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RIRDC Completed Projects in 2006-2007 & Research in Progress as at June 2007

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COMPLETED PROJECTS
 
 
COMPLETED PROJECTS
Flock Health
Project Title New diagnostic assays to improve control of coccidiosis in poultry
RIRDC Project No.: DAQ-316A
Researcher:  Dr Jess Morgan
Organisation: Department of Primary Industries and Fisheries (Qld)
Animal Research Institute
Locked Mail Bag No. 4
MOOROOKA QLD 4105
Phone: (07) 3362 9494
Fax: (07) 3362 9429
Email: jessica.morgan@dpi.qld.gov.au
Objectives ·1 To develop and validate new DNA-based quantitative assays for the diagnosis of poultry coccidiosis. 

·2 To develop and validate new serological assays for the diagnosis of poultry coccidiosis.
Background Coccidiosis is a major parasitic disease of poultry for which vaccines are becoming increasingly important as chemical control methods become less attractive due to the possibility of development of parasite resistance and concerns about environmental/food residues. The ability to accurately identify and quantify the species of Eimeria infecting birds, to monitor the development of immunity and to undertake epidemiological studies is essential to maximize the effectiveness of vaccination programs, but no appropriate tests exist currently. This shortfall can be overcome with the development of new species-specific molecular and serological tests. Real-time PCR uses species-specific DNA probes to both detect and quantify the species present. It has exceptional sensitivity and a higher throughput than traditional PCR assays. No available techniques (including DNA-based diagnostic probes) are suitable for use in real-time PCR assays. Preliminary work at ARI has demonstrated that ribosomal DNA (rDNA) should be suitable for development of assays. ELISAs using monoclonal antibodies can detect species-specific antibodies in blood samples and identify the species to which chickens have developed an immune response. Serological tests have the advantage of being able to detect exposure even when the parasites are no longer present. ELISAs are high throughput assays that would allow rapid screening of large numbers of birds.
Research  DNA sequences of the rDNA internal transcribed spacer 2 (ITS2) from each of the seven Australian species of Eimeria were obtained through a collaboration with Dr Robin Gasser’s laboratory at the University of Melbourne. PCR primers and species-specific real-time PCR probes were designed within the ITS2 region. Validation and specificity of the seven assays was determined by testing them on DNA stocks held at ARI (³ 2 strains of each of the seven species). Experimentally spiked faecal samples were used to assess the sensitivity of the assays. The assays were then used to detect and profile Eimeria infection in both vaccinated and unvaccinated flocks through time. An indirect ELISA was developed from E. tenella merozoite antigens after produced monoclonal antibodies were found to be unsuitable for the development of a competitive ELISA. The indirect ELISA was used to assess sera from vaccinated and unvaccinated birds from commercial farms.
Outcomes  Real-time PCR assays have been developed for each of the seven Australian species of Eimeria that infect domestic poultry. These intestinal parasites are difficult to distinguish morphologically but vary in their biology and pathogenicity. The assays are non-invasive and for the first time permit the accurate identification of species, and quantification of oocyst load, directly from faecal samples. The assays provide a reliable and sensitive technique for assessing bird exposure. They can be used to explain outbreaks, monitor and quality control vaccination programs, and follow the development of chemical resistance. 

A sensitive ELISA has been developed that clearly discriminates between sera from birds infected with wild or attenuated strains of E. tenella and sera from uninfected birds. The same ELISA also clearly distinguishes between birds inoculated with wild or virulent strains of E. necatrix and uninfected birds. Sensitivity and specificity of the test were both higher than 95%. This assay permits rapid and high throughput of samples and can detect current as well as historical exposure of birds to the parasite.

The assays developed in this project are already being requested by industry as contract research projects and discussions are underway to make the real time PCR and ELISA assays available from the Poultry Core Diagnostic Facility being established in Victoria.
Implications  This project has developed sensitive ELISA and real-time PCR assays for the detection of Australian Eimeria species in poultry. Using the real-time PCR assays it is now possible, for the first time, to identify and quantify oocyst load directly from faecal samples. The ELISA provides a rapid screen for live vaccine program quality control. The benefit to industry is better diagnostics of disease outbreaks and improved quality control of commercial live vaccines.
Publications Morgan, J.A.T., Morris, G., Anderson, G.R., Lew, A.E., Molloy, J.B., Gasser, R.B. and Jorgensen, W.K. (in prep). Multiplex real-time PCR assays for the detection and quantification of the seven Eimeria species that infect Australian domestic poultry.

Morgan, J.A.T., Jenner, M., Byrnes, R., Wlodek, B. and Jorgensen, W.K. (in prep). A protocol for direct DNA extraction from poultry faecal samples for detection and quantification of Eimeria species.

Morgan, J.A.T., Jenner, M., Byrnes, R., Wlodek, B. and Jorgensen, W.K. (in prep).Time series screening of poultry farms using real-time PCR to detect, quantify and profile Eimeria species.

Constantinoiu, C.C., Molloy, J.B., Jorgensen, W.K. and Coleman, G.T. (in prep). Development and validation of an ELISA for monitoring the vaccination programmes and exposure to Eimeria infections.

Constantinoiu, C.C., Molloy, J.B., Jorgensen, W.K. and Coleman, G.T. (in prep). Antibody response against endogenous life-cycle stages of an attenuated strain of E. tenella in chickens.

 
 
Project Title Characterisation and modulation of virulence of endemic IBDV strains using reverse genetics
RIRDC Project No.: CSA-30J
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 infectious bursal disease virus (IBDV) strains 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 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 the VP2 gene related to virulence and antigenicity and, in particular, to ascertain how these effect virulence of endemic strains.
Background Monitoring of IBDV strains over the last 14 years has demonstrated that Australia remains free of exotic IBDV strains, including very virulent IBDV (vvIBDV). Although the incursion of such strains would represent a serious threat to the local industry in terms of losses, the chance of local endemic strains increasing in virulence is also possible. Sequence analysis of vvIBDV strains has identified at least three highly conserved amino acid changes (I242, I256 and I294) within the viral coat protein VP2 which may be linked with an increase in virulence. As none of these genetic changes have been observed in an Australian isolate, it is unknown whether these substitutions would lead to an increase of virulence of local IBDV strains. 
Research  In an attempt to elucidate virulence determinants in IBDV, a fully functional reverse genetics system was developed for Australian classical strain 002/73 whereby genetic changes could be introduced into the genome. Using this system it was possible to introduce specific amino acid residues or regions associated with an increase in virulence of vvIBDV into the genome of strain 002/73. The genetically modified viruses were assessed in chickens alongside wild-type (genetically unmodified) viruses to determine whether the specific manipulations had influenced viral virulence.

For IBDV monitoring, field isolates were sourced from various commercial poultry sites across Australia. Isolates were characterized both genetically (VP2 sequences determined) and antigenically using monoclonal antibodies in an antigen ELISA. Sequences were aligned to enable a comparison to all previously isolated strains as well as selected overseas strains. 

 
 
Outcomes  The introduction of amino acid residues I242, I256 and I294, either individually or in various combinations, into the backbone of IBDV strain 002/73 did not increase the virulence of 002/73. Although a slight increase in virulence was observed when the entire VP2 gene of a vvIBDV was introduced into 002/73, it was significantly lower than that observed with the wild type vvIBDV control. 

Monitoring has indicated that the majority of strains circulating on broiler farms in Australia have not undergone any major changes in virulence or antigenicity compared to strains isolated previously in Australia. However, since 2002 there may have been an increase in the number of isolates showing unusual amino acids substitutions within the viral VP2 protein. 
Implications  Results obtained using reverse genetics would tend to suggest that strains similar to vvIBDV are unlikely to emerge in Australia due the high degree of genetic differences observed between Australian and overseas strains. 

Confirmation has been obtained that Australia remains free of exotic IBDV strains (including vvIBDV) and that local strains have not undergone any major changes in virulence or antigenicity.
Publications Sapats, S.I., Trinidad, L., Gould, G., Heine, H.G., van den Berg, T.P., Eterradossi, N., Jackwood, D., Parede, L., Toquin, D. and Ignjatovic, J. (2006). Chicken recombinant antibodies specific for very virulent infectious bursal disease virus. Archives of Virology, 151: 1551-1566. 

Ignatovic, J., Gould, G., Trinidad, L. and Sapats, S. (2006). Chicken recombinant antibodies against infectious bursal disease virus are able to form antibody-virus immune complex. Avian Pathology, 35: 293-301. 

Sapats, S., Gould, G., Trinidad, L., Parede, L.H., David, C. and Ignjatovic J. (2005). An ELISA for detection of infectious bursal disease virus and differentiation of very virulent strains based on single chain recombinant chicken antibodies. Avian Pathology, 34: 449-455. 

Ignatovic, J., Sapats, S., Reece, R., Gould, G., Gould, A., Selleck, P., Lowther, S., Boyle, D. and Westbury, H. (2004). Virus strains from a flock exhibiting unusually high mortality due to infectious bursal disease. Australian Veterinary Journal, 82: 763-768.

 
Project Title Rapid identification and pathotyping of virulent IBDV, NDV and AI isolates
RIRDC Project No.: CSA-24J
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 develop real-time PCR assays for detection of all serotype 1 infectious bursal disease virus (IBDV) strains and differentiation of very virulent IBDV from classical strains.

·2 To develop real-time PCR assays for detection of Newcastle disease virus (NDV) and differentiation of endemic low/non-pathogenic and vaccine strains to distinguish from highly pathogenic strains.

·3 To develop real-time PCR assays for detection of all avian influenza virus (AIV) strains and identification of subtypes of H5 (including H5N1) and Australian H7. 
Background Outbreaks of very virulent IBDV, virulent NDV or highly pathogenic AIV are major threats to the Australian poultry industry. Rapid index case diagnosis is essential for the swift implementation of disease control measures. New real-time RT-PCR methods based on gene-specific amplification and detection by TaqMan fluorescent probes are fast and highly sensitive, and able to differentiate between closely related isolates within a few hours.
Research  TaqMan real-time RT-PCR assays were designed to target nucleotide sequences specific for each of the virus families (IBDV, NDV, AIV) and for virulent and non-virulent isolates in each family. The assays were evaluated in the microbiologically secure laboratories at AAHL using virulent and non-virulent virus strains.
Outcomes  Rapid and highly sensitive molecular diagnostic tests based on TaqMan real-time RT-PCR have been developed and implemented to detect infectious bursal disease virus (IBDV), Newcastle disease virus (NDV) and avian influenza virus (AIV) and to differentiate harmless non-pathogenic isolates that may circulate in Australia from emerging or exotic virulent and pathogenic strains. 
Implications  The new TaqMan RT-PCR assays for diagnostic laboratories have been implemented at AAHL and will reduce the time for the identification of important poultry viruses. The early recognition of a potential emergence or outbreak of virulent or highly pathogenic strains will assist the affected industries in the rapid implementation of control measures to limit the spread of disease. 

 
 
Publications Heine, H.G., Trinidad, L., and Selleck, P. Influenza virus type A and subtype H5-specific real-time reverse transcription (RRT)-PCR for detection of Asian H5N1 isolates. Technical Report for Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease 2005. Download from http://www1.abcrc.org.au/

Rudd, M.F., Heine, H.G. and Ignjatovic, J. (2003). RT-PCR amplification and BmrI restriction digestion for the rapid detection of exotic strains of infectious bursal disease virus. Australian Veterinary Journal, 81: 162-164. 

Rudd, M.F., Heine, H.G., Sapats, S.I., Parede, L., and Ignjatovic, J. (2002). Characterisation of an Indonesian very virulent strain of infectious bursal disease virus. Archives of Virology, 147: 1303-1322. 

 
 
Project Title Molecular techniques for monitoring Marek's viraemias in broilers and layers
RIRDC Project No.: UJC-10J
Researcher:  Dr Graham Burgess
Organisation: James Cook University
Phone: (07) 4781 5472
Fax: (07) 4781 6833
Email: graham.burgess@jcu.edu.au
Objectives ·1 To assemble a comprehensive collection of DNA for Australian wild type Marek’s disease viruses.

·2 To develop and validate quantitative assays for detecting Marek’s disease viral DNA in chicken samples. 

·3 To transfer to the industry a panel of simple, cost-effective monitoring techniques to describe the effects of various management strategies on Marek’s disease control.
Background Marek's disease is a viral disease that is a major contributor to economic loss in both the egg and chicken meat industries. Vaccines and vaccination add significantly to the cost of production. There is an urgent need for diagnostics that can monitor vaccine efficacy, detect infection with wild type virus and evaluate viral vaccines in a timely and cost-effective manner. There are major limitations facing conventional diagnostics. The detection of viral DNA can provide innovative solutions to most of the problems presently being faced. 
Research  An extensive collection of viruses from international sources was acquired as purified viral DNA. Australian isolates were obtained from collaborators. Representative parts of the virus were purified. A number of the differences previously recorded as being useful for differentiating vaccine viruses and viruses capable of producing disease were shown to be unsuitable as reliable indicators of virus type in samples collected from infected or vaccinated birds. 

A comprehensive panel of diagnostics that could detect each of the three different vaccine viruses was developed for use in conventional laboratories and those equipped with the more advanced real-time detection equipment. Samples were collected and processed from Australian poultry flocks to provide proof of concept.
Outcomes  The collection of international and Australian viruses has been prepared in a way that they could be used to obtain information on the differences between Australian viruses and those being used as vaccines.

The panel of diagnostic techniques developed has been assessed to ensure that these procedures could be used on a routine basis for monitoring the effectiveness of vaccines and the presence of viruses capable of producing outbreaks.

 
 
Implications  The industry now has available a panel of techniques that can be applied in a range of diagnostic laboratories. These techniques can be used on a routine basis to monitor the effectiveness of vaccination against Marek's disease. The industry also has available diagnostics that will allow outbreaks of disease to be detected and monitored. It will now be possible to make logical management decisions based on the results of these assays. 

The panel of viruses that were acquired will be an important resource for the industry for many years to come. As new technologies become available it will be possible to use this panel to design the next generation of diagnostic tools for Marek’s disease.

 
 
Bird Nutrition and Feed Supply
Project Title Variability in performance and physiology of broilers fed wheat- and sorghum-based diets
RIRDC Project No.: US-137A
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 define what characteristics are critical to determining/predicting the feeding value of wheat and sorghum grains, with an emphasis on wheat.

·2 To provide an approach the will contribute to a more complete understanding of feeding value for these cereal grains, including the impact on gut physiology/microbiology.
Background In the past decade, attention has been drawn to limitations on voluntary feed intake of different feed grains by broiler chickens, and as a consequence, losses in growth and feed efficiency. Although feed processing and use of feed enzymes increases feed intake, these factors did not remove the variability between different sources of grain. It is imperative that the factor(s) in feed grains that lead to this variation in broiler performance be identified and then research can be designed to overcome the losses associated with variation in broiler performance and uniformity at market age. This will also increase the efficiency of the industry.
Research  A broiler chick bioassay was developed for assessing measurements of feed value of small amounts of test diet (~30kg). In the present study, a total of 45 feed grains (34 wheat, 8 triticale and 3 sorghum) were ground and provided 75% of each test diet; these diets were then split and one portion fed as is, the other supplemented with commercial feed enzymes (xylanase and phytase). The 90 diets were fed to four cages of six male broilers. The performance of the young broilers (4 to 17 days of age) were measured, as was their ability to digest the diets, and physiological assessments made of diet effects (in terms of immune response, gut tissue health, bone quality, and digesta or intestinal content viscosity). 
Outcomes  The preliminary study confirmed previous experiences in that when a number of sources of a feed grain (i.e. wheat) are compared using the broiler bioassay there are large differences in voluntary feed intake and these differences in intake are directly translated into differences in growth. Simply, if feed intake is limited then growth is limited. In the 34 wheat-based diets (each fed with or without enzyme, 68 diets) there was over a 20% difference in feed intake and likewise for growth. When examined further, those diets that facilitated higher feed intake actually had the lowest (best) feed conversion ratios; this is contrary to what is usually expected with higher feed intake. The nutrients of a diet are proportioned to supply the bird’s requirements for maintenance and growth, in that order. In the case of the wheat-based diets, with higher intakes (less limitation in intake) birds were able to supply a larger proportion of nutrients (above maintenance) for growth and, hence, birds grew markedly larger and feed conversions were lower. Similarly, as observed previously there was no relationship between feed intake and energy availability of diets. This again strongly supports that limitations in intake (i.e. digesta passage rate) are preventing the bird from adapting intake to meet energy requirements.

Moderate relationships were also observed between the measures of broiler performance and assessments of gut tissue health and immune response. This suggests that the bird responds directly to allergens in the diet or to gut bacteria populations (number or type of bacteria) that are influenced by the diets. To our knowledge, this is the first comparison of the impact of many sources of wheat fed in diets with or without enzyme on immune response and is worthy of further investigation; as is the need to identify those factors that limit feed intake.
Implications  The demonstrated limitations on feed intake bring into the question our present methods of formulating diets for nutrient balance. If there is a limitation in feed intake, there is minimal need to know the nutrient level or availability, as the bird will not be able to consume enough of these diets to attain its genetic potential and non uniform growth and market ages will be encountered. In a previous study, it was demonstrated that near infrared reflectance spectrometry calibrations could be developed to predict voluntary feed intake and feed conversion ratios of birds fed the source of grain measured. This technology is currently used by the plant breeders and feed industry and therefore may offer an opportunity to provide "tools" to select feed grain cultivars with no feed limitations or to purchase grains with no intake limitation for use in broiler chicken diets.

The study undertaken indicates that limitations in feed intake are real, and that differences between grains in this respect may be in part explained by the their differential effects on the bird in terms of stimulating an immune response and/or damaging gut tissue which in turn limits intake. However, other research projects have highlighted the concern that digesta passage rate (and thus amount of feed eaten) may be limited by physicochemical (physical and/or chemical) differences that restrict hydration time of the feed; digestion can not proceed until the diet is hydrated in the gut. Further research is required to resolve factors that limit feed intake.
Publications Scott, T.A. (2006). Feed value of wheat-, triticale- and sorghum-based diets supplemented with and without enzymes for broilers. Proceedings Australian Poultry Science Symposium, 18: 83-86.

Scott, T.A. (2006). Variability in feed value of Australian wheat-, triticale- and sorghum-based diets supplemented with and without enzymes for broilers. Poultry Science Association 95th Annual Meeting, Edmonton, Alberta, July. Poultry Science, 85 (Suppl 1), abstract 225.

Scott, T.A. and Muir, W.I. (2007). Variation in broiler performance due to wheat source and enzyme supplementation. Australian Poultry Science Symposium 19: 67.

 
Project Title Early dietary and management intervention on broiler breast meat yield
RIRDC Project No.: US-134A
Start Date: 01/07/2004
Finish Date: 30/12/2007
Researcher:  Prof Tom Scott
Organisation: The University of Sydney
Chair of Poultry Science
Faculty of Veterinary Science
PMB 3 University of Sydney
Poultry Research Unit
CAMDEN NSW 2570
Phone: (02) 4655 0612
Fax: (02) 4655 0693
Email: toms@camden.usyd.edu.au
Objectives ·3 To increase the yield of high value breast meat from broiler chicks by developing early dietary and/or management intervention programs to stimulate initial muscle cell fibre numbers and subsequent breast muscle yield at an optimum market weight.

·4 To maximise the capacity of ‘supply’ organs (gut, respiratory, curculatory systems) to supply the nutrients and metabolic homeostasis of the ‘demand’ tissues (meat, feathers and maintenance).

·5 To monitor (and expand methods to monitor) the effect of early dietary and/or management programs on the development of growth related metabolic disease, and on broiler flock uniformity and market efficiency.
Background The chicken meat industry has increased production to meet the steady demand by consumers; overall, this has been equal to 50% increases in production and consumption in each of the last several decades. Breast meat is the most valuable cut of the bird; although it accounts for approximately 20-25% of the live weight, it generates approximately 60% of the value of the bird. The amount of breast meat produced by a bird is based on a combination of both muscle fibre number and size. Typically in most animals muscle fibre number is fixed at birth. However, in the avian species there is believed to be a short period (approximately one week) after hatch when muscle fibre numbers can be increased. This may be an accommodation for the limited resources (physical space and nutrients for tissue development) that can be provided by the hatching egg. This study was designed to look at practical applications that may facilitate higher muscle fibre number development in the crucial first week post hatch.

Five possible early dietary intervention strategies for increasing breast muscle yield are (a) increasing reserves for the developing embryo via maternal routes (hatching egg); (b) increasing reserves for the developing embryo via in-ovo ‘feeding’ (delivery of nutrients into the egg by injection, similar to in-ovo vaccinations currently used in the industry); 

(c) provision of feed and/or water at hatch (in incubator or with ‘patio’ cage systems where hatching eggs are transferred from incubators to trays immediately above mesh floor cages with available feed and water and are brooded post-hatch for 7 to 21 days before transfer to conventional litter systems); (d) stimulation of gut maturation post hatch to facilitate nutrient uptake; and (e) increasing available nutrients in the first seven days post hatch.

Various combinations of the above are likely to act synergistically to improve nutrients available to stimulate muscle fibre numbers. 
Research  A series of experiments were conducted to examine the impact of delivery of bioactives (materials that have significant effect on tissue development, immune status, or other physiological factors) and/or nutrients to the developing embryo in ovo or directly into the egg. This system of delivery is now routinely used by the commercial industry to deliver vaccines to chicks and hence could be simultaneously employed to deliver bioactives or nutrients to the embryo. 

In a commercial hatchery situation eggs may begin to hatch as early as 19 days of incubation and hatching times may be spread out over a two to three day period. This is further complicated by delays in hatch removal, processing time for chicks at the hatchery as well as variable times for delivery and placement in the growing facilities. In some cases the first hatched chicks may be without feed or water for up to 96 hours (4 days). A study was conducted that separated chicks within 2 hr of hatch into four groups, those that were maintained as would normally happen (without feed or water) and then three groups given either feed, water or both feed and water. 
Outcomes  The data from in ovo feeding trials suggested that in ovo ‘feeding’ is a potential route for administration of nutrients, but it also may be a useful tool for identifying nutrients that may be supplied to the hatching egg by fortifying the diet of the breeder hen. Unfortunately this study was terminated before its due date as a consequence of the departure of key members of the research team. 

The study on provision of feed and/or water immediately post-hatch showed that groups given feed and water had significant growth advantages up to the end of the study (12 days post hatch). Practical means of applying this are being developed by supplement suppliers and equipment manufacturers. 
Implications  The study demonstrated the potential for use of in ovo ‘feeding’ as a direct strategy for increasing nutrients to the late developing embryo and potentially as a means of identifying dietary factors that could be added to the hatching egg by manipulating the maternal diets. Likewise, it is evident that very early hatching chicks are significantly advanced by providing access to both feed and water during the hatching process. 
Publications Wilkinson, S.J. and Scott, T.A. (2006). Preliminary results of in ovo strategies to increase breast meat yield in broiler chickens. Proceedings Australian Poultry Science Symposium, 18: 206-209.

Wilkinson, S.J. and Scott, T.A. (2005). Satellite cells: A review of their physiology, manipulation and importance to muscle development. Proceedings Australian Poultry Science Symposium, 17: 48-52.

 
 
Project Title Assessment of the anti-nutritive effects of phytate by dephytinisation
RIRDC Project No.: US-133A
Start Date: 01/01/2005
Finish Date: 31/01/2008
Researcher:  Dr Peter Selle
Organisation: The University of Sydney
Faculty of Veterinary Science
425 Werombi Road 
CAMDEN NSW 2570
Phone: (02) 9351 1697
Fax: (02) 9351 1693
Email: sellep@camden.usyd.edu.au
Objectives ·6 To define the negative ecological and nutritive effects of phytate on chicken meat production via the experimental use of dephytinised feed ingredients.
Background Phytate, the mixed salt of phytic acid, is found in all plant-sourced feedstuffs. Broiler diets typically contain 10.0 g/kg phytate or 2.82 g/kg phytate-bound phosphorus (phytate-P). Phytate-P is only partially available to broiler chicks because they lack sufficient endogenous phytase activity; therefore, phytase feed enzymes were introduced in The Netherlands in 1991, primarily to increase phytate-P availability and to reduce P excretion in pigs and poultry and the P load on the environment. It is becoming increasingly evident that phytate also depresses the utilisation of protein/amino acids and energy in broilers. However, the extent to which phytate depresses protein and energy utilisation is not clear because phytase feed enzymes degrade in the order of 35% of dietary phytate at standard inclusion rates. Pre-treatment of feed ingredients with phytase to eliminate phytate (or ‘dephytinisation’) was considered to be a viable approach to define accurately the negative impact of phytate on protein and energy utilisation. 
Research Sorghum and soyabean meal were selected as key feed ingredients and a hydrothermal dephytinisation procedure was developed. Sorghum was selected because, unlike wheat, it contains little intrinsic plant phytase activity. Essentially, the project methodology involved mixing the finely ground feedstuff with an equal quantity of water for 2 hours at 45° C as a slurry, followed by drying at 60° C for 70 hours. For dephytinisation, liquid exogenous phytase and citric acid were added to the slurry, which was not the case for placebo dephytinisation or sham-treatment. The effects of dephytinised and sham-treated sorghum and soyabean meal on nutrient utilisation and growth performance of broilers were evaluated. 
Outcomes The addition of citric acid to reduce slurry pH proved critical for dephytinisation. However, that sham-treatment needed to be innocuous was pivotal to the project, whereas sham-treatment negatively impacted on growth performance and nutrient utilisation to significant extents, which was clearly evident with sorghum and, presumably, any beneficial effects of dephytinisation were overwhelmed. That ‘moist-cooking’ adversely influences protein availability in sorghum is recognised. Therefore, it is relevant that pre-treatment of sorghum reduced protein (N) retention and growth performance in broilers offered sorghum-casein diets. It seems likely that pre-treatment of sorghum reduced protein digestibility by inducing disulphide cross-linkages and the formation of indigestible protein (kafirin) aggregates. Pre-treatment of soyabean meal negatively influenced N retention but the reasons for this are less clear. Also, dephytinised feedstuffs retain relatively large amounts of ‘residual phytase activity’, which may be another complicating factor.
Implications The hydrothermal dephytinisation procedure developed in this project was not suitable because sham-treatment is not sufficiently innocuous in respect of nutritional parameters other than phytate. However, citric acid facilitates phytate degradation by phytase, which may hold practical relevance. Also, there is the possibility that steam-pelleting sorghum-based broiler diets at high temperatures may have adverse effects on protein digestibility.
Publications Selle, P.H. (2006). Citric acid enhances enzymatic hydrolysis of phytate. Proceedings Australian Poultry Science Symposium, 18: 91.

 
 
 
 
Food Safety
Project Title Development of a sequence-based bacteriophage typing system for Salmonella
RIRDC Project No.: IMV-5A
Researcher:  Dr Michael Heuzenroeder
Organisation: Institute of Medical and 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.
Background Many cases of salmonellosis would be prevented if common outbreak sources could be identified rapidly. Rapid source identification would allow earlier intervention and thereby prevent a significant proportion of cases. Uninformed and sensational reports of food safety can affect retail consumption of implicated food/food products. In view of these problems which face the industry, development of DNA-based rapid typing methods for Salmonella in conjunction with classical methods was initiated in a number of successive projects.

Clusters of cases (or outbreaks) are difficult to recognise among common phage types and additional sub-typing is often needed. However, existing methods cannot always distinguish outbreak from non-outbreak strains within a phage type. Often, a single phage type predominates in one type of animal or geographic area, limiting the epidemiological value of phage typing. Pulsed-field gel electrophoresis (PFGE), that is the ‘gold standard’ of molecular typing techniques, takes 5-6 days to obtain a result and suffers from a range of other problems. Amplified fragment length polymorphism (AFLP) has been investigated as a potential alternative to PFGE in a previous project, but did not provide significant improvement. It has recently been shown that the major significant differences between chromosomes in members of clonal species like Salmonella are due to phages or phage-like sequences, therefore phage sequences are a potential target for a molecular typing. Development of a molecular phage typing system will draw upon the discoveries made in earlier projects to develop a typing system, which would be rapid, discriminatory, objective and cost effective when compared to the ‘gold standard’ typing methods in common use.
Research  Increasingly larger numbers of isolates have been subjected to classical serotyping and phage typing for the chicken meat industry. During the course of the project, no significant changes in the isolation rates of Salmonella serovars was observed, with the low virulence S. Sofia remaining the most commonly isolated serovar. S. Typhimurium and S Infantis are two other serovars consistently isolated from chickens. The phage type (DT) distribution in S. Typhimurium has changed during the course of the project, with DTs 108, 135 and 135a becoming predominant.. Changes in DT distribution have been observed before and are hard to explain.

Molecular typing techniques such as PFGE, AFLP and MLST have been shown to be slow and expensive. In addition they have been shown to be of limited use in certain clonal serovars such as S. Typhimurium, yielding low discriminatory power for epidemiological purposes. Low discriminatory power can also be a problem with classical phage typing. In view of this problem, a rapid PCR-based typing method based upon bacteriophage sequences was developed. This method, termed MAPLT (Multiple Amplification of Phage Loci Typing), showed itself in pilot studies in S. Typhimurium and S. Enteritidis to be superior to classical phage typing, PFGE and MLST. A second rapid typing method was also trialed in comparison to MAPLT and the other methods. This method, termed MLVA (Multi-Locus VNTR (Variable Number of Tandem Repeats). Analysis)), was comparable with MAPLT in discriminatory power, but often required the expense of DNA sequence analysis to fully analyse the results. MLVA potentially may not be as applicable to other serovars of significance as the amplification targets are relatively limited at present. It is possible that a combination of MAPLT and MLVA may be the best methods to discriminate Salmonella serovars.

Studies were also undertaken to determine the impact of phage immunity genes on the phage type DT, in which they reside. It was shown that the immC region of ST64B (defective phage characterised in a previous project that could mediate phage type conversion in a manner consistent with its predicted mechanism of action). It was concluded that although some predictions on specifies phage type could be made, the complexity of other confounding influences would make a molecular typing system that could be directly related to classical methods impractical. It was clearly demonstrated that strains having a similar DT were not necessarily genetically related.
Outcomes  A conventional serotyping and phage typing service was provided for the chicken meat industry.

A MAPLT typing system for Salmonella was developed and an Australian patent to cover the IP lodged.

MLVA was shown to be a practical and useful tool for typing Salmonella.
Implications  Classical typing of Salmonella will remain in use for some time yet, although it is clear that molecular based methods will replace classical methods in the medium term.

Given the discriminatory power and use of MLVA by a number of laboratories in Australia, a national database should be set up to collate epidemiological data.

Molecular typing methods (MAPLT and/or MLVA) should be expanded into other (non-S. Typhimurium) serovars of significance in Australia.
Publications Ross, I.L. and Heuzenroeder, M.W. (2005). Use of AFLP and PFGE to discriminate between Salmonella enterica serovar Typhimurium DT126 isolates from separate food-related outbreaks in Australia. Epidemiology and Infection, 133: 635-634.

Ross, I.L. and Heuzenroeder, M.W. (2005). Discrimination within Phenotypically Closely Related Definitive Types of Salmonella enterica Serovar Typhimurium by the Multiple Amplification of Phage Locus Typing Technique. J. Clin. Microbiol., 43: 1604-1611. 

Tucker, C.P. and Heuzenroeder, M.W. (2004) ST64B is a defective bacteriophage in Salmonella enterica serovar Typhimurium DT64 that encodes a functional immunity region capable of mediating phage-type conversion. Int. J. Med. Microbiol., 294: 59-63.

 
 
 
 

Project Title		 Campylobacter bio-replacement program to control food poisoning organisms in poultry
RIRDC Project No.: UG-7A
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. 
Background Bacteria of Campylobacter spp., in particular C. jejuni, are now the most common cause of food-borne disease in the developed world and have surpassed Salmonella and Shigella spp as causes of food related illness. It is estimated that the incidence of C. jejuni enteritis in Australia is at least 200,000 people each year. C. jejuni strains are widespread in animals and birds where they are commensal organisms and do not cause disease. The processing of animal food products, and poultry in particular, often leads to contamination of the end product which in turn can lead to the transfer of Campylobacter spp to humans if products are not adequately handled and/or cooked by the consumer. Campylobacter spp also have a relatively low infectious dose (can be as little as 500 organisms) and so it is essential that the risk of transfer of Campylobacter spp to humans via chicken meat products is minimised.

This project aimed to develop an alternative strategy to conventional biosecurity approaches for control of campylobacers in poultry. It was proposed that an avirulent strain of C. jejuni, which has been fully characterised, would be used to establish continuing colonisation in chickens. This strain would be a strong coloniser of the chicken intestinal tract with a capacity to displace other Campylobacter spp that could constitute a food poisoning risk.
Research  Four different chicken infection trials were carried to establish the validity of the "Biological Control" approach to colonisation of chickens with pathogenic campylobacters. 

The first trial aimed to establish an appropriate inoculation time and determine persistence of a ‘bio-replacement’ C. jejuni strain in the chickens during a 56 day period. 

The second trial aimed to establish whether persistence of colonisation by a C. jejuni bio-replacement’ strain throughout the life of the chicken could be affected by challenge by three other strains. Three C. jejuni strains were chosen as challenge strains on the basis of their belonging to a highly colonising phenotype similar to that of the ‘bio-replacement’ strain. 

In a third trial, colonisation of 7-day old chicks in a 100-bird flock was established by a C. jejuni ‘bio-replacement’ strain. The chicks were then challenged by other highly colonising strains. The chicks were then re-challenged with the ‘bio-replacement’ strain at various points in their 56 day production life cycle.

A fourth trial was designed to test whether the ‘bio-replacement’ C. jejuni strain was able to maintain itself in the commercial chicken flock in face of a challenge with highly colonising strains introduced at ‘high risk times’ and continue to dominate chicken gastrointestinal tract at key harvest dates – days 35 and 56. This study also examined the ability of C. jejuni ‘bio-replacement’ strain 331 to survive on chicken carcasses during carcass processing, the bacterial load of this strain being determined for freshly processed and frozen/thawed birds. In addition, the ability of the C. jejuni ‘bio-replacement’ strain to survive prolonged storage in chicken feed, water and Brucella broth was tested.

In order to be able to easily and conclusively identify the C. jejuni ‘bio-replacement’ strain and differentiate it from other C. jejuni stains, two sets of specific PRC primers were assessed for their ability to uniquely identify the ‘bio-replacement’ strain.
Outcomes  The four colonisation trials demonstrated that the candidate ‘bio-replacement’ C. jejuni strain can be successfully used to exclude other campylobacters from chicken flocks with a single inoculum. The maximum bacterial load of this strain was detected at the level of 102 bacteria per gram of chicken meat for birds sacrificed on day 28 following processing with available chlorine concentration of 1 PPM. This load was drastically reduced when available chlorine concentration was between 2 and 3 PPM for processing on days 35 and 55. Only 80 campylobacteria per gram of chicken meat was detected on fresh carcasses and no bacteria could be detected on frozen carcasses.

It was found that the candidate C. jejuni ‘bio-replacement’ strain should be stored in Brucella broth at 40 C and it will remain viable for up to 25 days.

Of 164 strains used to assess the specificity of the PCR primers for the candidate C. jejuni ‘bio-replacement’ strain, the first primer set showed cross reaction level of 3%, and the second set produced cross reaction at 1.2%.
Implications  Implementation of a ‘bio-replacement’ strategy could reduce contamination of poultry products with pathogenic campylobacters and minimise risk to the consumer, leading to higher consumer confidence in the poultry products. However, to obtain registration of such a product, work would need to be undertaken to prove that the ‘bio-replacement’ strain itself does not pose a food safety risk.

 
Project Title: Development and validation of Campylobacter microarrays for virulence detection and strain differentiation in poultry products
RIRDC Project No.: RMI-14A
Researcher:  Prof Peter J Coloe
Organisation: Royal Melbourne Institute of Technology
Phone: (03) 9925 7104
Fax: (03) 9925 7110
Email: pcoloe@rmit.edu.au
Objectives ·1 To utilise existing knowledge on Campylobacter spp. in order to select the most appropriate virulence factors to be used in the development of a Campylobacter microarray.

·2 To use the microarray results in a format that will enable the rapid identification and characterisation of Campylobacter species from poultry and poultry products.
Background Traditionally, the presence of Campylobacter spp. in food or in clinical specimens, is tracked by microbiological culture and isolate characterisation. Molecular techniques such as PCR have been used in some instances but with limited success. The use of DNA and oligonucleotide microchip technology is the ideal approach and will allow for the simultaneous analysis for multiple markers leading to the rapid identification and differentiation of campylobacters. Multiple specific genes can be used to identify and type a single microorganism, thus turning microbial identification into a pattern recognition process. The technology serves as a platform technology for the future incorporation of selected bacterial virulence genes so as to obtain a comprehensive analysis of the genetic makeup of a bacterial sample. Alternatively, specific gene markers could be used in a format that will allow screening of poultry flocks for high-risk strains allowing better targeting of intervention strategies at either the farm or processing plant.
Research  The project involved the assessment and selection of appropriate oligonucleotides for inclusion in a microarray through the use of bacterial genomic techniques and alignment of gene sequences to the Campylobacter genome. The proposed project also investigated the design of microchips for the immobilisation of oligonucleotides and hybridisation conditions for simultaneous detection of selected virulence factors.
Outcomes  Five key genes have been identified for the detection of potential GBS-associated Campylobacter strains and could be configured into a simple multiplex PCR. Similarly, key genes were identified and used in a grouping system that was then used in a limited survey of Campylobacter strains to determine if such a system had potential in grouping and identifying major poultry strains. The limited microarray was also used to investigate changes in virulence gene expression when poultry and human derived Campylobacter spp. were subjected to different environmental stresses, with poultry derived strains showing the least variation in virulence gene expression. 

 
 
Implications  This project has shown that molecular techniques could be used to select specific gene markers for the rapid identification of high risk Campylobacter strains and their potential virulence response when subjected to certain stressors. The information could be used to design tools to detect high risk Campylobacter strains in chicken flocks or on chicken carcasses so that interventions on-farm and in the processing plant can be better targeted.
Publications Liu, K., Nawagamuwa, V., Baxter, N.J., Wan, J., Coventry, M.J., Lee, A. and Coloe, P.J. (2005). Development of a multiplex PCR for the specific detection of Campylobacter jejuni GBS-associated strains. 13th International Workshop on Campylobacter, Helicobacter and Related Organisms, September 2005, Gold Coast, Queensland, Australia (Poster).

Liu, K., Nawagamuwa, V., Baxter, N.J., Wan, J., Coventry, M.J., Lee, A. and Coloe, P.J. (2005). Use of a PCR array to assess the influence of growth condition on virulence genes expression in Campylobacter jejuni. 13th International Workshop on Campylobacter, Helicobacter and Related Organisms, September 2005, Gold Coast, Queensland, Australia (Poster).

Baxter, N.J., Wan, J., Coventry, M.J., Lee, A. and Coloe, P.J. (2003). The sequence diversity of flaC and flaD from C. jejuni. 13th International Workshop on Campylobacter, Helicobacter and Related Organisms, September 2003, Denmark (Poster).

Environmental Management
 
Project Title Efficacy of windbreak walls for odour reduction
RIRDC Project No.: DAQ-321A
Researcher:  Mr Mark Dunlop
Organisation: Department of Primary Industries and Fisheries (Qld)
PO Box 102
TOOWOOMBA QLD 4350
Phone: (07) 4688 1280
Fax: (07) 4688 1192
Email: mark.dunlop@dpi.qld.gov.au
Objectives ·1 To investigate the value of windbreak walls for improving dispersion of exhaust air from tunnel ventilated sheds.

·2 To evaluate the use of windbreak walls as an odour reduction strategy for meat chicken sheds.
Background Odour emissions are the primary source of complaints about intensive poultry operations and there is increasing pressure on some poultry producers to reduce odour impacts. Windbreak walls have been identified as an inexpensive technology that may be capable of enhancing the dispersion of odorous emissions from tunnel ventilated poultry sheds. 

Windbreak walls operate by forcing air exhausted from sheds upwards, causing it to mix and be diluted, thereby theoretically reducing the concentration of odours and reducing odour impacts on nearby receptors. However, the efficacy of windbreak walls in terms of improving odour dispersion and reducing downwind odour concentrations has never been properly evaluated. Consequently, this project was developed to assess the efficacy of windbreak walls to improve odour dispersion. 
Research  Several methods were used to assess the efficacy of windbreak walls in improving dispersion. Tracer gas methods were initially trialled; however, results were inconclusive. Smoke observation methods were adopted and were found to be very effective. Smoke was release from two sheds, one with a windbreak wall and one without (the control). The movement and dispersion of the smoke was photographed and analysed. Finally, based on results seen during the smoke observations, computational fluid dynamics (CFD) modelling was used to quantify differences in dispersion from a control shed and one fitted with a windbreak wall. The scope of the CFD modelling was expanded to include an evaluation of short stacks. 

 
 
Outcomes  The efficacy of windbreak walls and short stacks in improving odour dispersion was highly dependent on atmospheric stability, weather conditions and shed operating conditions. Therefore, no definitive conclusion of overall performance could be reached. However, the results indicated that windbreak walls did not dramatically improve dispersion or reduce downwind ground level concentrations beyond a reasonably short distance (150 m to 500 m). Short stacks exhibited greater dispersion resulting in decreased downwind ground level concentrations.

Windbreak walls appeared to offer some additional benefits such as dust control, sunlight obstruction and maintaining fan performance with strong opposing winds. Short stacks would probably offer similar benefits. 
Implications  This project has shown that windbreak walls do not significantly improve the dispersion of exhaust air from tunnel ventilated sheds and would be unlikely to consistently reduce odour impacts. Short stacks show more promise than windbreak walls for improving dispersion. The performance of windbreak walls and short stacks is dependent on operating conditions such as atmospheric stability, weather and farm operations. As these conditions constantly change, the value of windbreak walls and short stacks for improving dispersion constantly changes. The efficacy of windbreak walls and short stacks in improving dispersion is therefore unreliable and unpredictable. As such, these devices should not be solely relied upon as an odour reduction strategy for meat chicken sheds.

 
 
Project Title Trials of odour control technologies for broiler farms
RIRDC Project No.: DAV-213A
Researcher:  Dr Julie Simons
Organisation: Department of Primary Industries (Vic)
Primary Industries Research Victoria, Attwood Site
475 Mickleham Road
ATTWOOD VIC 3049
Phone: (03) 9217 4358
Fax: (03) 9217 4111
Email: julie.simons@dpi.vic.gov.au
Objectives ·1 To identify, assess and quantify the performance of several technology options to control odour from broiler sheds.

·2 To define the conditions and application methods in which they work.

·3 To monitor the extent of odour emissions as they occur on a number of farms.

·4 To develop test protocols for the testing the efficacy of future odour control technologies as they become available.
Background The management of odour is a priority for the broiler industry in order to protect the amenity of surrounding neighbourhoods and to ensure that the future development of the industry is managed in an environmentally responsible way. Commercial technologies that act to reduce odour may provide effective options for the broiler industry to manage odour emissions. However few technologies have been proven to be effective within the Australian broiler industry environment. Thus research to establish the effectiveness of these technologies is important to ensure industry can adopt effective and feasible options.
Research  Overall, the project comprised of six sections, viz. (a) a preliminary farm survey; (b) a method development study; (c) a review of odour control products; (d) field trials on four products; (e) assessment of the relationships between odour emission and farm management factors; and (f) the development of Recommendations for odour and litter assessment methodologies.

Initially a preliminary survey of ten Victorian broiler farms was conducted to develop an understanding of typical farm and litter conditions. From this survey four farms were selected for ongoing experimental work. The project then examined odour emissions and litter conditions from four farms across Victoria over a two year period, encompassing 56 sheds from 22 farm visits in total. The testing was restricted to cover bird age from 26 – 36 days of age ie just prior to the first harvest of birds when the sheds are generally at their highest load (in terms of bird mass). The odour emissions were analysed by two laboratories with one laboratory (ETC) reporting a total of 47 average shed odour emission results, and the other (EPA) a total of 33 average odour emission results. Each odour emission result was matched with shed ammonia, litter moisture, litter pH, and other shed and environmental factors.

Four commercial odour control technologies were tested on farm for their efficacy. These four products were a litter additive of ‘beneficial micro-organisms’, a feed additive, based on a natural plant extract (Yucca), and two air additives (natural oils) applied through water based misting systems.
Outcomes  Baseline data were generated representing typical odour emission rates for Victorian broiler farms when birds are within the range of 26-36 days.

The four technologies tested in this study were not shown to be effective in reducing odour under the conditions in which they were applied and tested. These technologies, were selected because they were well recognised products internationally or had sufficient product information to support their claims. All the technologies considered in this study were selected, in part, because they were viewed as being potentially feasible options for industry. However, assessment by olfactometry did not result in a reduction in odour being observed. 

Method protocols were developed for the measurement of odour, and litter conditions on broiler farms.

The project resulted in a greater understanding of the types of odour control technologies available to the broiler industry and of the considerations that need to be taken into account prior to adoption of such technologies into a food producing environment such as a broiler shed. It also provided a greater understanding of olfactometry and the methodology under which it can be used to assess odour on farm and assess odour control technologies. The relationships between odour and some farm management conditions were also elucidated.
Implications  Odour control technologies will continue to become available to the broiler industry on the promise of providing opportunities for odour management and flexibility for new farm or farm extension planning options. The broiler industry must assess each technology for its suitability for use in the broiler and food production environment, as well as examine the effectiveness of the product to reduce odour. 

The baseline odour emission data will provide an indication of the expected odour emissions from sheds when they are most under load, and can be applied to odour modelling studies. 

This work should assist growers and other industry stakeholders in their decisions with respect to whether to adopt odour control technologies on farm, as well as providing guidance to manufacturers of such technologies about the testing required to substantiate the effectiveness of their products.

Further study is required to examine the nature of odour as well as total odour emissions and the various factors that impact upon them. 

Efficiency of Production
Project Title Developing novel biological management strategies for darkling beetle (Alphitobius diaperinus [Panzer]) in Australian broiler houses
RIRDC Project No.: DAQ-330A
Researcher:  Mr Trevor Lambkin
Organisation: Department of Primary Industries and Fisheries (Qld)
Entomology Building, Indooroopilly Research Centre
Locked Mail Bag No 4
MOOROOKA QLD 4105
Phone: (07) 3362 9606
Fax: (07) 3362 9429
Email: trevor.lambkin@dpi.qld.gov.au
Objectives ·1 To develop protocols for laboratory inoculation and assessment of virulence of entomopathogenic fungi (bio-pesticide) as a litter treatment for darkling beetle management.. 

·2 To evaluate entomopathogenic nematodes in the laboratory for their potential for beetle management as a litter or floor applicant. 

·3 To investigate in the laboratory the efficacy of entomopathogenic fungi in combination with diatomaceous earth.
Background Previous RIRDC funded research into the management of darkling beetle in Australian broiler houses indicated that high pest numbers predominately occur in broiler house litter and that treatments of earth floors of houses with the two currently registered insecticides (fenitrothion and cyfluthrin) are mostly ineffective. More importantly, this research also pointed out that, in most cases, applying control agents to fresh litter would be much more effective than application to floors. In light of these findings, the aim of this proposed project was to develop and trial in the laboratory a range of insecticide-free litter and earth treatments.
Research  The susceptibility of Alphitobius diaperinus to two fungal species, Beauveria bassiana and Metarhizium anisopliae, was evaluated in the laboratory and the most virulent isolates of each species were further tested in laboratory litter assays at 30°C and 55% RH, singly and in combination with diatomaceous earth. In addition, the susceptibility of larvae and adults of A. diaperinus was tested against four nematode species (Steinernema feltiae, S. carpocapsae, S. riobrave and Heterorhabditis bacteriofora) at a range of temperatures in the laboratory. The most virulent nematode species was further assessed in laboratory test boxes containing simulated litter and a sand substrate. 

 
 
Outcomes  Litter surface applications of fungal conidia were very effective, with 91% and 88% progeny reductions achieved by B. bassiana and M. anisopliae respectively at relatively low doses (0.49 x 108 and 0.87 x 109 conidia/m2 respectively). At almost all doses of M. anisopliae, combining the fungi with diatomaceous earth (at 0.25 kg/m3 and 0.5 kg/m3) saw an effect greater than using each agent singly. This was not evident with B. bassiana as its efficacy at the tested doses was very good and therefore overall efficacy was not notably enhanced by diatomaceous earth. The most effective and reliable nematode species was S. carpocapsae at 30°C. Results also indicated that a damp environment might be required for nematodes to be effective.
Implications  The results of these laboratory studies indicate that applications of native Australian isolates of B. bassiana, M. anisopliae, and S. carpocapsae to litter showed promising efficacy for control of A. diaperinus. With this in mind, field trialling of these agents should be undertaken to test their efficacy under conditions experiemced in broiler house.

 
 
 
 
Project Title Broiler performance on pearl millet based diets
RIRDC Project No.: DAQ-337A
Researcher:  Mr Danny Singh
Organisation: Department of Primary Industries and Fisheries (Qld)
Poultry Research and Development Centre (PRDC) 
PO Box 327
CLEVELAND Qld 4163
Phone: (07) 3820 0502
Fax: (07) 3824 4316
Email: Danny.singh@dpi.qld.gov.au
Objectives ·5 To ascertain the effect on broiler performance of a peal millet based diet.

·6 To ascertain the effect on chicken meat quality when pearl millet based diets are fed to birds.

·7 To promote the benefits and advantages of pearl millet as a feed grain to both grain growers and meat chicken industry.
Background RIRDC Project DAQ-302A characterised the nutritive value of pearl millet (PM) hybrids. PM hybrids were shown to have higher apparent metabolisable energy (AME) values and their digestible amino acid profile was superior than sorghum. The report recommended that further research was warranted to assess these hybrids in terms of broiler growth, feed efficiency and carcass quality.
Research  Two tonnes of two PM hybrids (NPM3 and NPM4) were harvested, cleaned and transported to the Poultry Research and Development Centre for evaluation in 2005. Twelve tonnes of hybrid PM35 were harvested in 2006. PM hybrids were analysed for nutrient content , AME and amino acid digestibility. Two growth experiments were conducted. The first experiment was undertaken to ascertain the maximum inclusion rates in broiler diets. This was conducted in cages under environmentally controlled conditions and the second experiment was conduc