens.
Publications To date no publications have been produced from this work.

Product Quality and Safety
 
Project Title: Baseline survey of Campylobacter and Salmonella during chicken processing
RIRDC Project No.: FSA-3A
Researcher:  Gary Dykes
Organisation: Food Science Australia
P O Box 3312
TINGALPA DC QLD 4173
Phone: (07) 3214 2037
Fax: (07) 3214 2050
Email:  gary.dykes@csiro.au
Objectives ·1 To establish levels of Campylobacter and Salmonella on chicken carcasses at ten points during processing in five Australian facilities and use these data to determine the impact and relative importance of the different processing points. 
Background There are currently a wide range of practices and levels of process control across the Australian industry with respect to the various steps in chicken processing. Information on the effectiveness of these steps in reducing pathogens during processing is available within individual companies. No independent survey of both prevalence and levels of Campylobacter and Salmonella across the range of industry practices has been previously undertaken in Australia.
Research  Facilities across three states, representing both medium and large processors (based on throughput) were selected for sampling. At each of ten processing points in each facility ten chicken carcasses from a single flock were taken. Whole bird rinses were prepared and the presence and levels of both Salmonella and Campylobacter on carcasses were determined by established methods. Environmental and processing parameters associated with each of the facilities, including water temperatures, overflow rate and chlorine levels, were recorded. Data were analysed and results between plants with respect to processing parameters were compared.
Outcomes  Despite variability in processing parameters, numbers of both Salmonella and Campylobacter on positive carcasses at the initial (~3 and ~7.5 log cfu carcass-1, respectively) and final (~1 and ~3 log cfu carcass-1, respectively) sampling points did not differ significantly between plants. Prevalence of both Salmonella and Campylobacter at the final sampling point did, however, vary substantially between plants. In most cases the reason for the effect on prevalence was not easily identified or consistent but some evidence for a role of temperature and chlorine levels in the washing/spin chilling step was apparent. The scalding and spin chilling steps were the only two points that clearly reduced prevalence and levels of Salmonella and Campylobacter. Higher temperatures in the scalder, in particular, appeared to result in a greater reduction in numbers of both pathogens. Subsequent processing neutralised the effect of scalding in most but not all cases, indicating that scalding may be a useful control point if reduced levels of pathogens can be subsequently maintained. 
Implications  This study demonstrated that most of the individual processing points, with two exceptions, appeared to have no influence on levels and prevalence of pathogens on carcasses. Control of parameters associated with scalding and washing/spin chilling are critical to achieving reduction in prevalence and levels of both Salmonella and Campylobacter on chicken carcasses. The results obtained represent variations on industry practice but it cannot be assumed that this is the same across the entire industry and resu